2 University by the Bay 2014: Biophysics of Amyloids and Prions

May 25-26, 2014 

Castel dell’Ovo - Naples, Italy 

Two Universities by the Bay 

Welcome to “Biophysics of Amyloids and Prions” 

Angela Lombardi and Jan Stöhr 

Co-Chairs of the Organizing Committee 

Dear Colleagues, Welcome to Naples! 

On behalf of the Scientific and Organizing Committees it is our great pleasure to welcome 

you to Napoli and to the 1 st meeting covering the “Biophysics of amyloids and prions”. 

This focused meeting has been designed to cover biophysical approaches aiming to understand 

protein misfolding into amyloids and prions. We are excited to have a diverse program 

with presentations covering 1) the mechanism of protein folding/misfolding; 2) the understanding 

of the physical basis for the pathological aggregation of peptides and proteins; 

and 3) the formation, propagation, and biology of prions and prion-like proteins. 

We would like to thank the invited speakers for accepting our invitation and we are excited 

to hear about their latest findings. 

We are thrilled about the number and quality of submitted abstracts and would like to 

thank all participants for their contribution to this exciting meeting. We are confident that 

a focused meeting in an absolutely stunning location will promote stimulating scientific 

exchange and we hope that these interaction lead to new ideas and collaborations. 

The meeting has been organized in an historical location, the beautiful Castel dell’Ovo, 

provided generously by the City of Naples. We are convinced that all participants will enjoy 

the cultural and historical heritage of Napoli. Historic buildings, beautiful churches, ancient 

fortresses as well as natural caves attract visitors worldwide to Naples. We hope that all 

participants find some time to enjoy this city and will take pleasant memories of Napoli 

back home. 

This meeting is an initiative by the University of California San Francisco and the Department 

of Chemical Science, University of Naples Federico II to facilitate international academic exchanges, 

scientific relationships, and to support collaborative research. This collaboration 

enjoys the high patronage of the mayor of Naples and the Italian Consulate in San Francisco. 

Finally, we would like to acknowledge the extremely generous funding by all our sponsors, 

which made possible the realization of this meeting. 

1 

We wish you a fantastic stay in Naples and hope that you are having a great conference.

Biophysics of Amyloids and Prions 




SCIENTIFIC COMMITTEE 

Stanley B. Prusiner 

University of California, San Francisco 

William F. DeGrado 


SCIENTIFIC PROGRAM 

Sunday, May 25 th 

9:00 - 9:45 Participants registration 

9:45 - 10:00 Introduction & welcome 

Room Compagna 

Jan Stöhr 


Angela Lombardi 

University of Naples Federico II 

ORGANIZING COMMITTEE 

OPENING LECTURE 

Chair: William F. DeGrado 

10:00 - 10:50 PL-1: Stanley Prusiner 

University of California, USA 

“Prions Causing Neurodegeneration: A Unifying Etiology and 

the Quest for Therapeutics” 

2 3 

Jan Stöhr (co-chair) 


Angela Lombardi (co-chair) 


10:50 - 11:30 Coffee break 

SESSION 1: STRUCTURAL STUDIES 

Chair: Detlev Riesner 

Flavia Nastri 


William F. DeGrado 




11:30 - 12:10 PL-2: Fabrizio Chiti 

University of Florence, Italy 

“The structural determinants of protein oligomer toxicity” 

12:10 - 12:30 OC-1: Stefano Ricagno 

University of Milano, Italy 

“D76N Beta-2 microglobulin, an amyloidogenic and pathologic 

mutant (clues on the native and on the fibrillar states)” 

12:30 - 12:50 OC-2: Holger Wille 

University of Alberta, Edmonton, Canada 

“A hybrid approach towards the structure of PrP Sc ” 

12:50 - 13:10 Exhibitor Talk (by Shimadzu) 

Roberto Castangia 

“Analysis and characterisation of Human Glycoproteins using 

MALDI TOF Mass Spectrometry” 

13:10 - 14:30 Lunch (Sponsored by Shimadzu)





SESSION 2: STRUCTURES, DYNAMICS 

AND INTERACTIONS BY NMR 

Chair: Koichi Kato 

14:30 - 15:10 PL-3: Astrid Gräslund 

Stockholm University, Sweden 

“Biophysical studies of the amyloid β peptide involved in 

Alzheimer´s disease: molecular interactions, secondary 

structure conversions and aggregation” 

15:10 - 15:30 OC-3: Giuliana Fusco 

University of Cambridge, United Kingdom 

“Unveiling the Nature of α-Synuclein in its Membrane-Bound 

State by Solid-State and Solution NMR” 

15.30 - 15:50 OC-4: Alfonso De Simone 

Imperial College London, United Kingdom 

“Order and disorder in amyloid formation” 

4 5 

15:50 - 16:40 Poster Session and Coffee break (Room Antro di Virgilio) 

SESSION 3: SPECTROSCOPIES IN FIBRILLATION PATHWAY 

Chair: Gaetano Irace 

16:40 - 17:20 PL-4: Daniel P. Raleigh 

Stony Brook University, USA 

“Islet Amyloid Formation From Basic Biophysics to Mechanisms 

of β- cell Death” 

17:20 - 17:40 OC-5: Andreas Barth 

Stockholm University, Sweden 

“Computational Infrared Spectroscopy of Proteins – 

Implications for the Spectrum of Amyloids” 

17.40 - 18:00 OC-6: Mauro Manno 

National Research Council of Italy (CNR), Palermo, Italy 

“Electrostatics promotes molecular crowding and selects the 

fibrillation pathway in fibril-forming protein solutions” 

Monday, May 26 th 

SESSION 4: AGGREGATION PATHWAY 

Chairs: Giuseppe Graziano and Jesus R. Requena 

09:00 - 9:40 PL-5: Tuomas Knowles 

University of Cambridge, United Kingdom 

“Biophysical insights into protein aggregation” 

Room Compagna 

09:40 - 10:00 OC-7: Clara Iannuzzi 

Seconda Università di Napoli, Naples, Italy 

“Insights into the molecular mechanism of glycation-stimulated 

protein aggregation” 

10:00 - 10:20 OC-8: Sanjeevi Sivasankar 

Iowa State University, Ames, USA 

“Resolving prion protein aggregation at the single 

molecule level” 

10:20 - 10:40 OC-9: Alessandro Marrone 

University “G. d’Annunzio”, Chieti, Italy 

“Investigation of the molecular electrostatic potential 

similarity in PrP model systems” 

10:40 - 11:10 Coffee break 

11:10 - 11:50 PL-6: Roland Riek 

ETH Zürich, Switzerland 

“Structure-Activity Relationship of protein aggregates in 

health and disease” 

11:50 - 12:10 OC-10: Maria Andreasen 

Interdisciplinary Nanoscience Center, Aarhus University, 

Aarhus, Denmark 

“Cytotoxic α-synuclein oligomers and their role in aggregation” 

12:10 - 12:40 Special Note (by CreAgri) 

Roberto Crea 

“Biotechnology, Mediterranean Diet and the Brain” 

20:30 - 23:00 Gala Dinner at Tennis Club Napoli 

12:40 - 14:00 Lunch (Sponsored by CreAgri)





SESSION 5: MECHANISM OF DISEASE, 

PREVENTION AND THERAPEUTICS 

Chairs: Renata Piccoli and Vincenzo Pavone 

14:00 - 14:40 PL-7: Stephen C Meredith 

The College of the University of Chicago, USA 

“Brain Seeded b-Amyoid Fibrils” 

14:40 - 15.00 OC-11: David W. Colby 

University of Delaware, Newark, United States 

“Detection of pathological tau conformations in CSF of 

patients with neurodegenerative diseases” 

15:00 - 15:40 PL-8: James Shorter 

University of Pennsylvania, Philadelphia, USA 

“Potentiated protein disaggregases to combat 

neurodegeneration” 

6 7 

15:40 - 16:00 OC-12: Annalisa Pastore 

King’s College of London, United Kingdom 

“Protein-protein interactions as a strategy towards proteinspecific 

drug design” 

16:00 - 16:20 OC-13: Andriy Kovalenko 

National Institute for Nanotechnology, University of Alberta, 

Edmonton, Canada 

“Molecular recognition and optimization of translocation of 

antiprion therapeutic agents from molecular theory of 

solvation” 

16:20 - 16:40 OC-14: Jan Bieschke 

Washington University in St Louis, USA 

“From molecular mechanisms to intervention strategies: 

Stabilizing fibrils to inhibit prion replication” 

16:40 - 17:00 Closing remarks 

PLENARY LECTURES 

PL-1 Prions Causing Neurodegeneration: A Unifying Etiology and the 

Quest for Therapeutics 

S.B. Prusiner 

PL-2 The structural determinants of protein oligomer toxicity 

F. Chiti 

PL-3 Biophysical studies of the amyloid b peptide involved in 

Alzheimer´s disease: molecular interactions, secondary structure 

conversions and aggregation 

A. Gräslund 

PL-4 Islet Amyloid Formation From Basic Biophysics to Mechanisms 

of beta-cell Death 

P. Cao, A. Abedini, C. T. Middleton, L.-H. Tu, H. Wang, A.M. Schmidt, 

M.T. Zanni, D.P. Raleigh 

PL-5 Biophysical insights into protein aggregation 

T. Knowles 

PL-6 Structure-Activity Relationship of protein aggregates in health 

and disease 

R. Riek 

PL-7 Brain Seeded b-Amyoid Fibrils 

S. Meredith 

PL-8 Potentiated protein disaggregases to combat 

neurodegeneration 

J. Shorter





ORAL PRESENTATIONS 

OC-1 D76N Beta-2 microglobulin, an amyloidogenic and pathologic 

mutant (clues on the native and on the fibrillar states) 

L. Halabelian, C. Santambrogio, E. Barbet-Massin, S. Giorgetti, A. Barbiroli, 

R. Grandori, V. Bellotti, G. Pintacuda, M. Bolognesi, S. Ricagno 

OC-2 A hybrid approach towards the structure of PrPSc 

H. Wille, E. Vázquez-Fernández, M. Vos, L. Cebey Zas, P.J. Peters, 

J.-J. Fernández, H. Young, J. R. Requena 

OC-10 Cytotoxic α-synuclein oligomers and their role in aggregation 

M. Andreasen, W. Paslawski, N. Lorenzen, S.B. Nielsen, K. Thomsen, J.D. 

Kaspersen, J.S. Pedersen, D.E. Otzen 

OC-11 Detection of pathological tau conformations in CSF of 

patients with neurodegenerative diseases 

O. Morozova, Z. March, D. Colby 

OC-12 Protein-protein interactions as a strategy towards proteinspecific 

drug design 

C. de Chiara, R.P. Menon, G. Kelly, A. Pastore 

OC-3 Unveiling the Nature of α-Synuclein in its Membrane-Bound 

State by Solid-State and Solution NMR 

G. Fusco, A. De Simone, G. Tata, V. Vostrikov, M. Vendruscolo, C.M. Dobson, 

G. Veglia 

8 9 

OC-4 Order and disorder in amyloid formation 

A. De Simone 

OC-13 Molecular recognition and optimization of translocation of 

antiprion therapeutic agents from molecular theory of solvation 

A. Kovalenko, N. Cashman, N. Blinov 

OC-14 From molecular mechanisms to intervention strategies: 

Stabilizing fibrils to inhibit prion replication 

J. Bieschke 

OC-5 Computational Infrared Spectroscopy of Proteins – Implications 

for the Spectrum of Amyloids 

E.-L. Karjalainen, A. Barth 

OC-6 Electrostatics promotes molecular crowding and selects the 

fibrillation pathway in fibril-forming protein solutions 

S. Raccosta, V. Martorana, M. Manno 

OC-7 Insights into the molecular mechanism of glycation-stimulated 

protein aggregation 

C. Iannuzzi, R. Maritato, G. Irace, I. Sirangelo 

OC-8 Resolving prion protein aggregation at the single molecule level 

C.-F. Yen, A. Kanthasamy, S. Sivasankar 

OC-9 Investigation of the molecular electrostatic potential similarity 

in PrP model systems 

R. Paciotti, L. Storchi, A. Marrone 

EXHIBITOR TALK 

ET Analysis and characterisation of Human Glycoproteins using 

MALDI TOF Mass Spectrometry 

R. Castangia, M.S.F. Choo, O. Belgacem, A. Dell 

SPECIAL NOTE 

SN Biotechnology, Mediterranean Diet and the Brain 

R. Crea





POSTER PRESENTATIONS 

P-1 Anti-amyloidogenic Property of Human Gastrokine 1 

F. Altieri, C.S. Di Stadio, G. Miselli, E. Rippa, P. Arcari 

P-2 Novel approaches in the study of the Aβ peptide oligomers: 

shining light by time-resolved Fourier-transform infrared 

spectroscopy 

M. Baldassarre, A. Barth 

P-3 Structural restraints for early Ab-oligomers from propensity for 

aggregation predicted by molecular theory of solvation 

N. Blinov, N. Cashman, A. Kovalenko 

P-4 Investigating the aggregation properties of β-synuclein and its 

effect on the aggregation of α-synuclein 

J. Brown, C. Galvagnion, A. Buell, C. Dobson 

10 11 

P-5 Molecular chaperones suppress the toxicity of misfolded protein 

oligomers 

R. Cascella, E. Evangelisti, B. Mannini, B. Tiribilli, A. Relini, J.N. Buxbaum, 

C.M. Dobson, M.R. Wilson, F. Chiti, C. Cecchi 

P-6 Computational design of Peptides that target the Amyloid 

precursor protein Transmembrane domain 

T. Lemmin, M. Chino, A. Lombardi, W.F. DeGrado 

P-7 Infrared Microspectroscopy as a Method of Choice for Structural 

Analysis of Minute Amounts of Prions 

M.L. Daus, M. Beekes, P. Lasch 

P-10 Structural properties of amyloid-like fibers unveiled by 

molecular dynamics studies 

L. Esposito, A. De Simone, L. Vitagliano 

P-11 Protein misfolded oligomer binding to membrane ganglioside 

GM1: a real-time single particle tracking study 

E. Evangelisti, R. Cascella, M. Calamai, F. Chiti, C. Cecchi, M. Stefani 

P-12 Interaction of N-methylated compounds with Aβ (25-35) 

Amyloid Peptide 

M. Grimaldi, A. Iuliano, S. Di Marino, M. Scrima, A. Polverino, G. Sorrentino, 

A.M. D’Ursi 

P-13 Oligomerisation of alpha-synuclein at nearly-physiological 

concentrations 

M. Iljina, M. Horrocks, D. Klenerman 

P-14 Flavone derivatives as inhibitors of insulin amyloid-like fibril 

formation 

R. Mališauskas, A. Botyriute, D. Dargužis, V. Smirnovas 

P-15 Nucleophosmin a-helical regions spontaneously undergo 

conformational transitions toward β-sheet-rich amyloid-like 

aggregates 

C. Di Natale, P.L. Scognamiglio, M. Leone, V. Punzo, G. Morelli, L. Vitagliano, 

D. Marasco 

P-16 Transglutaminase: an enzymatic approach to influence the 

Amyloid fibril formation 

A. Sorrentino, C.V.L. Giosafatto, I. Sirangelo, P. Di Pierro, R. Porta, 

L. Mariniello 

P-8 Environmental factors affecting the aggregation state of Ab(25- 

35): role of unsaturated omega-3 fatty acid 

M. Sublimi Saponetti, M. Grimaldi, M. Scrima, S.L. Nori, F. Bobba, A. Cucolo, 

A.M. D’Ursi 

P-9 Interaction between amyloid peptide Aβ(1-42) with model 

phospholipid bilayers 

A. Emendato, G. D’Errico, A. Falanga, S. Galdiero, D. Picone, R. Spadaccini 

P-17 The length distribution of protein biofilaments 

T.C.T. Michaels, G.A. Garcia, T.P.J. Knowles 

P-18 HGA-induced aggregation and fibrillogenesis of amyloidogenic 

proteins: implications in alkaptonuria 

L. Millucci, D. Braconi, G. Bernardini, S. Gambassi, M. Laschi, M. Geminiani, 

L. Ghezzi, A. Santucci





P-19 Comparative analysis of two amyloidogenic variants of ApoA-I 

R. Del Giudice, D.M. Monti, A. Arciello, F. Itri, R. Piccoli 

P-20 Secondary structures and conformational stability of 

β-2microglobulin mutants in solution, in single crystals and in form 

of fibrils: an FTIR study 

A. Natalello, S. Ricagno, D. Ami, L. Halabelian, M. Bolognesi, S.M. Doglia 

P-21 Examining disease-associated Aβ mutants as conformational 

strains 

M.C. Nick, J. Stöhr, W.F. DeGrado 

P-22 Interaction between Prion Protein and G-Quadruplex-Forming 

Nucleic Acids: a Biophysical Study 

B. Pagano, P. Cavaliere, V. Granata, S. Prigent, H. Rezaei, E. Novellino, 

C. Giancola, A. Zagari 

12 13 

P-23 Cu(II) induced oligomerization of amyloid-β on the millisecond 

time scale 

J. T. Pedersen, C.B. Borg, K. Teilum, L. Hemmingsen 

P-24 Fibril formation of a 3D domain swapping ribonuclease: a model 

based on the crystal structure of the protein 

A. Pica, A. Merlino, A.K. Buell, T. P.J. Knowles, E. Pizzo, G. D’Alessio, F. Sica, 

L. Mazzarella 

P-25 Evidence for a role of hydroxytyrosol in decreasing 

oligomerization in a model system of AD 

R. Crea, C. Bitler, P. Pontoniere 

P-26 Secondary nucleation in stirring induced alpha-lactalbumin 

amyloid fibril formation 

E. Rao, V. Vetri, V. Foderà, V. Militello, M. Leone 

P-27 Tetracycline and Epigallocatechin-3-Gallate differently affect the 

Ataxin-3 Fibrillogenesis and Toxicity 

M. Bonanomi, C. Visentin, A. Natalello, A. Penco, G. Colombo, A. Relini, 

S.M. Doglia, M.E. Regonesi 

P-28 Interactions of the Glial Fibrillary Acidic Protein with 

Ceftriaxone revealed through SRCD 

P. Ruzza, G. Siligardi, R. Hussain, B. Biondi, A. Calderan, G.P. Sechi 

P-29 Early determinants of human prion protein conversion 

investigated by solution-state NMR, XAFS and nanobody-assisted 

crystallography 

G. Giachin, G. Salzano, G. Ilc, I. Biljan, R.N.N. Abskharon, A. Wohlkonig, 

S.H. Soror, E. Pardon, J. Steyaert, F. Benetti, P. D’Angelo, J. Plavec, G. Legname 

P-30 Elongation of mouse Prion Protein Amyloid-like Fibrils: effect of 

temperature and denaturant concentration 

K. Milto, K. Michailova, V. Smirnovas 

P-31 α-synuclein aggregation: effect of protein charge on fibril 

elongation and membrane interaction 

A.I.M. van der Wateren, A.K. Büll, C. Galvagnion, C.M. Dobson 

P-32 Chaperon-like activity of intrinsically disordered caseins in Aβ 

fibrillogenesis 

S. Vilasi, R. Carrotta, G.C. Rappa, M.G. Ortore, C. Canale, P.L. San Biagio, 

D. Bulone 

P-33 Insights into structural features of intermediate states along 

the fibrillogenesis pathway of amyloid-like systems 

L. Vitagliano, A. De Simone, L. Esposito 

P-34 Assembly mechanism of hereditary amyloid-β variants 

promoted on their specific gangliosides 

M. Yagi-Utsumi, K. Kato, C.M. Dobson





GENERAL INFORMATION 

DATES AND VENUE 

Naples (Italy), May 25 and 26, 2014 

Castel dell’Ovo – Room Compagna 1 st floor 

Via Eldorado 3, Naples (Italy) 

ORGANIZING SECRETARIAT 

Rione Sirignano, 5 - 80121 Naples (Italy) 

Ph.: +39 081 7611086 – 668774 

Fax +39 081 664372 

Email: acanfora@mcmcongressi.it 

A desk of the Organizing secretariat will be available on site at the following 

date and time: 

• Sunday, May 25: from 8.00 to 18.00 

• Monday, May 26: from 8.30 to 15.00 

14 15 

REGISTRATION FEES 

The on-site registration fee will be as follows: 

• Participant: Euro 170,00 

• Student: Euro 120,00 

• Accompanying person: Euro 100,00 

(VAT 22% included) 

The registration fee for participants and students includes: 

• Congress badge and kit 

• Admission to scientific sessions and Exhibition 

• Conference kit 

• Coffee breaks and lunches as per the scientific program 

• Gala dinner on May 25 

The accompanying persons fee includes: 

• Gala Dinner on May 25 

• Half day excursion to the Ancient Naples on May 25 from h. 10 to 13. 

Meeting point at h. 10 at the secretariat desk at Castel dell’Ovo. 

PAYMENTS 

Payments can be made onsite by credit card or cheque 

COFFEE BREAKS AND LUNCHES 

Coffee breaks and lunches will be served at Castel dell’Ovo to all registered 

participants 

GALA DINNER 

The Gala dinner is organized on May 25 at h. 20.30 for all registered participants. 

It will be held at the exclusive Tennis Club Napoli located in Viale Dohrn, Villa 

Comunale. The Club is located at walking distance from Castel dell’Ovo (about 

15 minuts walk) 

Jacket and tie are compulsory 

ATTENDANCE CERTIFICATE 

An attendance certificate will be provided to all registered participants at the 

end of the meeting at the secretariat desk. 

SCIENTIFIC INFORMATION 

Oral presentations 

Presenters are kindly requested to deliver their presentation on a USB pen to 

the technician available in the Room Compagna (Castel dell’Ovo 1 st floor) at 

least one hour prior to their presentation. Presentation should be in Power 

Point. 

Time at presenters’ disposal is 15 minutes + 5 minutes discussion. 

Poster presentations 

Posters can be fixed on the dedicated boards located in the Room Antro di 

Virgilio (Castel dell’Ovo 1 st floor) on May 25 in the morning and removed 

on May 26 at the end of the Meeting. Boards will be numbered as per the 

scientific program, so Presenters are kindly requested to fix their poster on the 

corresponding board. 

Pins are provided by the organizing secretariat. 

TECHNICAL EXHIBITION 

An exhibition of technical devices will be available during the Meeting days in 

the Room Antro di Virgilio.





ABSTRACTS 

16 17


“Biophysics of Amyloids and Prions” 




May 25-26, 2014 - Naples, Italy 

Prions Causing Neurodegeneration: A Unifying Etiology and the 

Quest for Therapeutics 


University of California, Institute for Neurodegenerative Diseases, 

San Francisco, CA 94158, USA, stanley@ind.ucsf.edu 

PL-1 

Mounting evidence argues that prions feature in the pathogenesis of many, if not 

all, neurodegenerative diseases. Such disorders include Alzheimer’s, Parkinson’s, 

Lou Gehrig’s and Creutzfeldt-Jakob diseases as well as the fronto-temporal 

dementias. In each of these illnesses, aberrant forms of a particular protein 

accumulate as pathological deposits referred to as amyloid plaques, neurofibrillary 

tangles, Lewy bodies, as well as glial cytoplasmic and/or nuclear inclusions. The 

heritable forms of the neurodegenerative diseases are often caused by mutations 

ABSTRACTS 

in the genes encoding the mutant, prion proteins that accumulate in the CNS of 

18 19 

PLENARY LECTURES 

patients with these fatal disorders. The late onset of the inherited 

neurodegenerative diseases seems likely to be explained by the protein quality 

control systems being less efficient in older neurons and thus, more permissive for 

prion accumulation. To date, there is not a single drug that halts or even slows one 

neurodegenerative disease. 

REFERENCES 

Prusiner, S. B. (2013). Biology and genetics of prions causing neurodegeneration. Annu. Rev. Genet. 

47, 601–623. 

Jucker, M., and Walker, L. C. (2013). Self-propagation of pathogenic protein aggregates in 

neurodegenerative diseases. Nature 501, 45–51.









PL-2 

The structural determinants of protein oligomer toxicity 

Fabrizio Chiti 

Biophysical studies of the amyloid peptide involved in 

Alzheimer´s disease: molecular interactions, secondary 

structure conversions and aggregation. 

PL-3 

Department of Experimental and Clinical Biomedical Sciences, Section of Biochemistry, 

University of Florence, Italy, fabrizio.chiti@unifi.it 

Astrid Gräslund 

The conversion of soluble proteins into highly structured fibrils is associated with a 

number of human diseases, including Alzheimer’s disease, spongiform 

encephalopathies, amyloidosis and many others. Although it is widely recognized 

that misfolded protein oligomers forming as on- or off-pathway species early in the 

process or released from the fibrils are toxic to cells, the structural and biological 

factors responsible for such toxicity are poorly understood. 

Using different solutions conditions, molecular chaperones and sitedirected 

mutagenesis we have converted a sample protein, namely HypF-N, from 

its denatured state to protein oligomers featuring different levels of toxicity, ranging 

from highly toxic to nontoxic. HypF-N oligomer toxicity was found to arise from the 

ability of the oligomers, added to the extracellular medium of cultured 

neuroblastoma cells, to interact with the cell membrane and cause a dramatic 

calcium influx with consequent apoptosis. When added to cultured rat primary 

neurons, perfused into rat hippocampus slices or injected into rat brains the 

oligomers were found to colocalise with presynaptic densities 95 (PSD95), inhibit 

long term potentiation (LTP) and cause a cognitive impairment in the water maze, 

thus reproducing all the effects of A oligomers associated with Alzheimer’s 

disease. 

By comparing HypF-N oligomers formed under different solution 

conditions, we show that exposure and flexibility of hydrophobic residues on the 

oligomer surface is a key determinant of their toxicity, with the most toxic 

aggregates displaying the highest hydrophobic exposure. Using molecular 

chaperones as a tool to modulate the size of the oligomers we show that oligomer 

toxicity correlates inversely with oligomer size, with the most toxic oligomers having 

a small size. Finally, using a number of mutants of HypF-N that are able to form 

oligomers with different size, hydrophobic exposure and toxicity, we show that 

oligomer toxicity correlates with a combination of both small size and high 

hydrophobicity. 

Overall, the data indicate that the ability of protein misfolded oligomers to 

interact with the membrane and cause cell toxicity depends on two different 

determinants: (i) flexibility and solvent exposure of hydrophobic regions on the 

oligomer surface and (ii) small size of the oligomers. 

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden, 

astrid.graslund@dbb.su.se 

The amyloid (A) peptide consists of 39-43 residues and is the major 

component of neuritic plaques in the brains of Alzheimer's disease patients. We 

study the structure conversions and aggregation properties of the A(1-40) peptide 

using mainly high resolution NMR spectroscopy. At low concentrations, low 

temperatures and low ionic conditions in an aqueous solution, A(1-40) is 

monomeric. Metal ions like Cu 2+ and Zn 2+ bind to amino acid ligands in the N- 

terminus of the peptide and induce increased order in the N-terminus 1 . The 

interactions of A(1-40) with small molecules that modulate the aggregation can be 

studied in semi-stationary states by NMR. The kinetic effects on the aggregation 

process can be followed by optical spectroscopies, such as circular dichroism or 

fluorescence using suitable labels. 

By gradually adding a detergent (membrane mimetic) such as lithium 

dodecyl sulphate (LiDS) or SDS to a dilute aqueous solution of A(1-40), 

secondary structure conversions of A(1-40) can be observed 2 . An initial transition 

involves conversion of the weakly structured peptide to -sheet structure, 

concomitant with formation of co-aggregates. These co-aggregates, formed 

relatively slowly on a min/hr timescale, are in much faster dynamic exchange with 

free peptide 3 . The dynamic process, on a ms time scale, can be studied by NMR 

relaxation dispersion. Parallel studies with Small Angle X-ray Scattering (SAXS) 

give information about the size and slow growth of the co-aggregates. At LiDS 

concentrations close to the CMC or above, a new kind of structure transition makes 

the peptide rearrange to form a partly -helical structure, concomitant with 

disaggregation and formation of normal LiDS micelles which apparently partly 

dissolve the aggregates. This -helical structure is similar to that previously 

observed by NMR at high detergent concentrations 4 . A β-hairpin structure can be 

induced in A(1-40), in a specific complex with a small designed protein 

(Affibody) 5 . -structure in dynamic co-aggregates is also induced by interactions 

with certain small molecules such as Congo red 6 . The β-hairpin structure with the 

weakly structured N-terminus appears to be a basic unit for formation of higher 

ordered amyloid structures. 

Taken together, the results give evidence of a “chameleon” A peptide, whose 

structures and aggregation propensities in solution are strongly influenced by 

20 21

Biophysics “Biophysics of Amyloids of and Amyloids Prions and Prions” 




Two Universities by the BayMay 25-26, 2014 - Naples, Italy 

PL-3 


molecular interactions with e.g. metal ions, small molecules and/or 

biomembranes 7 . This behavior is important for the formation of the amyloid states 

of the peptide in vivo and for its neurotoxic activities, which are associated with 

Alzheimer´s disease. 

Islet Amyloid Formation From Basic Biophysics to Mechanisms 

of beta-cell Death 

Ping Cao 1 , Andisheh Abedini 2 , Chris T. Middleton 3, Ling-Hsien Tu 1 , 

Hui Wang 1 , Ann Marie Schmidt 2 , Martin T. Zanni 3 , 

Daniel P. Raleigh 1 

1 Department of Chemistry, Stony Brook University, Stony Brook, NY, 11794-3400, USA, 

daniel.raleigh@stonybrook.edu 

2 Diabetes Research Program, Department of Medicine, NYU School of MedicineNew York, NY, USA. 

3 Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706-1393, USA. 

REFERENCES 

1. Danielsson, J., Pierattelli, R., Banci, L. and Gräslund, A. FEBS J. 274, 46-59 (2007). 

2. Wahlström, A., Hugonin, L., Peralvarez-Marin, A., Jarvet, J. and Gräslund, A. FEBS J. 275, 5117- 

5128 (2008). 

3. Abelein, A., Kaspersen, J.D., Nielsen, S.B., Vestergaard Jensen, G., Christiansen, G., Pedersen, 

J.S., Danielsson, J., Otzen, D.E. and Gräslund, A. J. Biol. Chem. 288 (2013), 23518-23528. 

4. Jarvet, J., Danielsson, J., Damberg, P., Oleszczuk, M. and Gräslund, A. J. Biomol. NMR 39, 63-72 

(2007). 

Amyloid formation by the hormone, islet amyloid polypeptide (IAPP or amylin), 

5. Lindgren, J., Segerfeldt, P., Sholts, S., Gräslund, A., Eriksson Karlström, A., Wärmländer, S. J. Inorg. 

causes pancreatic beta-cell death, consequences of which include diabetes and 

Biochem. 120, 18-23 (2013). 

6. Abelein, A., Lang, L., Lendel, C., Gräslund, A. and Danielsson, J. FEBS Lett. 586, 3991-3995 (2012). 

islet transplant failure. The nature of the toxic species produced during IAPP 

7. Wärmländer, S., Tiiman, A., Abelein, A., Luo, J., Jarvet, J., Söderberg, K., Danielsson, J. and 

amyloid formation and the cellular mechanisms by which it elicits cell death are 

Gräslund, A. ChemBioChem 14, 1692-1704 (2013). 

unknown. We define the toxic entity produced during IAPP amyloidosis: transient, 

22 23 

soluble, loosely packed, low order, pre-fibrillar, oligomeric lag phase intermediates 

with modest beta-sheet structure, which unregulated pro inflammatory markers and 

are ligands for the pattern recognition receptor RAGE. We demonstrate how the 

emerging technique of two dimensional IR spectroscopy can provide unique site 

specific time resolved information about the pathway of amyloid assembly. 

PL-4

“Biophysics of Amyloids of and Amyloids Prions and Prions” 





PL-5 


Biophysical insights into protein aggregation 

Tuomas Knowles 

Department of Chemistry, University of Cambridge, UK, tpjk2@cam.ac.uk 

The molecular basis for protein aggregation has been challenging to probe since 

many of the conventional approaches that have been highly successful in 

biophysics work best for homogeneous preparations of soluble proteins. However, 

protein aggregation is characterised by heterogeneity, with structures formed 

ranging from soluble oligomers to insoluble high molecular weight aggregates, 

including amyloid fibrils. This talk outlines some of the approaches that we have 

developed and applied to the problem of protein aggregation, including 

microfluidics and kinetic analysis. Using such techniques we aim to access the 

fundamental molecular level events that underlie the transition of proteins from 

their normal soluble states into aggregated forms, and find ways of controlling and 

curtailing the onset and progression of the aggregation process. 


Structure-activity relationship of protein aggregates in health 

and disease 

Roland Riek 

ETH Zürich, Physical Chemistry, Switzerland, roland.riek@phys.chem.ethz.ch 

Amyloids are highly ordered cross--sheet protein aggregates that are associated 

with many diseases including Alzheimer’s, Creutzfeldt-Jakob and Parkinson’s 

disease, but also have significant biological functions such as hormone storage in 

secretory granules and skin pigmentation in mammals. 

The cross--sheet entity, composed of an indefinitely repeating inter-molecular - 

sheet motif, is unique among protein folds. It can grow by recruitment of the 

corresponding amyloid protein, while its repetitiveness can translate what would be 

a non-specific activity as monomer into a potent one through cooperativity. 

Furthermore, the one-dimensional crystal-like repeat in the amyloid provides a 

structural framework for polymorphisms that arise from energetically similar but 

kinetically uncoupled alternative -sheet packings or even side chain 

conformations. Because of these properties, the activities of amyloids are manifold 

and include peptide storage, template assistance, loss of function, gain of function, 

generation of toxicity, membrane binding, infectivity etc. In the presentation we 

summarise structure activity relationships of the HET-s prion, peptide hormone 

storage, and -synuclein aggregation. 

24 25 

PL-6

PL-7 



Brain Seeded -Amyoid Fibrils 

Stephen Meredith 


Departments of Pathology, and Biochemistry and Molecular Biology, The University of Chicago, 

Chicago, IL (USA), scmeredi@uchicago.edu 

-Amyoid (A) peptides aggregate into soluble oligomers and fibrils, and these 

aggregates are the putative neurotoxins believed to cause neuronal damage and 

death in Alzheimer’s disease. A, like other aggregating peptides, contrasts with 

“competent” proteins. Whereas competent proteins fold into one, or a small 

number of unique three-dimensional structures, determined by amino acid 

sequence, the three-dimensional structure of A aggregates is not uniquely 

determined by amino acid sequence. Thus, A peptide forms polymorphic 

aggregates, whose structures depend on fibril growth conditions. This seminar will 

describe the structure of A fibrils, both those made in vitro from synthetic A 

peptide, and those formed using human Alzheimer’s Disease brain A fibrils. The 

latter fibrils are made by extracting amyloid from brains of patients with Alzheimer’s 

disease at autopsy, and then growing seeded fibrils from this extract. Such 

seeding procedures yield replicate fibrils, which can be studied by biophysical 

methods, especially solid-state NMR spectroscopy and electron microscopy. We 

will focus on experiments using tissue from two Alzheimer’s disease patients with 

distinct clinical histories. Fibrils seeded using brain amyloid from each of these 

patients each showed a single predominant A40 fibril structure. Furthermore, 

despite the typical polymorphism of purely synthetic amyloid fibrils made in vitro, 

for each patient, brain amyloid from three different brain regions showed a single 

structure per patient. The structures of the fibrils from the two patients, however, 

were different from each other. A molecular structural model has been developed 

for A40 fibrils seeded from the first of these patients. This structure reveals 

features that distinguish in vivo- from in vitro-produced fibrils. The finding of a 

single structure in different parts of the brain from each patient strongly suggests 

that fibrils may spread from a single nucleation site to other parts of the brain. 

Furthermore, the fact that the two patients had different fibril structures indicates 

that fibril structure may reflect different fibrillization conditions in different patients. 

Since these patients had different clinical presentations, these data also suggest 

that structural variations may correlate with phenotypic variations in AD. Structurespecific 

amyloid imaging and therapeutic agents may be an important future goal. 

More recently, we have examined replicate fibrils seeded from the brains of 

several additional patients with Alzheimer’s Disease, Cerebral Amyloid Angiopathy, 

and Alzheimer’s Disease lesions at autopsy but no clinical history of cognitive 






dysfunction. As in some of our earlier studies, we observed that some patients 

have two or more (up to four, thus far) fibril structures, as indicated by resonances 

in solid-state NMR spectra. Structure-specific amyloid imaging and therapeutic 

agents may be an important future goal. Hence, our ultimate goal is to 

characterize a possible structure-malfunction relationship that will give us greater 

understanding of the relationship between aggregate structure and disease 

phenotype. 

26 27 

PL-7







PL-8 

Potentiated protein disaggregases to combat 

neurodegeneration 

James Shorter 

Department of Biochemistry & Biophysics, University of Pennsylvania, Philadelphia, PA (USA), 

jshorter@mail.med.upenn.edu 

There are no therapies that reverse the deleterious protein-misfolding events that 

underpin various fatal neurodegenerative diseases including amyotrophic lateral 

sclerosis (ALS) and Parkinson disease (PD). Hsp104, a conserved hexameric 

AAA+ prion disaggregase from yeast, solubilizes disordered aggregates and 

amyloid, but has no metazoan homologue and only limited activity against various 

human neurodegenerative disease proteins. Here, I will present our efforts to 

reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by 

mutating single residues in the middle domain or first AAA+ nucleotide-binding 

ABSTRACTS 

28 domain. Surprisingly, loss of amino acid identity at specific, but disparate positions, 

29 

rather than mutation to specific side chains, unleashes Hsp104 activity against 

ORAL PRESENTATIONS 

diverse substrates. These potentiated Hsp104 variants effectively restore proper 

protein localization and clear protein aggregates. They also suppress dopaminergic 

neurodegeneration in a C. elegans PD model. Potentiating mutations rewire how 

Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, eliminate 

dependence on Hsp70, and empower ATPase, translocation, and unfoldase 

activity.









OC-1 

D76N Beta-2 microglobulin, an amyloidogenic and pathologic 

mutant 

(clues on the native and on the fibrillar states) 

Levon Halabelian1, Carlo Santambrogio2, Emeline Barbet-Massin4, Sofia 

Giorgetti5 Alberto Barbiroli3, Rita Grandori4, Vittorio Bellotti6, Guido 

Pintacuda4, Martino Bolognesi1 Stefano Ricagno1 

1 Dept. of Bioscience, Univ. of Milan (Italy); Stefano.ricagno@unimi.it 

2 Dept. of Biotech and Biosc. Univ. of Milan Bicocca (Italy) 

3 Dept. DeFENS, Univ. of Milan (Italy); 

4 CRMN, CNRS/ENS Lyon/UCB Lyon 1 Lyon (France); 

5 Inst. of Biochemistry "Castellani", Univ. of Pavia (Italy) 

6 Center for Amyloidosis and Acute Phase proteins, UCL, London (UK). 

Beta-2 microglobulin (b2m) is an aggregation prone protein that is responsible for a 

human disorder known as dialysis related amyloidosis. In patient with kidney failure 

b2m is accumulated to high serum level eventually aggregates as amyloid fibrils. In 

2012 a new systemic familial amyloidosis was reported: an unreported b2m mutant 

(D76N) is the etiological agent of such disease. In vitro from the biophysical point 

of view the D76N b2m is much less stable and more amyloidogenic than wt b2m; 

however, its crystal structure reveals few minor conformational changes compared 

with the wt protein [1]. 

30 31 

Such an unstable protein should be targeted for degradation by the Unfolded 

Protein Response in the ER, however this is not the case. Physiologically, b2m is 

part of the Major Histocompatibility Complex (MHC). In order to understand what is 

the role of the MHC complex in the protection of D76N variant against degradation, 

the biophysical and structural aspects of MHC complex bearing the D76N mutation 

have been investigated and evaluated. The crystal structure of the mutated MHC 

was determined and several in solution techniques showed that the MHC assembly 

exerts a remarkable stabilisation effect on the D76N variant so that the mutated 

MHC has structural and biophysical properties utterly similar to the wild type 

complex [2]. 

Finally some preliminary solid state NMR data on D76N in crystal and fibrillar will 

be shown. 

[1] Valleix et al. Hereditary systemic amyloidosis due to Asp76Asn variant β2-microglobulin. NEJM 2012 

Jun 14;366(24):2276-83 

[2] Halabelian et al. Class I Major Histocomapatibility Complex: the Trojan horse for secretion of 

amyloidogenic β2-microglobulin. J Biol Chem. 2014 Feb 7;289(6):3318-27. 

A hybrid approach towards the structure of PrP Sc . 

H. Wille, 1,2 E. Vázquez-Fernández, 1,2,3 M. Vos, 4,5 L. Cebey Zas, 1,2 P. J. 

Peters, 5,6 J.-J. Fernández, 7 H. Young, 1 J. R. Requena, 3 

1 Department of Biochemistry, University of Alberta, Edmonton (Canada) 

2 Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton (Canada) 

3 CIMUS Biomedical Research Institute & Department of Medicine, University of Santiago de 

Compostela-IDIS (Spain) 

4 FEI Company, Eindhoven (The Netherlands) 

5 Netherlands Cancer Institute, Amsterdam (The Netherlands) 

6 Institute of Nanoscopy, Maastricht University, Maastricht (The Netherlands) 

7 National Centre for Biotechnology - CSIC, Madrid, (Spain) 

The structure of the infectious prion protein (PrP Sc ) and that of its proteolytically 

truncated homologue (PrP 27-30) have eluded experimental determination due to 

their insolubility and propensity to aggregate. Molecular modelling has been used 

to predict their structures, but the various modelling approaches produced 

conflicting models indicating the limitations of this method. In absence of a threedimensional 

(3D) structure, a variety of experimental techniques have been used 

to gain insights into the fold of PrP Sc . Negative stain electron microscopy, X-ray 

fiber diffraction, and molecular modelling indicated that the β-sheets of PrP Sc form 

a β-helix or β-solenoid structure with a height of four β-strands (rungs) per 

molecule (= 19.2 Å). 

In our current efforts to analyze the structures of delta-GPI PrP Sc and PrP 

27-30 we have employed a hybrid approach. In particular, the helical periodicity 

that is inherent to most amyloid fibrils can be used to generate a three-dimensional 

structure from two-dimensional electron micrographs and electron tomograms. 

Cryo low-dose electron micrographs of delta-GPI PrP 27-30 amyloid fibrils 

routinely exhibit a 4.8 Å spacing, confirming the presence of β-strands in a cross-β 

configuration. The 3D reconstructions of individual fibrils show two protofilaments 

coiled around a common axis with 4.8 Å striations running perpendicular to the 

fibrils axis, suggesting that the β-strands of PrP Sc are being visualized. The lack of 

α-helices in these reconstructions is notable, strengthening earlier arguments 

against any remaining α-helical structure. Furthermore, single-particle analyses of 

15,942 individual fibril segments show a repeating pattern of ~40 Å densities with 

~20 Å sized subunits, confirming earlier measurements regarding the molecular 

height of PrP Sc . Moreover, the observed ~40 Å periodicity suggests a head-to-head 

arrangement of the PrP Sc molecules along the fibril axis. 

ACKNOWLEDGEMENTS 

Generous funding was provided by grants from the Alberta Prion Research Institute (APRI), the Alberta 

Livestock & Meat Agency (ALMA), and EU FP7 222887 (Priority). 

OC-2









OC-3 

Unveiling the Nature of α-Synuclein in its Membrane-Bound 

State by Solid-State and Solution NMR. 

Giuliana Fusco 1 , Alfonso De Simone 2,* , Gopinath Tata 3 , Vitaly Vostrikov 3 , 

Michele Vendruscolo 1 , Christopher M. Dobson 1,* , Gianluigi Veglia 3,* 

Order and disorder in amyloid formation 

Alfonso De Simone 

NMR centre and Department of Life Science, Imperial College London (UK), adesimon@imperial.ac.uk 

OC-4 

1 

Department of Chemistry, University of Cambridge, Cambridge (UK). 

2 

Department of Life Sciences, Imperial College, London (UK). 

3 

Department of Chemistry & Department of Biochemistry, Molecular Biology & Biophysics, University of 

Minnesota, Minneapolis (USA). vegli001@umn.edu; cmd44@cam.ac.uk; adesimon@imperial.ac.uk 

α-synuclein (αS) is a protein involved in neurotransmitter release in presynaptic 

terminals, and whose aberrant aggregation is associated with Parkinson’s disease. 

In dopaminergic neurons, αS exists in a tightly regulated equilibrium between 

water-soluble and membrane-associated forms. Understanding the structure and 

dynamics of the membrane-bound state of αS is a major priority to clarify how for 

this protein the balance between functional and dysfunctional processes can be 

regulated. Using a combination of solid-state and solution-state NMR 

spectroscopy, we characterised the conformations of αS bound to lipid membranes 

mimicking the composition and physical properties of synaptic vesicles. Our 

approach proved to be highly effective in enabling the fine tuning between 

structural order and disorder in the membrane-bound state of αS to be probed 

directly without requiring any chemical modification of the protein or changes to its 

amino acid sequence. The study evidences key regions of the protein possessing 

distinct structural and dynamical properties and having specific roles in determining 

the way the protein partitions between membrane-bound and unbound states. 

Taken together, our data define the nature of the interactions of αS with biological 

membranes and provide insights into their function as well as in the processes of 

αS aggregation under pathological conditions. 

Protein molecules adopt well-defined functional and soluble states under 

physiological conditions. In some circumstances, however, proteins can aggregate 

into fibrillar assemblies designated as amyloid fibrils. In vivo these processes are 

normally associated with severe pathological conditions. It is now clear that the 

primary mechanism for controlling protein aggregation involves the existence of 

intrinsic free energy barriers that govern the propensity to engage functional 

protein-protein interactions by disfavouring unwanted aggregation events. Our 

understanding of these barriers is currently limited. By using novel methods at the 

interface between NMR experiments and statistical mechanics, we investigated 

how conformational free energy landscapes of proteins control the population of 

dangerous aggregation-prone species [1, 2] and how intrinsically disordered 

regions modulate the propensity of proteins to aggregate [3]. Our studies identified 

the nature of the balance between structural order and disorder as a crucial 

modulator in the processes of protein aggregation into amyloids. 

32 33 


Financial support from the Leverhulme Trust, EMBO, EPSRC and EU FP7 is acknowledged for this 

works 

REFERENCES 

[1] Camilloni C, Schaal D, Schweimer K, Schwarzinger S, De Simone A, 2012, Biophys J 102(1):158- 

167. 

[2] De Simone A, Dhulesia A, Soldi G, Vendruscolo M, Hsu ST, Chiti F, Dobson CM, 2011, Proc Natl 

Acad Sci USA 108(52):21057-62. 

[3] De Simone A, Kitchen C, Kwan A, Sunde M, Dobson CM, Frenkel D, 2012, Proc Natl Acad Sci USA 

109(18):6951-6. 


Financial support from Parkinson’s UK, Wellcome Trust, MRC, Leverhulme Trust, NIH









OC-5 

Computational Infrared Spectroscopy of Proteins - Implications 

for the Spectrum of Amyloids 

Electrostatics promotes molecular crowding and selects the 

fibrillation pathway in fibril-forming protein solutions. 

OC-6 

E.-L. Karjalainen, A. Barth 

Samuele Raccosta, 1 Vincenzo Martorana, 1 Mauro Manno 1 

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 

barth@dbb.su.se 

1 National Research Council of Italy (CNR), Institute of Biophysics (IBF), Via Ugo La Malfa 153, 

Palermo (Italy), mauro.manno@cnr.it. 

The protein backbone gives rise to the amide I absorption in the infrared spectrum 

of proteins. It is sensitive to secondary structure but also to additional structural 

features, some of which are discussed here. We have developed a Matlab program 

to calculate the amide I absorption of proteins [1]. It gives good agreement 

between simulation and experiment although this task was deliberately made more 

difficult by the use of resolution enhanced spectra. These spectra contain more 

distinct features than unprocessed absorption spectra, which have to be simulated 

adequately. The simulation protocol includes (i) the effects of transition dipole 

coupling for long range interaction between amide vibrations, (ii) nearest neighbour 

coupling from density functional theory data, and (iii) influences on the intrinsic 

frequency by (iiia) the local conformation, (iiib) hydrogen bonding to other amide 

groups, and (iiic) hydrogen bonding to water. 

Earlier calculations which included only long-range coupling (i) indicate that 

stacking of beta sheets, as it occurs in the formation of amyloid fibrils, shifts the 

amide I band maximum upwards by a few cm -1 [2]. Such shifts can easily be 

detected experimentally and the effect should be considered in the interpretation of 

experimental spectra. A similar upshift has been calculated for the vibrational 

coupling between helices, which explains the high amide I maximum of 

bacteriorhodopsin [3]. 

34 35 

REFERENCES 

[1] E-L. Karjalainen, T. Ersmark, A. Barth (2012), J. Phys. Chem. B, 2012, 116, 4831−4842, 

(http://dx.doi.org/10.1021/jp301095v). Optimization of model parameters for describing the amide I 

spectrum of a large set of proteins. 

[2] E-L. Karjalainen, H. Kumar, A. Barth, J. Phys. Chem. B, 2011, 115, 749–757, 

(http://dx.doi.org/10.1021/jp109918c). Simulation of the amide I absorption of stacked beta-sheets. 

[3] E-L. Karjalainen, A. Barth, J. Phys. Chem. B, 2012, 116, 4448−4456, 

(http://dx.doi.org/10.1021/jp300329k). Vibrational coupling between helices influences the amide I 

infrared absorption of proteins. Application to bacteriorhodopsin and rhodopsin. 

The role of intermolecular interaction in fibril-forming protein solutions and its 

relation with molecular conformation is a crucial aspect for the control and inhibition 

of amyloid structures. Here, we use optical spectroscopies, x-ray, neutron and light 

scattering to study the fibril formation and the protein-protein interactions of 

different model proteins: lysozyme, insulin and alpha-chymotrypsinogen. In the 

case of lysozyme, the monomeric solution is kept in a thermodynamically 

metastable state by strong electrostatic repulsion, even in denaturing conditions. At 

high temperature proteins are driven out of metastability through conformational 

sub-states, which are kinetically populated and experience lower activation energy 

for fibril formation. This explains how electrostatic repulsion may act as a 

gatekeeper in selecting the appropriate pathway to fibrillation. [1] 

The protein density fluctuations are measured by light scattering and analyzed in 

terms of classical second virial coefficient approach and a new model-free 

approach based on Kirkwood-Buff integrals. [2] The latter allows taking into 

account the role of density fluctuations even at high concentrations and to highlight 

a regime dominated by interaction and a regime dominated by concentrations. 

Such a “crowding” effect is observed at moderately high concentrations due to 

repulsion. Further, by photon correlation spectroscopy (PCS) and fluorescence 

correlation spectroscopy (FCS) we measure protein collective and self-diffusion. 

Both PCS and FCS evidence how an increase of concentration hinders protein 

self-diffusion, likely due to both hydrodynamics and crowding effect. [3] 

REFERENCES 

[1] S. Raccosta, V. Martorana, M. Manno J. Phys. Chem. B, 2012, 116, 12078-12087. 

[2] M. Blanco, T. Perevozchikova, V. Martorana, M. Manno, C. J. Roberts, 2014, submitted. 

[3] S. Raccosta, V. Martorana, M. Manno J. Phys. Chem. B, 2014, submitted.









OC-7 

Insights into the molecular mechanism of glycation-stimulated 

protein aggregation 

Resolving prion protein aggregation at the single molecule level 

Chi-Fu Yen 1,2 , Anumantha Kanthasamy 3 and Sanjeevi Sivasankar 1,2 

OC-8 

C. Iannuzzi, R. Maritato, G. Irace, I. Sirangelo 

Dept. of Biochemistry, Biophysics and General Pathology, Seconda Università di Napoli, Naples, Italy, 

clara.iannuzzi@unina2.it. 

Reducing sugars play important roles in modifying proteins, forming advanced 

glycation end-products (AGEs) in a non-enzymatic process named glycation. 

Several proteins linked to neurodegenerative diseases, such as ß-amyloid, tau, 

prions and transthyretin have been found glycated in vivo. 

Although it is now accepted that there is a direct correlation between AGEs 

formation and amyloidosis, several questions still remain unanswered: whether 

glycation is the triggering event or just an additional factor acting on the 

aggregation pathway. In this study we have investigated the effect of glycation on 

the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although 

this protein is not related to any amyloid disease, it represents a good model to 

resemble proteins that intrinsically evolve toward the formation of amyloid 

aggregates in physiological conditions. Thus, this protein allows biophysical studies 

without the interference of denaturing agents [1-3]. We show that D-ribose rapidly 

induces the W7FW14F apomyoglobin to generate AGEs and this strongly 

contributes to accelerate its aggregation kinetics. We propose that ribosylation of 

the W7FW14F apomyoglobin induces the formation of intermolecular cross-links 

that strongly reduce protein flexibility thus promoting fibril formation [4]. On the 

other side, glycation of the wild-type apomyoglobin promotes conformational 

changes without inducing amyloid aggregation. These results indicate that AGEs 

formation cannot be considered a trigger factor but an active player in later stages 

of the amyloid aggregation. 

36 37 

REFERENCES 

[1] I. Sirangelo, C. Malmo, C. Iannuzzi, A. Mezzogiorno, M. R. Bianco, M. Papa, G. Irace J. Biol. 

Chem., 2004, 279, 13183-13189. 

[2] C. Malmo, S. Vilasi, C. Iannuzzi, S. Tacchi, C. Cametti, G. Irace, I. Sirangelo Faseb Journal, 2006, 

20, 346-347. 

[3] C. Iannuzzi, S. Vilasi, M. Portaccio, G. Irace, I. Sirangelo Protein Science, 2007, 16, 507-516. 

[4] C. Iannuzzi, R. Maritato, Irace, I. Sirangelo. PLoS ONE, 2013, 8, e80768. 

1 Dept. of Physics and Astronomy, Iowa State University, Ames, IA (USA) 

2 Dept. of Electrical and Computer Engineering, Iowa State University, Ames, IA (USA) 

3 College of Veterinary Medicine, Iowa State University, Ames, IA (USA) 

Corresponding Author (Sanjeevi Sivasankar): sivasank@iastate.edu 

In Transmissible Spongiform Encephalopathies, cellular prion proteins (PrP) 

misfold to form proteinase-K resistant, neurotoxic, aggregates. Cellular PrP consist 

of an unstructured, N-terminal region containing four octapeptide repeats that bind 

divalent ions and an α-helix rich, C-terminal domain. However, biophysical studies 

of PrP aggregation have primarily focused on PrP lacking the N-terminal region; 

the role of octapeptide repeats in PrP binding remains unresolved. Furthermore, 

while divalent metal ions are known to play an important role in PrP aggregation, 

the underlying mechanisms are poorly understood at molecular level. 

Here we use single molecule force measurements with an Atomic Force 

Microscope (AFM) and single molecule fluorescence imaging with a Confocal 

Fluorescence Microscopy (CFM) to (i) identify the role of the N-terminal 

octapeptide repeats in PrP aggregation, (ii) determine how divalent metal ions 

promote aggregation in vitro, and (iii) isolate a monomeric form of PrP that is 

resistant to Proteinsae-K digestion. Using single molecule AFM force 

measurements, we measure the rate constants for the aggregation of PrP with and 

without octapeptide repeats and in the presence of different divalent metal ions. 

We show that while Cu 2+ ions increase the affinity for full length PrP aggregation 

almost a thousand fold, exposure to Ni 2+ , Zn 2+ , and Mn 2+ does not significantly 

enhance PrP binding affinity. The effect of Cu 2+ ions is eliminated in PrP truncation 

mutants that lack the N-terminal domain. Finally, using CFM we demonstrate that 

the binding of Cu 2+ to the N-terminal octapeptide repeats induces a conformational 

change that makes PrP monomers resistant to Proteinase-K digestion. Our results 

provide the first quantitative, biophysical characterization of PrP aggregation at the 

single molecule level.









OC-9 

Investigation of the molecular electrostatic potential similarity in 

PrP model systems 

Roberto Paciotti 1 , Loriano Storchi 1 , Alessandro Marrone 1 

Cytotoxic α-synuclein oligomers and their role in aggregation 

M. Andreasen 1,2 , W. Paslawski 1,2 , N. Lorenzen 1 , S. B. Nielsen 1,2 , K. 

Thomsen 1 , J. D. Kaspersen 1,3 , J. S. Pedersen 1,3 and D. E. Otzen 1,2 

OC-10 

1 Dept. of Pharmacy, Università “G d’Annunzio” di Chieti-Pescara, Italy, amarrone@unich.it 

Misfolding can be considered the structural alteration with the most cogent 

pathogenic impact on protein functionality due to the involvement of both 

secondary and tertiary structure impairment. Prion diseases are fatal 

neurodegenerative disorders characterized by the post-translational misfolding of 

the cellular prion protein, PrP C , yielding the pathological form PrP Sc . [1,2] Recently, 

the role of high [Ca 2+ ] in catalyzing the formation of PrP Sc has been evaluated. [3] It 

was found that the binding of one Ca 2+ ion between the H1 helix and the H2-H3 

loop of PrP C may induce a specific pattern of charges on the protein surface 

significantly similar to that detected on the pathogenic E200K mutant. [3] 

Here, a multi-level 

computational workflow was 

designed to study in major 

details the effect of Ca 2+ 

binding on the PrP C 

molecular properties. The 

specific patterns of either 

charged or hydrophobic 

groups on the surface of 

either wild type (I), E200K (II) 

or Ca 2+ -bound (III) PrP C were 

obtained by the use of 

multiple structure subsets. By 

the analysis of molecular 

electrostatic potential (MEP) and hydrophobic fields, we obtained a measure of the 

electrostatic and hydrophobic similarity within the considered PrP C models as a 

function of the protein space. Interestingly, we found that Ca 2+ binding at PrP C 

increased the MEP similarity with the pathogenic E200K mutant which parallels the 

catalyzing effect of [Ca 2+ ] in the formation of PrP Sc . [3] 

38 39 

[1] S. B. Prusiner N. Engl. J. Med., 2001, 344, 1516–1526. 

[2] J. Collinge Annu. Rev. Neurosci., 2001, 24, 519–550. 

[3] S. Sorrentino, T. Bucciarelli et al. PLoSONE, 2012, 7, e38314. 

1 Interdisciplinary Nanoscience Center (iNANO), Aarhus University (Denmark) 

2 Dept. of Molecular Biology and Genetics, Aarhus University (Denmark) 

3 Department of Chemistry, Aarhus University (Denmark) 

Many neurodegenerative diseases (ND) are connected with formation of amyloid 

deposits with cytotoxic oligomers as the main suspected culprit of pathology.[1,2] 

These oligomers are generally viewed as transient species present in very small 

amounts making them hard to study.[3] Here we report on two α-synuclein (αSN) 

oligomeric species and their role in aggregation. 

The two oligomers of αSN display same overall morphology and are found to be 

ellipsoid with a high degree of flexibility as seen by small angle X-ray scattering. 

They also have the same degree of β-sheet structure which is intermediate to that 

of the disordered monomer and the fibrils, and both oligomers are capable of 

permeabilizing lipid vesicles in vitro.[4] However one of the oligomers (oligo I) is in 

equilibrium with the monomer and is probably an on-pathway oligomer. This 

oligomer show a higher degree of protection against hydrogen/deuterium exchange 

(HDX) for the back-bone amide groups but it is a short lived species. The other 

oligomer (oligo II) is less protected against HDX but displays higher stability.[5] 

This second type of oligomer is an off-pathway oligomer which does not elongate 

fibril seeds. Furthermore oligo II resist dissociation at extreme pH values, elevated 

temperatures up to 120 °C, in the presence of SDS and using urea dissociation a 

ΔG dis of 2.5 ± 0.1 kcal/mol is found. Even after prolonged incubation at 37 °C no 

dissociation of monomers from oligo II are seen. However oligo II associate to form 

non-fibrillar aggregates which retain the secondary structure of the oligomers. 

Hence these oligomers are neither elusive nor short-lived making it even more 

important to further understand their role in αSN aggregation in vivo and the 

relationship between the two oligomer types. 


Financial support from the Michael J. Fox Foundation, The Danish Council for Independent Research | 

Natural Sciences , the Danish Research Foundation (inSPIN) and Lundbeckfonden is acknowledged. 

REFERENCES (STYLE LIGHT REFER) 

[1] Chiti, F.; Dobson, C. M. Annual review of biochemistry 2006, 75, 333 

[2] Lashuel, H. A.; Hartley, D.; Petre, B. M.; Walz, T.; Lansbury, P. T., Jr. Nature 2002, 418, 291 

[3] Uversky, V. N. Febs J 2010, 277, 2940 

[4] Lorenzen, N.; Nielsen, S.; Buell, A.; Kaspersen, J.; Arosio, P.; Vad, B.; Paslawski, W.; Christiansen, 

G.; Hansen, Z.; Andreasen, M.; Enghild, J.; Pedersen, J.; Dobson, C.; Knowles, T.; Otzen, D. JACS 

2014, 136, 3859 

[5] Paslawski, W.; Mysling, S.; Thomsen, K.; Jørgensen, T.J.D.; Otzen, D.E. Angew. Chem. In press

OC-11 




Detection of pathological tau conformations in CSF of patients 

with neurodegenerative diseases 

O. Morozova 1 , Z. March 1 , D. Colby 1 

1 Dept. of Chemical and Biomolecular Engineering, University of Delaware (USA), colby@udel.edu 

The formation of tau amyloids is associated with more than twenty 

neurodegenerative diseases, including Alzheimer’s disease (AD). Although it is 

established that tau protein concentrations are elevated in CSF of patients with 

many of these diseases, its conformational state there has been largely 

unexplored. We have developed a method for the detection and amplification of tau 

amyloid strains that preserves their underlying structures. We have performed 

ultrastructural analysis on these tau strains using electron microscopy, circular 

dichroism, and chemical denaturation. Using this method, we can detect 

amyloidogenic tau in the CSF of patients with AD, Corticobasal Degeneration 

(CBD), and Progressive Supranuclear Palsy (PSP). Tau amyloids present in CSF 

consist of strains which appear to be distinct for each disease. When recombinant 

tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, 

the amyloid formed shares many of the structural features of the PHF seeds. In 

contrast, tau amyloids formed with heparin as an inducing agent—a common 

biochemical model of tau misfolding—are structurally distinct from brain-derived 

PHFs. Our findings have implications for the prion-like propagation of misfolded tau 

protein in the pathogenesis of neurodegenerative diseases and provide new insight 

into the tau amyloid strains present in disease states. 






Protein-protein interactions as a strategy towards proteinspecific 

drug design 

Cesira de Chiara, Ramesh P. Menon, G. Kelly, A. Pastore 1 

1 Dept. of Clinical Neurosciences, King’s College LondonI, London (UK), 

apastor@nimr.mrc.ac.uk. 

Protein aggregation and amyloid formation seems to be a universal structural 

solution that most if not all proteins may be able to adopt. It is therefore important 

to understand the mechanism that protect proteins from aggregating and allow 

correct protein function. I shall discuss different examples that suggest the 

importance, Here, we show how partner recognition of the AXH domain of the 

transcriptional co-regulator ataxin-1 is fine-tuned by a subtle balance between selfand 

hetero-associations. Ataxin-1 is the protein responsible for the hereditary 

spinocerebellar ataxia type 1, a disease linked to protein aggregation and 

transcriptional dysregulation. Expansion of a polyglutamine tract is essential for 

ataxin-1 aggregation, but the sequence-wise distant AXH domain plays an 

important aggravating role in the process. The AXH domain is also a key element 

for non-aberrant function as it intervenes in interactions with multiple protein 

partners. Previous data have shown that AXH is dimeric in solution and forms a 

dimer of dimers when crystallized. By solving the structure of a complex of AXH 

with a peptide from the interacting transcriptional repressor CIC, we show that the 

dimer interface of AXH is displaced by the new interaction and that, when blocked 

by the CIC peptide AXH aggregation and misfolding is impaired. This is a unique 

example in which palindromic self- and hetero-interactions within a sequence with 

chameleon properties discriminate the partner. We propose a drug design strategy 

for the treatment of SCA1 that is based on the information gained from the 

AXH/CIC complex. 

40 41 

OC-12









OC-13 

Molecular recognition and optimization of translocation of 

antiprion therapeutic agents from molecular theory of solvation 

Andriy Kovalenko, 1,2 Neil Cashman, 3 and Nikolay Blinov, 1,2 

From molecular mechanisms to intervention strategies: 

Stabilizing fibrils to inhibit prion replication 

OC-14 

1 National Institute for Nanotechnology, Edmonton (Canada), andriy.kovalenko@nrc-cnrc.gc.ca. 

2 Dept. of Mechanical Engineering, University of Alberta, Edmonton (Canada), nblinov@ualberta.ca. 

3 Dept. of Medicine, University of British Columbia, Vancouver (Canada), Neil.Cashman@vch.ca. 

Elucidation of the mechanisms of recognition and inhibition of neurotoxic 

aggregates by the therapeutic agents as well as their efficient delivery to the 

central nervous system is important for development of therapy against 

neurodegenerative diseases.[1,2] Experimental screening can be time consuming 

and expensive, thus molecular modelling can be useful for initial selection of drug 

candidates and optimization of anti-prion drugs and conformational antibodies 

targeting neurotoxic aggregates for efficient delivery.[3] 

Solvation is a major factor in biomolecular processes, 

including slow exchange and localization of solvent, ions, 

protein-ligand recognition, and membrane translocation. 

Statistical-mechanical, 3D-RISM-KH molecular theory of 

solvation [4] accurately describes solvation effects in 

protein-ligand recognition protocols.[5,6] In a single 

formalism, the 3D-RISM-KH method efficiently accounts 

for electrostatic and non-polar effects, including hydrogen bonding, hydrophobicity, 

structural solvation / desolvation in crowded inter- and intracellular environments. 

42 43 

The new multiscale modeling platform for optimization of molecular recognition and 

translocation of antiprion therapeutic agents is based on the implementations of the 

3D-RISM-Dock protocol in AutoDock suite,[5] 3D-RISM-KH solvent analysis in 

Molecular Operating Environment package,[6] and multi-time-step MD steered with 

3D-RISM-KH effective solvation forces in Amber package.[7,8] We apply the new 

platform to study binding modes of antiprion compounds, molecular recognition at 

the initial stages of oligomerization of Aβ peptides, structural solvation effects on 

stability of amyloid fibrils, and optimization of antiprion agents for efficient delivery. 

REFERENCES 

[1] B. Frost and M. I. Diamond Nat. Rev. Neurosci., 2010, 11,155-9. 

[2] Y. Biran et al. J. Cell Mol. Med., 2009, 13, 61-6. 

[3] G. Subramaniana and D. B. Kitchen J. Comput. Aid. Mol. Des., 2003,17, 643–4. 

[4] A. Kovalenko, in: Molecular Theory of Solvation, Hirata(ed.), Kluwer,Dordrecht 2003,169-275. 

A. Kovalenko. Pure Appl. Chem., 2013, 85, 159-99. 

[5] D. Nikolic, et al. J.Chem. Theory Comput., 2012, 8, 3356-2. N. Blinov, et al. Molec.Simul., 2011, 37, 718-8. 

[6] http://www.chemcomp.com/MOE-Structure_Based_Design.htm 

[7] T. Luchko, et al. J. Chem. Theory Comput., 2010, 6, 607-4. 

[8] I. Omelyan and A. Kovalenko. J. Chem. Phys., 2013, 139, 244106-23. 

Bieschke, Jan 1 

1 Department of Biomedical Engineering, Washington University in St. Louis, One Brookings 

Drive, St. Louis, MO 63130, USA, bieschke@wustl.edu 

In protein misfolding disorders, such as Alzheimer's disease (AD) and Parkinson's 

disease (PD), and prion diseases proteins accumulate in insoluble aggregates in 

the affected tissue. Recently, it became clear that protein misfolding in prion 

diseases, AD and PD follows a common mechanism of seeded polymerization. 

Prion-like autocatalytic replication of seeds are found on a molecular, if not 

organismal level are also observed in alpha-synuclein (aS) and Amyloid beta 

(Abeta) misfolding in PD and AD, respectively. These findings suggest that 

inhibiting prion replication could be a promising therapeutic strategy not just in 

prion disease, but also in AD and PD. 

We have recently found that derivatives of Orcein, a phenoxazine dye that can be 

isolated from the lichen Rocella tinctoria, accelerate fibril formation 1 . At the same 

time these compounds deplete toxic oligomeric and protofibrillar forms of the 

peptide reducing toxic oligomers. The theory of nucleated polymerization posits 

that stabilizing amyloid fibrils should decrease their seeding potential and thus 

should choke off prion replication. We have tested this hypothesis in vitro and in 

cell culture for a number of amyloidogenic proteins and peptides, including Abeta, 

aS, and tau. 

Drug treatment stabilized fibrils against fragmentation, decreased their seeding 

efficacy and reduced their replication rate in serial protein misfolding cyclic 

amplification (serial PMCA) experiments. At the same time it decreased cellular 

uptake and seeding in neuronal cell models and rescued Abeta and aS toxicity. 

These results serve as proof-of-principle for a new therapeutic strategy of 

stabilizing mature amyloid fibrils in order to prevent prion replication in AD, PD and 

other protein msifolding diseases. 


Financial support from NIH Grant No. 5 P30 DK020579, DFG, BI 1409/1-1, BMBF GERAMY 

01GM1107C is acknowledged. 

REFERENCES 

[1] Bieschke, J.; Herbst, M.;et al., Small-molecule conversion of toxic oligomers to nontoxic beta-sheetrich 

amyloid fibrils. Nature chemical biology 2012, 8 (1), 93-101.







Analysis and characterisation of Human Glycoproteins using 

MALDI TOF Mass Spectrometry 

R. Castangia 1 , Matthew S. F. Choo 2 ; Omar Belgacem 1 and 

ET 

Anne Dell 2 

1 Global MALDI Applications Development Group, Manchester, UK roberto.castangia@kratos.co.uk 

2 Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, UNITED 

KINGDOM 

The diversity of protein structures and functions can be considered a direct 

consequence of the posttranslational modifications (PTMs) variability. Among 

them, glycosylation represents a key process that is yet to be fully understood 

due to its combinatorial nature and biological implications. 

In this respect, mass spectrometry gives a profound impact in the investigation 

ABSTRACTS 

44 of glycoprotein structure and functions, unveiling structural details not 45 

EXHIBITOR TALK 

accessible with other analytical methods. 

This presentation will outline a full approach in the analysis of human 

glycoproteins using MALDI TOF mass spectrometry. The analytical method 

relies on desorption of molecules by direct laser irradiation. Intact molecules 

are ionised producing fragment-less m/z (mass over charge) signals in a broad 

mass range (typically 100-500kDa). Moreover, the combination with a highenergy 

collision, allows the fragmentation of the molecules unveiling detailed 

structural properties. 

We have applied MALDI TOF analysis to a broad set of glycoproteins including 

IgGs, Factor IX, and Erythropoietin. Glycans were also investigated and 

identified by means of a proprietary database able to distinguish between 

structural isomers. In addition, a de-novo approach to glycans identification will 

be presented. This method employs a quick and easy access to signal 

assignment based a sensitive MS n analysis.





Two Universities by the BayMay 25-26, 2014 - Naples, Italy 

Biotechnology, Mediterranean Diet and the Brain 

Roberto Crea 

SN 

President and CSO. CreAgri, Inc., Hayward, California 94545. 

The cellular mechanism of inflammation, one the most important scientific 

discoveries of the past twenty years in cell and molecular biology, has been pivotal 

to the development of numerous drugs (biologics) both by the Pharma and the 

Biotechnology industry to help manage the devastating effects of uncontrolled 

inflammation [1]. 

Chronic inflammation plays a key role in the pathogenesis of some of today’s major 

public health epidemics including diseases such as Alzheimer’s, Parkinson, heart 

failure and arthritis [2]. Especially injurious among the aging population, the 

outcome of uncontrolled inflammation is causing a widespread health emergency 

by utterly taxing family budgets and emotions. By causing an incalculable loss of 

working hours is also becoming an economic emergency and is fueling potential 

ABSTRACTS 

46 47 

SPECIAL NOTE 

societal instability. 

The Mediterranean diet, consisting of high concentration of natural polyphenols 

and flavonoids, is associated with a low incidence of neurodegenerative diseases, 

cancer, diabetes and cardiovascular disorders [3]. Olive polyphenols, in particular 

hydroxytyrosol, have been associated with one of the strongest anti-inflammatory 

activity among many naturally occurring antioxidants [4]. CreAgri Inc., and other 

laboratories have discovered that hydroxytyrosol, and other minor olive 

polyphenols obtained from the vegetation water of olives during the production of 

olive oil, exercise their anti-inflammatory activity by down-modulating the activation 

of the cellular pathway Nuclear Factor Kappa - Beta (“NF-kb”) [5]. 

In recent research and scientific literature the NF-kb cellular complex, a five protein 

complex of transcription factors, has emerged as a central regulator of chronic 

inflammatory activity [6]. Its activation by a number of toxic stimuli leads to the 

expression of more than 500 proteins with a strong pro-inflammatory activity. 

Cellular NF-kb activation seems to be remarkably modulated by hydroxytyrosol in 

inactivating the expression of numerous pro-inflammatory cytokines, chemokines 

and other pro-inflammatory factors [5]. 

Present in small concentration in olive oil, Hydroxytyrosol is an orally bioavailable 

phenolic compound, particularly active as anti-inflammatory agent. Its low 

molecular weight, high bioavailability, chemical similarity to dopamine and high 

perfusion through tissues and cellular membranes, have made it a strong 

candidate in addressing the management of brain inflammation caused by a variety 

of pathologies, including the gradual deposition of protein units that form amyloid 

plaques and tau tangles (Pasinetti, G. et al., unpublished study).





SN 


We propose here that a balanced and healthy diet, further enriched in polyphenols 

and hydroxytyrosol, might mitigate the onset of neurodegenerative and cognitive 

diseases and provide a non toxic, inexpensive and natural solution to brain ageing 

and chronic inflammation. Hydroxytyrosol is now readily available by the use of an 

aqueous process that produces it in large quantities from the vegetation water of 

olives (juice) without any use of solvents or toxic chemicals. Combined with its 

strong safety profile [6-7], increasing clinical evidence supports its therapeutic 

potential in the management of chronic inflammatory diseases in the brain. 

REFERENCES 

[1] Makarov, S et al. (2001), Arthritis Res. 3, 200-206 

[2] Manabe, I. et al. (2011) Circ. J. 75, 2739-2748 

[3] Lourida et al. , Epidemiology, 2013, 4,479-89 

[4] Bitler, C. et al. , J. of Nutritional 2005,135, 1475-1479 

ABSTRACTS 

[5] Richard N. et al. , Planta Medica, 2011, 77, 1890-97 

[6] Killeen, M et al., Drug Discovery Today, 2014, 383-38 

[7] Christian M.S., et al. Drug and Chemical Toxicology, 2004, 27-4, 309-330 

48 49 

POSTER PRESENTATIONS









P-1 

ANTI-AMYLOIDOGENIC PROPERTY OF HUMAN GASTROKINE 1 

F. Altieri, C.S. Di Stadio, G. Miselli, E. Rippa, P. Arcari. 

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, 

Naples, Italy. 

Gastrokine 1 (GKN1) is a stomach-specific protein that plays an important role in 

maintaining the physiological function of the gastric mucosa [1]. The protein 

contains the BRICHOS domain of about 100 amino acids, present also in several 

unrelated proteins associated with major human diseases like BRI2, related to 

familial British and Danish dementia; chondromodulin-I (ChM-I), linked to 

chondrosarcoma; surfactant protein C (SP-C), associated with respiratory distress 

syndrome; and gastrokines linked to gastric cancer [2]. Recent researches have 

shown that recombinant Bri2 and SP-C precursor (proSP-C) BRICHOS domains 

were able to prevent fibril formation of amyloid-beta peptide (Aβ). Aβ is the major 

component of extracellular amyloid deposits in Alzheimer's disease and derives 

from the partial hydrolysis of the amyloid precursor protein (APP) catalyzed by β- 

and γ-secretase. 

Having available purified recombinant GKN1 (rGKN1) [3], we investigated on the 

interaction of the protein versus Aβ(1-40). Human rGKN1 was incubated in 

presence or absence of Aβ at 1:10 molar ratios. Chicken cystatin was used as 

negative control. SDS-PAGE was then used to highlight Aβ solubility. Thioflavine T 

binding assay was also used to evaluate the aggregation of Aβ. Blue Native Page 

(BN-PAGE), BIAcore technique and mass spectrometry analysis were performed to 

characterize the interaction. 

The results showed that rGKN1 prevented in vitro amyloid aggregation and fibrils 

formation by inhibiting Aβ(1-40) aggregation. BN-PAGE, ITC and mass 

spectrometry showed the formation of rGKN1/Aβ complex. BIAcore kinetics of 

rGKN1/Aβ interaction led to calculate a dissociation constant (k D ) of 34 µM. Taking 

advantage from the presence in the recombinant protein of the His 6 -Tag sequence, 

we also investigated the interaction by confocal microscopy and NiNTA pull-down 

assay and probing with anti-APP antibody. 

The preliminary data obtained strongly suggested that GKN1 is endowed by antiamyloid 

activity thus it might play a role as chaperone directed against unfolded 

peptide segments. In particular, GKN1 showed the ability to recognize APP and to 

bind amyloid-beta peptide preventing its aggregation. 

Novel approaches in the study of the Aβ peptide oligomers: 

shining light by time-resolved Fourier-transform infrared 

spectroscopy 

Maurizio Baldassarre, Andreas Barth 

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden 

Although amyloid fibrils represent the hallmark in the onset and evolution of 

Alzheimer's disease, the focus on the actual pathogenic species in this brain 

disorder has shifted from fibrils to soluble oligomers of the Aβ peptide. Our efforts 

aim at elucidating the following aspects. (a) Validity of the β-hairpin model for the 

Aβ peptide within the oligomers. (b) Structural organisation of the oligomers, 

allowing to confirm/confute the main models proposed by other authors (stacks of 

β-hairpins, β-barrels, Y-shaped dimers). (c) Extent of β-sheet in the monomer. (d) 

Residue-level resolution of the kinetics of oligomer and fibril formation. Our 

research brings together several cutting-edge approaches often used separately in 

infrared spectroscopy: (1) high-sensitivity rapid scanning infrared spectrometers 

capable of acquiring spectra with millisecond resolution; (2) UV flash-induced 

photolysis of “caged” sulfate, inducing instant (~100 μs) pH drops and triggering in 

situ aggregation of the Aβ peptide; and in future (3) 13 C-labelling of peptide 

carbonyls in selected peptide groups, allowing to study secondary structure 

transitions, as well as formation of intra- and inter-molecular contacts with singleresidue 

resolution; (4) Calculation of theoretical infrared absorption spectra for 

candidate structures, allowing for straightforward comparison with infrared spectra 

obtained experimentally. The poster will present our approach and our recent 

results with Aβ 42. 

50 51 

P-2 

REFERENCES 

[1] TE Martin, CT Powell, Z Wang, S Bhattacharyya, MM Walsh-reitz, K Agarwal, FG Toback AJP 

Gastrointesinal and Liver Physiology 2003, 285, G332–G343. 

[2] H Willander, J Presto, G Askarieh, H Biverstål, B Frohm, SD Knight, J Johansson, S Linse J Biol 

Chem 2012, 287, 31608-31617. 

[3] LM Pavone, P Del Vecchio, P Mallardo, F Altieri, V De Pasquale, S Rea, NM Martucci, CS Di 

Stadio, P Pucci, A Flagiello, M Masullo, P Arcari, E Rippa Mol Biosyst 2013, 9, 412-421. 

Figure 1. Theroretical scheme showing how transitions of Aβ1-40 from statistical random coil 

conformations (blue circles) to β-hairpins (orange circles), induced by a pH drop from high to low pH, 

are expected to change the infrared absorption of the peptide in the amide I region. (A) Unlabelled 

peptide. (B) Peptide 13C-labelled at two positions, showing the expected effect of coupling (red arrow).









P-3 

Structural restraints for early A-oligomers from propensity for 

aggregation predicted by molecular theory of solvation 

Investigating the aggregation properties of β-synuclein and its 

effect on the aggregation of α-synuclein 

P-4 

Nikolay Blinov, 1,2 Neil Cashman, 3 and Andriy Kovalenko 1,2 

1 National Institute for Nanotechnology, Edmonton (Canada), andriy.kovalenko@nrc-cnrc.gc.ca. 

2 Dept. of Mechanical Engineering, University of Alberta, Edmonton (Canada), nblinov@ualberta.ca. 

3 Dept. of Medicine, University of British Columbia, Vancouver (Canada), Neil.Cashman@vch.ca. 

James Brown, Céline Galvagnion, Alex Buell, Chris Dobson 

Small A oligomers have been linked to neurodegeneration in Alzheimer's 

disease.[1] Information about 3D structure of the oligomers can provide a basis for 

interference with the pathways of aggregation and for inhibition of neurotoxicity. 

Experimental characterization of the oligomers has been complicated by their 

transient nature and structural flexibility. In this 

situation, molecular modelling can provide a 

valuable insight into the mechanisms of 

oligomerization and structural characteristics of 

oligomers and amyloid fibrils.[2] 

52 53 

Here we use a novel approach providing 

prediction of the propensity for aggregation of 

amyloidogenic peptides to identify possible 

structural restraints for A-oligomers at the initial 

stages of oligomerization. The approach is 

based on the 3D-RISM-KH molecular theory of 

solvation which efficiently and accurately 

describes solvation effects with consistent account for solvent molecular 

specificities, composition, and thermodynamic state.[3] The theory has been 

recently used to study thermodynamics and solvation structure of -sheet 

oligomers, A and prion amyloid fibrils, and to predict binding modes of antiprion 

compounds.[4] We use this methodology to build/screen plausible models of A 

(toxic) oligomers with account for restraints derived from conformational antibodyoligomer 

binding experiments. 

REFERENCES 

[1] C. Haass and D. J. Selkoe Nat. Rev. Mol. Cell Biol., 2007, 8, 101-112. 

[2] J. E. Straub and D. Thirumalai Curr. Opin. Struct. Biol., 2010, 20, 187-195. 

[3] A. Kovalenko, in: Molecular Theory of Solvation, Hirata (ed.), Kluwer, Dordrecht, 2003,169-275. 

[4] N. Blinov, et al. Biophys. J., 2010, 98, 282-296; N. Blinov, et al. Mol. Sim., 2011, 37, 718-728; T. 

Yamazaki, et al. Biophys. J., 2009, 95, 4540-8, D. Nikolic, et al., J. Chem. Theory Comput., 2012, 8, 

3356-3372. 

Dept. of Chemistry, Cambridge University, Cambridge (UK), jb882@cam.ac.uk 

α-synuclein is an intrinsically disordered protein known to be directly involved in the 

pathogenesis of Parkinson's disease through the formation of toxic oligomers and 

amyloid fibrils. 1,2 It localises to synaptic vesicles in vivo and the presence of certain 

phospholipid vesicles modulates aggregation in vitro. 3 β-synuclein has very similar 

structural properties to α-synuclein however has a greatly reduced aggregation 

propensity. 4 Experiments were performed to investigate the difference between α- 

and β-synuclein aggregation kinetics in a phospholipid vesicle aggregation model 

and, further, the effect of β-synuclein on α-synuclein aggregation kinetics (Fig. 1). 

Our strategy consists of trying to resolve the effect of the sequence difference 

between α- and β-synuclein on the various mechanistic steps that are involved in 

amyloid formation, such as primary nucleation of aggregates, aggregate growth 

and proliferation. We find that β-synuclein interferes with α-synuclein primary 

nucleation and secondary nucleation processes, but has no significant effect on 

fibril elongation. 

a b c 

Fig. 1. A set of experiments to determine the effect of βsyn on different αsyn aggregation processes. 

(a) Lipid-nucleated aggregation (350 uM DMPS small unilamellar vesicles, pH 6.5, 30 °C) of 100 uM 

(dark purple) and 50 uM (light purple) αsyn in the presence of a 1:1 molar ratio of βsyn (dark and 

light green, respectively). (b) Elongation from 1% αsyn seed fibrils (pH 6.5, 30 °C). 100 uM αsyn 

(purple) in the presence of 50 uM (light green) and 100 uM (green) βsyn. βsyn (100 uM, darkest 

green) does not elongate from these seeds. (c) Secondary nucleation 5 (pH 4.8, 30 °C, 10 nM αsyn 

seed) of 55 uM αsyn (purple) in the presence of 55 uM (light green) and 110 uM (dark green) βsyn. 

REFERENCES 

[1] M.R. Cookson Annu. Rev. Biochem. 2005 

[2] N. Cremades et. al. Cell 2012 

[3] C. Galvagnion et. al. Submitted 

[4] C. Roodveldt et.al. Biochemistry 2012 

[5] A.K. Buell et. al. PNAS Accepted

P-5 



Molecular chaperones suppress the toxicity of misfolded 

protein oligomers 






COMPUTATIONAL DESIGN OF PEPTIDES THAT TARGET THE 

AMYLOID PRECURSOR PROTEIN TRANSMEMBRANE DOMAIN 

P-6 

R. Cascella 1 , E. Evangelisti 1 , B. Mannini 1 , B. Tiribilli 2 A. Relini 3 , J.N. 

Buxbaum 4 , C.M. Dobson 5 , M.R. Wilson 6 , F. Chiti 1 and C. Cecchi 1 

1 Dept. of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 

50134 Florence, Italy, roberta.cascella@unifi.it. 

2 Consiglio Nazionale delle Ricerche (CNR), Istituto dei Sistemi Complessi, Via Madonna del Piano 10, 

50019 Sesto Fiorentino, Florence, Italy 

3 Dept. of Physics, University of Genoa, 16146 Genoa, Italy 

4 Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, 

USA 

5 Dept of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom 

6 School of Biological Sciences, University of Wollongong, Wollongong 2522, Australia 

Molecular chaperones are proteins known to assist the folding process of newly 

54 55 

synthesised or temporarily misfolded proteins, inhibit protein aggregation and 

promote disaggregation and clearance of misfolded aggregates inside cells. We 

have tested the effects of different chaperones (human αB-crystallin, Hsp70, 

clusterin, haptoglobin, α 2 -macroglobulin, and three different types of TTR) on the 

toxicity of misfolded oligomers preformed from different amyloidogenic 

peptides/proteins (HypF-N and Aβ 42 ) added extracellularly to cultured cells. 

Chaperones were found to decrease oligomer toxicity significantly, even at very low 

chaperone/protein molar ratios. Infrared spectroscopy and site-directed labeling 

experiments using pyrene ruled out structural reorganizations within the discrete 

oligomers following incubation with chaperones. Rather, confocal microscopy, 

SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding 

between oligomers and chaperones. Moreover, atomic force microscopy (AFM) 

indicated that larger assemblies of oligomers are formed in the presence of the 

chaperones, suggesting that the chaperones bind to the oligomers and promote 

their assembly into larger species. Overall, the data indicate a generic ability of 

chaperones to efficiently neutralize the toxicity of oligomers formed by misfolded 

proteins and reveal that further assembly of protein oligomers into larger species 

can be an effective strategy to neutralize such species. 

T. Lemmin 1 , M. Chino 2 , A. Lombardi 2 , W. F. DeGrado 1 

1 Dept. of Pharmaceutical Chemistry, University of California, San Francisco (USA), 

thomas.lemmin@ucsf.edu 

2 Dept. of Chemical Sciences, University Federico II, Napoli (Italy), marco.chino@unina.it 

The Amyloid Precursor protein (APP) proteolytic cleavage by γ-secretase of the 

transmembrane (TM) domain leads to the formation of amyloid-β (Aβ) peptides, the 

deposition of which is an early indicator of Alzheimer’s disease (AD). [1] Even though 

the APP pathological degradation is well established, there is still debate about its 

biological function. The simultaneous presence of two (small)xxx(small) motifs 

(G 700 xxxG 704 xxxG 708 ; G 709 xxxA 713 ), as found in glycophorin A, suggests the 

existence of biological partners in homo- or hetero-dimerization. While mutation of 

these residues dramatically influences the cleavage positions, these mutations also 

affect the cholesterol regulation in the brain, in which APP may be found as bound 

to sterol regulatory proteins and to the cholesterol itself. [2,3] 

Computed helical anti-membrane proteins (CHAMP) have proven to be useful tools 

in the study of protein-protein interactions of TM domains both in vitro and in vivo. 

[4] A fully automated de novo design protocol was implemented within the Rosetta 

software by means of a structural-based pairwise potential. The protocol 

development and the design of anti-TM-APP peptides, which would target 

specifically each motif will be reported. The strategy adopted aims to predict, 

identify and biophysically evaluate the dimerization partners of TM-APP by 

selectively targeting only one binding site. 


Financial support for T.L. from the Swiss National Science Foundation is acknowledged. 

Financial support for M.C. from Regione Campania STRAIN program and from University Federico II 

Short Term Mobility program are acknowledged. 

REFERENCES 

[1] R.E. Tanzi, J.F. Gusella, P.C. Watkins, G.A. Bruns, P. St George-Hyslop, M.L. Van Keuren, D. 

Patterson, S. Pagan, D.M. Kurnit, R.L. Neve. Science, 1987, 235, 880-884. 

[2] T. Lemmin, M. Dimitrov, P. C. Fraering, M. Dal Peraro. Journal of Biological Chemistry, 2014, jbc- 

M113. 

[3] Y. Song, A.K. Kenworthy, C.R. Sanders. Protein Science, 2013, 23, 1-22. 

[4] H. Yin, J.S. Slusky, B.W. Berger, R.S. Walters, G. Vilaire, R.I. Litvinov, J.D. Lear, G.A. Caputo, J.S. 

Bennett, W.F. DeGrado. Science, 2007, 315, 1817-1822.









P-7 

Infrared Microspectroscopy as a Method of Choice for Structural 

Analysis of Minute Amounts of Prions 

Martin L. Daus, Michael Beekes and Peter Lasch 

ZBS6-Centre for Biological Threats and Special Pathogens, Proteomics and Spectroscopy, 

Robert Koch-Institut, Berlin (Germany) 

Environmental factors affecting the aggregation state of 

Aβ(25-35): role of unsaturated omega-3 fatty acid. 

Matilde Sublimi Saponetti 1 , Manuela Grimaldi 2 , Mario Scrima 2 , 

Stefania Lucia Nori 3 , Fabrizio Bobba 1 , Annamaria Cucolo 1 , 

Anna Maria D’Ursi 2* 

P-8 

Infrared spectroscopy allows the structural analysis of proteins. Oligomers of 

misfolded prions form rather large protein aggregates with poor crystal forming 

properties. Therefore no high resolution structure by NMR or X-ray diffraction of a 

complete misfolded prion protein is so far available. Infrared spectroscopy gives 

structural information mainly about secondary structural elements and can help to 

discriminate different prion strains. There are several advantages of this technique 

as i) no sample labelling or staining is needed, ii) spectra can be obtained from tiny 

amounts of purified proteins, iii) it is relatively simple in use and iv) can provide 

data within a very short time. We will present a technical advance that allows 

spectroscopy with unprecedented sensitivity using dried protein extracts that have 

been analysed by linking an IR-microscope to an IR-spectrometer. Spectroscopy 

was applied on protein extracts of as little as about 3 nanograms. This may help to 

discriminate differentially folded prions on a structural level even when just minute 

amount of tissue/sample material is available. Biochemical and biological assays 

may additionally enhance the validity of biophysical results. 

56 57 


Financial support from the Alberta Prion Research Institute 

1 SPIN-CNR and Department of Physics, “E.R. Caianello” University of Salerno, 

2 Department of Pharmacy, University of Salerno, 

3 Department of Medicine, University of Salerno, 

*Department of Pharmacy, University of Salerno, dursi@unisa.it 

Aβ amyloid peptide has an important role in the manifestation and in the 

progression of Alzheimer disease. It has tendency to aggregate in low–molecular 

weight soluble oligomers, higher-molecular weight protofibrillar oligomers and 

insoluble fibrils. The relative importance of these single oligomeric-polymeric 

species in relation with the morbidity of the disease is nowadays debated. We have 

previously analyzed by EPR spectroscopy the effect of unsaturated omega- 

3 fatty acid (DHA) on the ability of (25–35) to be interfaced with DPPC 

bilayers.[2] Here we present an atomic force microscopy (AFM) study of (25–35) 

aggregation in three different environmental conditions: phosphate buffer, lipid 

dioleoylphosphocholine (DOPC) bilayers and lipid DOPC bilayer containing 

unsaturated omega-3 fatty acid (DHA). (25–35) is the smallest fragment 

retaining much of the biological activity of the full-lenght peptide, whereas 

phosphate buffer, lipid DOPC and lipid DHA/DOPC bilayers have been selected as 

paradigm of aqueous physiological solution and cell membrane mimic 

environments characterized by different fluidity. Our study shows that the 

aggregation process of (25–35) on lipids is always characterized by the 

presence of annular oligomers. Their conformational evolution during time, as 

monitored by in-liquid AFM experiments, show that on DOPC bilayers they cause 

damages in the membrane inducing pores and delipidation. On the opposite the 

addition of DHA to the lipid, making the lipid substrate more fluid, helps to preserve 

the membrane integrity against the protein aggression. 

REFERENCES 

[1] Sorrentino P, Iuliano A, Polverino A, Jacini F, Sorrentino G FEBS Lett., 2014, 588(5), 641-652. 

[2] Vitiello G, Di Marino S, D'Ursi AM, D'Errico G. Langmuir, 2013, 29(46),14239-45.









P-9 

Interaction between amyloid peptide Aβ(1-42) with model 

phospholipid bilayers 

A. Emendato 1 , G. D’Errico 1,2 , A. Falanga 3 , S. Galdiero 3 , D. Picone 1 , R. 

Spadaccini 4 

1 Dept. of Chemical Science, University “Federico II”, Napoli (Italy), alexeme@fastwebnet.it 

2 CSGI: Consorzio interuniversitario per lo Sviluppo dei sistemi a Grande Interfase, Firenze (Italy) 

3 Dept. of Molecular Diagnostic and Pharmaceutics, University “Federico II”, Napoli (Italy) 

4 Dept. of Science and Technology, University of Sannio, Benevento Italy 

Alzheimer disease is an irreversible neurodegenerative pathology that causes loss 

of memory and cognitive processes, and after few years death. The biochemical 

hallmark of this pathology is the occurrence, in neuronal cells, of insoluble 

aggregates formed by short peptides, derived from proteolytic cleavage of a transmembrane 

protein, the Amyloid Precursor Protein (APP). The interaction of these 

peptides with phospholipid bilayers is a very important topic in current Alzheimer 

literature, although not well understood.[1] The major component of Amyloid 

aggregates, the fragment 1-42, called Aβ (1-42), is a highly hydrophobic peptide 

that shows a sequence and a three-dimensional structure similar to viral fusion 

peptides.[2] Recent literature data report experimental evidences of membrane 

fusion and permeabilization induced by this peptide.[3,4] In this work we have 

studied, with an integrated experimental approach, the interaction between this 

peptide and model phospholipid bilayers: a simple one, formed only by POPC, and 

a more complex biomimetic one, formed by other components of neuronal cell 

membrane. The conformational preference of the peptide interacting with the 

membranes was studied using circular dichroism spectroscopy (CD); the effect of 

this interaction on the microstructure of the model bilayers was studied by electron 

paramagnetic resonance spectroscopy (EPR). Our data show that the Aβ (1-42) is 

readily incorporated in the simpler and fluid bilayer, and CD spectra shows the 

occurrence of soluble aggregates; in contrast, with more rigid and complex 

membrane, the interaction is slower and superficial, and the aggregates are 

detectable only after a prolonged incubation. The leakage activity of the peptide 

towards these lipid compositions was investigated by a FRET-based experiment; 

the data show a very strong permeabilization effect towards the liposomes 

composition studied. These data represent a further step in the understanding of 

the Aβ peptide-membrane interaction. 

58 59 

ACKNOWLEDGEMENTS: Financial support from F.A.R.O. 

REFERENCES 

[1] K. Matsuzaki, Biochimica et Biophysica Acta, 2007, 1768, 1935-1942 

[2] O. Crescenzi, et al. European Journal of Biochemistry, 2002, 269, 5642-5648 

[3] S. Dante, T. Hauß, A. Brandt, N. A. Dencher, Journal of Molecular Biology, 2008, 376, 393-404 

[4] M.C. Vestergaard, et al., Biochimica et Biophysica Acta, 2013, 1828, 1314-1321 

Structural properties of amyloid-like fibers unveiled by 

molecular dynamics studies 

L. Esposito 1 , A. De Simone 2 , L. Vitagliano 1 

1 Institute of Biostructures and Bioimaging, CNR, Napoli (Italy), luciana.esposito@unina.it. 

2 Division of Molecular Biosciences, Imperial College South Kensington Campus, London (UK) 

Neurodegenerative diseases are widespread and severe pathologies whose 

etiology is largely unknown. In this scenario, it is not surprising that therapeutic 

approaches for these diseases are largely ineffective. A complete understanding of 

the molecular basis of these diseases requires detailed information on the factors 

that destabilize/stabilize the globular state as well as the amyloid-like fibrils. The 

structural characterization of these fibrils has long been hampered by their very low 

solubility and by the non-crystalline nature of these aggregates. Major advances in 

this field have been recently accomplished by the use of model peptides, whose 

solid aggregates exhibit most of the features of amyloid fibrils.[1] High resolution 

crystallographic analyses of these amyloidogenic peptides have shown that they 

have a tendency to adopt a common motif denoted as cross-β spine steric 

zipper.[1] In order to obtain further insights into the structure determinants of 

amyloid fiber structure/formation and to analyse the effects of crystal packing on 

aggregate structure, we have undertaken molecular dynamics (MD) simulations on 

a variety of different models arranged in a cross-β spine structure.[2-5] These 

studies, by analyzing peptides assemblies in a crystalline-free context, have 

provided information on the intrinsic propensities of peptide fragments to associate 

in amyloid-like states. They have also provided reliable estimates of the energetic 

factors involved in the aggregation process. More recently, we evaluated the 

dynamic properties of a model peptide which is able to form two different steric 

zipper assemblies in the crystalline state.[6] The structural features of these two 

polymorphic structures have been related to the occurrence of strain in 

neurodegenerative diseases. 

REFERENCES 

[1] D. Eisenberg, M. Jucker Cell. 2012, 148, 1188-203. 

[2] L. Esposito, C. Pedone, L. Vitagliano PNAS, 2006, 103, 11533-11538. 

[3] A. Merlino, L. Esposito, L. Vitagliano Proteins, 2006, 63, 918-927. 

[4] L. Esposito, A. Paladino, C. Pedone, L. Vitagliano Biophysical J., 2008, 94, 4031-4040. 

[5] L. Vitagliano, F. Stanzione, A. De Simone, L. Esposito Biopolymers, 2009, 91, 1161-1171. 

[6] F. Stanzione, A. De Simone, L. Esposito, L. Vitagliano Protein Pept.Lett., 2012, 19, 846-51. 

P-10









P-11 

Protein misfolded oligomer binding to membrane ganglioside 

GM1: a real-time single particle tracking study 

E. Evangelisti 1 , R. Cascella 1 , M. Calamai 2 , F. Chiti 1 , C. Cecchi 1 , M. Stefani 1 

1 Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence 

(Italy), elisa.evangelisti@unifi.it. 

2 European Laboratory for Non-linear Spectroscopy (LENS), University of Florence, Sesto Fiorentino, 

Italy 

Interaction of N-methylated compounds with Aβ(25-35) Amyloid 

Peptide 

M. Grimaldi 1 , A. Iuliano 2,3 , S. Di Marino 1 , M. Scrima 1 , A. Polverino 2,3 , G. 

Sorrentino 2 , A. M. D’Ursi 1 

1 Department of Pharmacy, University of Salerno, Fisciano (SA), Italy 

2 Department of Sciences of movement, University of Naples Parthenope, Napoli, Italy. 

3 Istituto di Diagnosi e Cura Hermitage Capodimonte, Napoli, Italy 

P-12 

Recent results suggest an important role of ordered lipid domains of the plasma 

membrane, in terms of protein and lipid content, as modulators of the formation of 

amyloid fibrils and their precursor oligomers, and of the cytotoxicity of these 

species. We investigated the contribution to the toxicity of the membrane 

ganglioside GM1 in SH-SY5Y neuroblastoma cells exposed to oligomeric 

conformers of Aβ1-42 and HypF-N endowed with different ultrastructural properties 

and cytotoxicities. By means of real-time single particle tracking, we show that 

oligomers interact with GM1 decreasing its lateral diffusion on the plasma 

membrane of living cells. In turn, the cell biochemical response to the oligomeric 

species results from the membrane content of GM1 and its clustering. 

Deposition of senile plaques composed of fibrillar aggregates of Aβ-amyloid 

peptide is a characteristic hallmark of Alzheimer’s disease. Amyloid plaques are 

primarily composed of β-amyloid peptides Aβ(1-40) and Aβ(1-42). Aβ(25-35) 

represents the biologically active region of Aβ, as it includes the shortest fragment 

capable to form large β-sheet aggregates. Depending on conditions, amyloid 

peptides undergo a conformational transition from random coil or α-helical 

monomers to the highly toxic β-sheet oligomers, which form the mature fibrils. [1] A 

widely employed approach in the research of anti-Alzheimer agents involves the 

identification of substances able to prevent amyloid aggregation, or to disaggregate 

the amyloid fibrils through a direct interaction with either soluble or aggregated 

peptide. A selective mode of interaction of these compounds with soluble oligomers 

or amyloid aggregates has still not been clearly established. The development of 

small molecules able to interact with amyloid peptides is considered strategic in 

view of identifying the structural parameters responsible for Aβ stabilization and/or 

aggregation. 

In the present contribution, we study the interaction of Aβ(25-35) with a panel of N- 

methylated compounds using a combined approach based on circular dichroism, 

nuclear magnetic resonance and thioflavin fluorescence spectroscopy. Aβ(25-35) 

Amylod peptide is known to be able to activate Phospholipases. [2] 

To investigate the consequence of the modulation of Aβ aggregation in a biological 

context we evaluated the amyloid induced phosphorylation of phospholipase A2 

(cPLA2) in a cholinergic cell line (LAN-2) with or without N-methylated compounds. 

Our data show that N-methylated compounds are able to preserve the soluble form 

of the peptide; moreover given that the activation of phosphorilated form of cPLA is 

dependent on the aggregation state of Aβ, in presence of specific N-methylated 

compunds, cPLA2 phosphorylation is lost. 

60 61 

REFERENCES 

[1] P. Sorrentino, A. Iuliano, A. Polverino, F. Jacini, G. Sorrentino, FEBS Lett., 2014, 588(5), 641-652. 

[2] J.N. Kanfer, G. Sorrentino and D. Sitar, Neurosci Lett., 1998, 257: 93-96.









P-13 

Oligomerisation of alpha-synuclein at nearly-physiological 

concentrations 

Marija Iljina 1 , Mathew Horrocks 1 , David Klenerman 1 

1 Dept. of Chemistry, University of Cambridge, UK, mi291@cam.ac.uk. 

Alpha-synuclein is a small intracellular protein naturally abundant in the brain at 

low-micromolar concentrations. Its fibrillar aggregates are the major constituents of 

intracellular inclusions, known as Lewy bodies, which are the pathological 

hallmarks of Parkinson disease and related neurodegenerative disorders. 

However, increasing evidence suggest that oligomers, rather than fibrils, are the 

most toxic and damaging to brain neurons. In this presentation, I will discuss the 

recent results of in-vitro studies of alpha-synuclein oligomer formation at 

physiologically-relevant concentrations using single-molecule FRET spectroscopy. 

Flavone derivatives as inhibitors of insulin amyloid-like fibril 

formation 

Ričardas Mališauskas 1,2 , Akvilė Botyriūtė 1 , Domantas Dargužis 2 , Vytautas 

Smirnovas 1 

1 Dept. of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology, Vilnius 

(Lithuania), vytautas.smirnovas@bti.vu.lt 

2 Dept. of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius (Lithuania) 

ricardas.malisauskas@vgtu.lt 

Several natural and synthetic flavone derivatives were reported as inhibitors of 

amyloid fibril formation, discriminating inhibition potential by the number and the 

position of hydroxyl groups. In most of studies the main value used to rate the 

inhibition potential of flavones is fluorescence intensity of Thioflavin T (ThT) binding 

assay. 

P-14 

62 63 


Tayyeb-Hussain Scholarship, Christ’s College Cambridge 

We studied impact of 260 commercially available flavone derivatives on insulin fibril 

formation. In addition to ThT fluorescence intensity we also fitted kinetic curves to 

obtain halftime of aggregation (t 50 ). A third of all derivatives changed final ThT 

fluorescence at least two fold, however most of these derivatives had much smaller 

impact on t50, with no clear correlation between these two values.









P-15 

Nucleophosmin -helical regions spontaneously undergo 

conformational transitions toward β-sheet-rich amyloid-like 

aggregates 

C. Di Natale 1,2 , P. L. Scognamiglio 1,2 , M. Leone 3 , V. Punzo 1 , G Morelli 1 , L. 

Vitagliano 3 and D. Marasco 1 

1 Dep. of Pharmacy, CIRPEB, DFM, University of Naples “Federico II”, Naples, (Italy), 

daniela.marasco@unina.it 

2 Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia (IIT), Naples, 

(Italy) 

3 Institute of Biostructures and Bioimaging, National Research Council, 80134, Naples, Italy 

Conformational alterations of proteins and peptides are very relevant because of 

their association with many diseases [1]. Nucleophosmin (NPM1) is a 

multifunctional protein that is involved in a variety of fundamental biological 

processes and its deregulation is implicated in the pathogenesis of several human 

64 Q101 

65 

malignancies. Notably, NPM1 has been identified as the most frequently mutated 

gene in acute myeloid leukemia (AML) patients, accounting for approximately 30% 

of cases [2]. AML-linked mutations affect the NPM1 C-terminal domain (CTD) 

which adopts a globular structure consisting of a three helix bundle motif, whose 

destabilization abolishes the correct nucleolar localization of the protein. In recent 

biophysical characterizations of CTD folded state we highlighted the mutual 

influence of NPM1 distinct domains [3]. Moreover, through a protein dissection 

approach, we have also identified the role of specific protein regions involved in G- 

quadruplex DNA recognition mechanism [4]. These studies also showed that the 

first helix of the bundle is intrinsically endowed with an unusual thermal stability. 

Here we have further investigated the structural determinants of CTD folding 

process focusing on the region encompassing the second helix of the bundle. To 

this purpose we have designed and synthesized several peptides corresponding to 

extended and truncated variants of the native helix. Unexpectedly, biophysical 

characterizations of these peptides demonstrate that they spontaneously undergo 

structural transitions toward β-sheet-rich amyloid-like states. 

REFERENCES 

[1] F. Chiti, C. M. Dobson Ann Rev Biochem, 2006, 75, 333-366 

[2] B. Falini, N.Bolli, A. Liso, M.P. Martelli, R. Mannucci, S. Pileri, I. Nicoletti Leukemia, 2009, 10,1731- 

43. 

[3] D. Marasco, A. Ruggiero, C. Vascotto, M. Poletto, P.L. Scognamiglio, G. Tell, L. Vitagliano Biochem 

Biophys Res Commun, 2013, 430, 523-528. 

[4] P.L. Scognamiglio, C. Di Natale, M. Leone, M. Poletto, L. Vitagliano, G. Tell, D. Marasco Biochim 

Biophys Acta 2014, 6, 2050-2059. 

TRANSGLUTAMINASE: AN ENZYMATIC APPROACH TO 

INFLUENCE THE AMYLOID FILBRIL FORMATION 

A. Sorrentino 1 , C. V. L. Giosafatto 2 , I. Sirangelo 3 , P. Di Pierro 2 , 

R. Porta 2 , L. Mariniello 2 

1 Dept. of Agriculture, University Federico II, Napoli (Italy), angela.sorrentino@unina.it. 

2 Dept. of Chemical Sciences, University Federico II, Napoli (Italy), loredana.mariniello@unina.it. 

3 Dept. of Biochemistry and Biophysics “F. Cedrangolo”, Second University of Naples (Italy), 

ivana.sirangelo@unina2.it. 

Transglutaminases (E.C. 2.3.2.13, TG) are enzymes that catalyze the formation of 

isopeptide bonds inside the proteins between glutamine (acyl-donor) and lysine 

(acyl-acceptor) residues, as well as the incorporation of small primary amines into 

proteins, such as biogenic amines [1]. 

In our studies we have demonstrated that a 

microbial isoform of transglutaminase is able to 

modify a recombinant ovine Prion Protein (PrP) 

[2] and other amyloid proteins such as 

apomyoglobin (Apo) (Figure). In particular, 

intramolecular crosslinks and enzymatic 

polymerization were obtained for PrP and Apo 

respectively, while several primary amines and 

biogenic polyamines (hydroxylamine, 

monodansylcadaverine, putrescine, spermidine 

and spermine) effectively counteracted both 

transglutaminase-mediated PrP intracrosslinking 

and Apo polymerization [2]. 

Furthermore, TG-crosslinked PrP showed a 

higher proneness in forming amyloid 

aggregates and was less susceptible to 

Proteinase K digestion, compared to the 

unmodified one [2]. 

Apomyoglobin 

(Apo) 

K 19 

Q 6 

Finally, we suggest a possible use of both transglutaminase and biogenic primary 

amines in elucidating the molecular basis of the amyloid aggregation. 

REFERENCES 

[1] J. E. Folk. Ann. Rev. Biochem., 1980, 49,517-531. 

[2] A. Sorrentino, C. V. L. Giosafatto, I. Sirangelo, C. De Simone, P. Di Pierro, R. Porta and L. 

Mariniello. Biochim. Biophys. Acta, 2012, 1822,1509-1515. 

G96 

K 10 

Q 17 

A 

B 

K104 

A136 

K107 

K109 

K197 

K113 

Q189 

Q163 

Prion Protein 

(PrP) 

K188 

Q215 

K207 

R154 

S-S 

Q171 

Q175 

Q220 

C 

Q222 Q226Q230 

P-16

P-17 

 


 

 

 

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66 67 

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HGA-induced aggregation and fibrillogenesis of amyloidogenic proteins: 

implications in alkaptonuria 

L. Millucci, D. Braconi , G. Bernardini, S. Gambassi, M. Laschi, M. Geminiani, L. Ghezzi 

and A. Santucci 

Dipartimento diBiotecnologie, Chimica e Farmacia, UniversitàdegliStudi di Siena, Siena (Italy), annalisa.santucci@unisi.it. 

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, 

causing homogentisic acid (HGA) accumulation that producesochronotic pigment, of unknown composition, 

responsible of organ damage. There is no therapy for AKU. We provided experimental evidence that AKU is 

a secondary serum amyloid A (SAA)-based amyloidosis. [1–3]. Ochronotic pigment and amyloid co-localized 

in AKU tissues indicating a close structural correlation between these two types of deposits. Aberrant 

expression of proteins involved in amyloidogenesis, folding and proteases was found in AKU cells 

suggesting an increase in protein oxidation/aggregation [4]. In the present work we evaluated the HGAmediated 

induction of protein aggregation and fibrillogenesis of amyloidogenic proteins. Our in vitro models 

were based on well-known amyloidogenic proteins and peptides such as Aβ(1-42) peptide, transthyretin 

(Ttr), α-synuclein (α-Syn) and SAA incubated at 37°C with HGA. Our results indicate that HGA can 

significantly accelerate the aggregation of proteins/peptides leading to the production of Congo Red- positive 

aggregates (Figure, panelsA, B) up to formation of oligomers, protofibrils and fibrils (Figure, panels C, D). 

Our findings indicate a role of the key metabolite in AKU, HGA, in the SAA-amyloid production in this 

disease. We hypothesize that HGA auto-oxidation to benzoquinone (BQA) can be at the basis of 

amyloidogenesis in AKU, as supported by our findings on the binding of BQA to α-Syn and SAA. Overall, the 

results obtained, helping the clarification of AKU pathogenesis and related amyloidogenesis, could lay the 

basis to set up more appropriate pharmacological interventions for the disease. 


Financial support from the FMPS 2008-2009-2010, TLSF-Orphan 0108, Telethon GGP10058, FP7-304985, is acknowledged. 

The authors thank P. Lupetti for TEM analysis. 

REFERENCES 

[1] L. Millucci L, A. Spreafico, L. Tinti, D. Braconi, L. Ghezzi, E. Paccagnini, G. Bernardini, L. Amato, M. Laschi, E. Selvi, M. Galeazzi, A. 

Mannoni, M. Benucci, P. Lupetti, F. Chellini, M.Orlandini, A. Santucci. BiochimBiophysActa., 2012, 1822(11):1682-9. 

[2] A. Spreafico, L.Millucci, L. Ghezzi, M. Geminiani, D. Braconi, L. Amato, F. Chellini, B. Frediani, E. Moretti, G. Collodel, G. Bernardini, 

A. Santucci. Rheumatology (Oxford)., 2013, 52(9):1667-73. 

[3] D. Braconi, M. Laschi, A. Taylor, G. Bernardini, A. Spreafico, L. Tinti, JA. Gallagher, A. Santucci. J Cell Biochem.,2010,111:922–932. 

[4] D. Braconi, G. Bernardini, C. Bianchini, M. Laschi, L. Millucci, L. Amato, L. Tinti, T. Serchi, F. Chellini, A. Spreafico, A. Santucci. J 

Cell Physiol. ,2012, 227(9):3333-43. 

P-18






P-19 


A comparative analysis of two amyloidogenic variants of ApoA-I 

R. Del Giudice 1 , D. M. Monti 1 , A. Arciello 1 , F. Itri 1 , R. Piccoli 1 

1 Dept. of Chemical Sciences, University Federico II, Napoli (Italy), mdmonti@unina.it. 


Secondary structures and conformational stability of 

β-2microglobulin mutants in solution, in single crystals and in 

form of fibrils: an FTIR study 

P-20 

Nineteen variants of apolipoprotein A-I (ApoA-I) are associated with hereditary 

systemic amyloidoses, characterized by amyloid deposition in peripheral organs of 

patients [1-3]. As these are heterozygous for the amyloidogenic variants, their 

isolation from plasma is impracticable and recombinant expression systems are 

needed. We report the physicochemical characterization of recombinant ApoA-I 

amyloidogenic variant Leu75 with Pro (L75P) and Leu174 with Ser (L174S) 

expressed and isolated from bacterial cells, with respect to wild-type ApoA-I. 

Proteins were isolated from the cell lysate following a one-step procedure and 

characterized by circular dichroism (CD) and fluorescence analyses. At 

physiologic-like conditions (pH 6.4) all proteins were characterized by a high α- 

helix content, whereas upon incubation at 37°C for 48h both ApoA-I variants 

underwent a conformational transition to a β-sheet rich structure with a concomitant 

increase of thioflavin T fluorescence. On the contrary, conformational changes of 

wt ApoA-I occurred more slowly (1 week). Fluorescence analyses demonstrated 

that at pH 8.0 the L75P variant conformation was altered with respect to wt ApoA-I, 

along with a decreased chemical and thermal stability. On the other hand, even 

though the presence of L174S mutation has no effect on protein conformation, this 

variant undergoes denaturation at lower temperature and denaturant concentration 

with respect to wt ApoA-I. 

pH-induced destabilization of amyloidogenic proteins is known to induce protein 

aggregation. Following the transition from pH 8.0 to 4.0, we observed that L75P 

variant undergoes an immediate transition towards a β-sheet rich structure, along 

with a strong decrease of CD signal, while the α-helix content of L174S variant 

decreases over time, suggesting protein aggregation. Our results clearly indicate 

that amyloidogenic ApoA-I variants are more susceptible to environmental changes 

and are more prone to aggregate when destabilizing conditions occur. 

68 69 

REFERENCES 

[1] L. Obici, G. Franceschini, L. Calabresi, S. Giorgetti, M. Stoppini, G. Merlini, V. Bellotti Amyloid, 

2006,13,191 205. 

[2] M. Eriksson, S. Schonland, S. Yumlu, U. Hegenbart, H. von Hutten, Z. Gioeva, P. Lohse, et al J. Mol. 

Diagn., 2009, 11, 257 262. 

[3] D. Rowczenio, A. Dogan, J.D. Theis, J.A. Vrana, H.J. Lachmann, A.D. Wechalekar, J.A. Gilbertson, 

et al Am. J. Pathol., 2011, 179, 1978-1987. 

A. Natalello 1 , S. Ricagno 2 , D. Ami 1 , L. Halabelian 2 , M. Bolognesi 2 , S.M. 

Doglia 1 

1 Dept. of Physics and Dept. of Biotechnology and Biosciences, University of Milano-Bicocca (Italy); 

2 Dept. of Bioscience, University of Milan (Italy), antonino.natalello@unimib.it 

β-2 microglobulin (β2m) is an amyloidogenic protein involved in the dialysis related 

amyloidosis. 

Here, we characterized by Fourier transform infrared (FTIR) spectroscopy the wild 

type protein and its mutants Asp59Pro, Trp60Gly, and Trp60Val. Although they 

displayed very similar 3D structures, the FTIR measurements in solution pointed to 

significant differences in thermal stability and aggregation propensity. To 

comprehend these features, we studied each β2m variants in form of single 

crystals by FTIR microscopy. Significant spectral differences were observed 

between the protein in solution and in the crystalline state involving mainly the main 

β-sheet band. Furthermore, appreciable differences in secondary structures were 

found among the variants [1]. 

We investigated also the β2m fibrils obtained after incubation for one week of each 

variant under fibrillogenic conditions. Attenuated total reflection (ATR) 

measurement displayed a comparable hydrogen/deuterium exchange behaviour of 

the intermolecular β-sheets, indicating similar accessibility and dynamics of these 

structures. 

This study illustrates the potential of FTIR (micro)spectroscopy to obtain 

complementary structural information on the protein under different physical states, 

namely in solution, in form of insoluble aggregates, and as single protein crystals. 

REFERENCES 

[1] D. Ami, S. Ricagno, M. Bolognesi, V. Bellotti, S.M. Doglia, A. Natalello 

Biophys. J., 2012, 102, 1676-1684.







P-21 

EXAMINING DISEASE-ASSOCIATED Aβ MUTANTS AS 

CONFORMATIONAL STRAINS 

Mimi C Nick 1 , Jan Stöhr 2 , William F DeGrado 3 


Mimi.Nick@ucsf.edu 

2 Institute for Neurodegenerative Diseases, University of California, San Francisco (USA), 

JStoehr@ind.ucsf.edu 


Bill.DeGrado@ucsf.edu 

INTERACTION BETWEEN PRION PROTEIN and 

G-QUADRUPLEX-FORMING NUCLEIC ACIDS: 

a BIOPHYSICAL STUDY 

Bruno Pagano 1 , Paola Cavaliere 2 , Vincenzo Granata 3 , Stephanie Prigent 4 , 

Human Rezaei 4 , Ettore Novellino 1 , Concetta Giancola 1 , Adriana Zagari 3 

1 Dept. of Pharmacy, University of Naples Federico II, Napoli (Italy), bruno.pagano@unina.it. 

2 Dept. of Microbiology, Institut Pasteur, Paris (France). 

3 CEINGE-Biotecnologie Avanzate, Napoli (Italy). 

4 Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, 

Jouy-en-Josas (France). 

P-22 

Alzheimer’s Disease (AD), the most common form of dementia, cruelly strips 

patients of memory, cognitive ability, and independence, and is a leading cause of 

death for people aged 65 and older.[1] The major pathological hallmark of AD is 

the accumulation of β-amyloid (Aβ) peptide within amyloid plaques in the brain.[2,3] 

When forming the cross-β structure typical of amyloid fibrils, there is some inherent 

flexibility regarding the intermolecular contacts that the Aβ peptide can adopt. As a 

result, the fibril structure may vary, resulting in distinct conformational strains.[4-6] 

While a specific primary sequence is able to adopt multiple strain conformations, it 

is not known how disease-associated mutations occurring within that sequence 

may alter the fibril structure. We are using biophysical methods to determine 

whether certain Aβ mutants form unique fibril strains that propagate their particular 

structure irrespective of primary sequence. 

70 71 


Financial support from the Glenn Foundation is acknowledged. 

REFERENCES 

[1] W Thies, L Bleiler, Alzheimer’s Association. Alzheimers Dement, 2013, 9(2), 208-245. 

[2] GG Glenner, CW Wong. Biochem Biophys Res Commun, 1984, 120(3), 885-890. 

[3] RE Tanzi, JF Gusella, PC Watkins, GA Bruns, P St George-Hyslop, ML Van Keuren, D Patterson, S 

Pagan, DM Kurnit, RL Neve. Science, 1987, 235, 880-884. 

[4] M Tanaka, P Chien, N Naber, R Cooke, JS Weissman. Nature, 2004, 428, 323-328. 

[5] AT Petkova, RD Leapman, Z Guo, WM Yau, MP Mattson, R Tycko. Science, 2005, 307, 262-265. 

[6] AK Paravastu, RD Leapman, WM Yau, R Tycko. Proc Natl Acad Sci USA, 2008, 105, 18349-18354. 

Several studies have been recently carried out about the Prion protein (PrP) 

interaction with nucleic acids, looking for the biological relevance that these 

interactions may hold inside cell environment.[1] G-quadruplex-forming nucleic 

acids have been found to specifically bind to both cellular and pathological PrP 

isoforms.[2] To clarify the relevance of these interactions, thermodynamic, kinetic 

and structural studies have been performed, using isothermal titration calorimetry, 

surface plasmon resonance and circular dichroism methodologies.[3] 

Three G-quadruplex-forming sequences and various 

forms of PrP were selected for this study. Our results 

showed that the investigated G-quadruplexes exhibit a 

high affinity and specificity toward PrP, with K d values 

within the range 62÷630 nM, and a weaker affinity 

toward a PrP-β oligomer, which mimics the pathological 

isoform. We demonstrated that the G-quadruplex 

architecture is the structural determinant for the 

recognition by both PrP isoforms. Furthermore, we 

spotted both PrP N-terminal and C-terminal domains as the binding regions 

involved in the interaction with DNA/RNAs, using several PrP truncated forms. 

Interestingly, a reciprocally induced structure loss was observed upon PrP-nucleic 

acid interaction. Our results indicate the formation of dynamic complexes between 

PrP and G-quadruplex-forming nucleic acids, that may have a feedback in vivo. 

REFERENCES 

[1] (a) Y. Cordeiro, F. Machado, L. Juliano, M. A. Juliano, R. R. Brentani, D. Foguel, J. L Silva J. Biol. 

Chem., 2001, 276, 49400–9. (b) S. Gilch, H. M. Schatzl Cell. Mol. Life Sci., 2009, 66, 2445-55. (c) M. P. 

Gomes, T. C. Vieira, Y. Cordeiro, J. L. Silva Wiley Interdiscip. Rev. RNA, 2012, 3, 415-28. 

[2] K. Murakami, F. Nishikawa, K. Noda, T. Yokoyama, S. Nishikawa Prion, 2008, 2, 73-80. 

[3] P. Cavaliere, B. Pagano, V. Granata, S. Prigent, H. Rezaei, C. Giancola, A. Zagari Nucleic Acids 

Res., 2013, 41, 327-39.









P-23 

Cu(II) induced oligomerization of amyloid-β on the millisecond 

time scale 

J. T. Pedersen 1 , C.B. Borg 2 , K. Teilum 2 , L. Hemmingsen 3 

1 Dept. of Pharmacy, University of Copenhagen, Copenhagen (Denmark), jeppe.trudslev@sund.ku.dk 

2 Dept. of Biology, University of Copenhagen, Copenhagen (Denmark) 

3 Dept. of Chemistry, University of Copenhagen, Copenhagen (Denmark) 

Many neurodegenerative disorders (ND) including Alzheimer’s (AD) and 

Parkinson’s disease (PD) are associated with the accumulation of amyloid fibrils. 

Metal ion binding to these amyloid proteins together with metal dyshomeostasis in 

the body are implicated in both AD and PD. [1] Moreover, recent data demonstrate 

that metal ions such as Cu(II) and Zn(II) can induce misfolding and oligomerization 

of amyloid-β on the biologically relevant millisecond–second time-scale [2,3]. 

Hence, rapid metal–Aβ interactions may play a role in the pathology of AD (Fig. 1). 

It is well-established that a range of soluble Aβ–Cu species co-exist in a dynamic 

equilibrium depending on the pH.[4] It is reasonable to think that distinct Aβ–Cu(II) 

species have distinct oligomerization propensities. Here, we study the mechanism 

of Cu(II) binding to Aβ and the subsequent metal induced oligomerization at 

different pH using stopped-flow fluorescence and light scattering in combination 

with NMR relaxation. This approach allows us to study the properties of the Aβ–Cu 

species formed milliseconds upon metal ion binding. 

72 73 

Fig. 1 Potential mechanism of metal induced amyloid formation 


Financial support by the Villum Foundation is acknowledged. 

FIBRIL FORMATION OF A 3D DOMAIN SWAPPING 

RIBONUCLEASE: A MODEL BASED ON THE CRYSTAL 

STRUCTURE OF THE PROTEIN 

A. Pica 1,2 , A. Merlino 1,2 , A.K. Buell 3 , T.J. Knowles 3 , E. Pizzo 4 , G. 

D’Alessio 4 , F. Sica 1,2 , L. Mazzarella 1,2 

1 Dept. of Chemical Sciences, University of Naples Federico II, Naples (Italy), andrea.pica@unina.it. 

2 Institute of Biostructures and Bioimaging, CNR, Naples, Italy 

3 Nanoscience Centre, University of Cambridge, Cambridge CB3 0FF, UK 

4 Dept. of Biology, University of Naples Federico II, Naples, Italy 

The deletion of five residues in the loop connecting the 

N-terminal helix to the core of monomeric human 

pancreatic ribonuclease leads to the formation of an 

enzymatically active domain-swapped dimer 

(desHP).[1] The crystal structure of desHP reveals the 

generation of an intriguing fibril-like aggregate of 

desHP molecules that extends along the c 

crystallographic axis. Dimers are formed by threedimensional 

domain swapping.[2] Tetramers are 

formed by the aggregation of swapped dimers with 

slightly different quaternary structures. The tetramers 

interact in such a way as to form an infinite rod-like 

structure that propagates throughout the crystal. The 

observed supramolecular assembly captured in the 

crystal predicts that desHP fibrils could form in solution; 

this has been confirmed by atomic force microscopy. 

These results provide new evidence that threedimensional 

domain swapping can be a mechanism for 

the formation of elaborate large assemblies in which the 

protein, apart from the swapping, retains its original 

fold.[3-5] 

P-24 

REFERENCES 

“Biophysics 

[1] Viles, J. H. Coord. 

of 

Chem. 

Amyloids 

Rev. 2012 256, 

and 

2271-2284 

Prions” 

[2] Pedersen, J. T., Teilum, K., Heegaard, N. H. H., Østergaard, J., Adolph, H. W., and Hemmingsen, L. 

Angew. Chem. Int. Ed. 2011 50, 2532-2535 

[3] Noy, D., Solomonov, I., Sinkevich, O., Arad, T., Kjaer, K., and May Sagi, 25-26, I. J. Am. 2014 Chem. - Naples, Soc. 2008 Italy 130, 

1376-1383 

[4] Drew S.C., Noble C.J., Masters C.L., Hanson G.R., Barnham K.J. J. Am. Chem. Soc. 2009 

131,1195-1207 

REFERENCES 

[1] N. Russo, A. Antignani, and G. D'Alessio. Biochemistry, 2000, 39, 3585-3591. 

[2] M.J. Bennett, M.P. Schlunegger, and D. Eisenberg. Protein Sci, 1995, 4, 2455-2468. 

[3] Y. Liu, and D. Eisenberg. Protein Sci, 2002, 11, 1285-1299. 

[4] S. Sambashivan, Y. Liu, M.R. Sawaya, M. Gingery, and D. Eisenberg. Nature, 2005, 437, 266-269. 

[5] A. Pica, A. Merlino, A.K. Buell, T.P.J. Knowles, E. Pizzo, G. D’Alessio, F. Sica, and L. Mazzarella. 

Acta Cryst D, 2013, 69, 2116-2123.

P-25 




Evidence for a role of hydroxytyrosol in decreasing 

oligomerization in a model system of AD 

R. Crea, C. Bitler, P. Pontoniere 

CreAgri, Inc., 25565, Whitesell Street, Hayward, CA 94545 (USA), rcrea@creagri.com, 

cmbitler@aol.com, ppontoniere@creagri.com 






of HT on demyelination and re-myelination, we evaluated its effect on cuprizoneinduced 

demyelination in zebra fish, a model system of multiple sclerosis. 

Treatment of zebra fish with HT olive extract significantly reduced cuprizoneinduced 

demyelination thus significantly protecting neurons from eventual cell 

death (McGrath et al., unpublished study). 

P-25 

Toxic changes occur in the brain during the early stage of Alzheimer’s disease, 

including abnormal deposits of proteins that form amyloid plaques and tau tangles, 

mitochondrial dysfunction, and once-healthy neurons begin to work less efficiently. 

In time, neurons lose their ability to perform and eventually die. 

Lack of physical activity, cognitive stimulation, unhealthy diet and nutrition are all 

thought to contribute to the outcome of the AD phenotype. On the contrary, high 

adherence to the Mediterranean Diet has been shown to be associated with a 

reduced risk of developing both cognitive impairment and Alzheimer’s disease [1]. 

Although the Mediterranean diet combines several foods, micronutrients, and 

macronutrients, one molecule in particular stands out for its medicinal properties, 

3,4 dihydroxyphenylethanol or hydroxytyrosol (HT), a bio-phenolic molecule found 

almost exclusively in the olive plant [2]. Recently interest on HT has been focused 

on its anti-inflammatory [3,4], and brain protecting [5] activities. 

74 75 

In one study of hypoxia/re-oxygenation, HT was shown to decrease cell death, 

reduce stress parameters, and increase enzyme activities associated with 

glutathione production [6]. In another study, HT attenuated the cytotoxic effects of 

Fe 2+ and nitric oxide-induced toxicity in murine brain cells. The cytotoxic effects 

included a severe loss of cellular ATP, lipid peroxidation, and a markedly 

depolarized mitochondrial membrane potential. Additionally, oral administration of 

HT resulted in a significant decrease in oxidative stress, mitochondrial membrane 

potential reset, and neuroprotection [7]. Finally, in another study HT was shown to 

prevent Tau fibril aggregation in vitro. Tau aggregation into fibrillary tangles 

contributes to intra-neuronal and glial lesions in Alzheimer’s disease [8]. In this 

study, the effectiveness of HT was significantly greater than the reference inhibitor. 

Prolonged neuroinflammation is associated with many diseases including 

Parkinson’s, Alzheimer’s and multiple sclerosis. Our lab has recently shown that 

HT in its natural milieu of the olive fruit significantly inhibited production and 

secretion of TNF-a, IL-6 and IL-1b in both microglia and astrocyte cells [9]. 

Increasing evidence suggests that cognitive deterioration in AD is directly linked to 

the accumulation of extracellular soluble oligomers of β-amyloid protein rather than 

amyloid plaque deposition in the brain. In one study, we have shown that HT 

interferes with the formation of β-amyloid oligomer formation as well as Tau protein 

oligomer formation (Pasinetti et al., unpublished study). Finally, to study the effects 

REFERENCES 

1. Lourida et al., Epidemiology, 2013, 4, 479-89. 

2. Mulinacci et al.., JAFC, 2001, 49 (8), 3509-14. 

3. Bitler et al., J Nutrition, 2005, 135, 1475–1479. 

4. Richard et al., Planta Med., 2011, 77, 1890–1897. 

5. DeNicolo et al., Nutrition, 2013 Apr., 29 (4), 681-7 

6. Cabrerizo et al., J Nutr Biochem, 2013 Dec., 24 (12), 2152-7. 

7. Schaffer et al., JAFC, 2010, 55, 5043-5049. 

8. Daccache et al., Neurochem Int., 2011 May, 58 (6):700-7. 

9. Crea et al., AG Food Technology, 2010 March/April, 23 (2), 26-9.









P-26 

Secondary nucleation in stirring induced alpha-lactalbumin 

amyloid fibril formation. 

E. Rao 1 , V.Vetri 1 , V. Foderà 2,3 , V. Militello 1 and M. Leone 1 

1 Dip. di Fisica e Chimica, Università di Palermo, Palermo, Italy 

2 Dept. of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark 

3 Dept. of Physics, Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 

email: estella.rao@gmail.com 

Protein aggregation is one of the most challenging topics in recent biomedical and 

biotechnological research. Several neurodegenerative pathologies like Parkinson's 

and Alzheimer's disease or type-II diabetes are associated with the formation and 

deposition, in organs and tissues, of large amounts of insoluble and highly ordered 

aggregates, known as amyloid fibrils. The interest in amyloid fibrils is also related 

to their potential application as biomaterials due their peculiar and tunable physicochemical 

properties. Recently, since proteins are often subjected to mechanical 

stress both in vivo and during procedures in pharmaceutical processing, specific 

interest has been focused on shear-induced aggregation. Stirring, shaking, or 

mechanical agitation are known to accelerate supramolecular assembly rate and to 

modify fibril morphology. 

We here report an experimental study on stirring-induced amyloid formation of 

alpha-lactalbumin (α-La). Multitechnique approach consisting in spectroscopy and 

microscopy techniques is used to analyse mechanisms underlying aggregation. 

α-La amyloid formation was observed as a function of time by means of light 

scattering and in situ Thioflavin T (ThT) fluorescence measurements in different 

environmental conditions. With the aim of probing the secondary structure and 

morphology, final aggregates were characterized by FTIR absorption spectroscopy 

and by microscopy. The obtained results allow us to describe the effect of stirring 

on α-La amyloid formation process. Under the presented experimental conditions, 

amyloid formation is induced or critically accelerated by stirring. At defined 

temperature, different stirring velocities appear to not critically change the 

assembly mechanisms,, final aggregates structure and morphology, while the 

number of formed amyloid structures is enhanced at increasing stirring velocities. 

The analysis of obtained results suggests that α-lactalbumin aggregation, in the 

used conditions, underlines a complex interconnection of assembly mechanisms 

regulated by secondary nucleation which causes inherent irreproducibility of 

observed aggregation kinetics. These mechanisms are enhanced by stirring which 

favours the exposure of new accessible surfaces through fibrils fragmentation or by 

bringing back into solution aggregates formed at liquid-air interfaces. 

TETRACYCLINE AND EPIGALLOCATECHIN-3-GALLATE 

DIFFERENTLY AFFECT THE ATAXIN-3 FIBRILLOGENESIS AND 

TOXICITY 

M. Bonanomi 1 , C. Visentin 1 , A. Natalello 1 , A. Penco 2 , G. Colombo 1 , A. 

Relini 2 , S.M. Doglia 3 , M.E. Regonesi 4 

1 Dept. of Biotechnologies and Biosciences, University of Milano-Bicocca, Milan (Italy) 

2 Dept. of Physics, University of Genoa, Genoa (Italy) 

3 Dept. of Physics “Giuseppe Occhialini”, University of Milano-Bicocca, Milan (Italy) 

4 Dept. of Statistics and Quantitative Methods, University of Milano-Bicocca, Milan (Italy), 

mariaelena.regonesi@unimib.it 

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers the inherited 

neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3) when its 

polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. The 

expansion results in misfolding and other structural rearrangements in the protein 

product, which lead to aberrant interactions of the expanded protein and to the 

consequent formation of fibrillar amyloid-like aggregates [1,2]. AT3 consists of the 

N-terminal globular Josephin domain (JD) and the C-terminal disordered one [3,4]. 

Regarding its physiological role, it has ubiquitin hydrolase activity implicated in the 

function of the ubiquitin-proteasome system, but also plays a role in the pathway 

that sorts aggregated protein to aggresomes via microtubules [5,6]. In recent years, 

therapeutic strategies aimed to prevent or control amyloid-related diseases were 

developed, based on molecules that inhibit protein deposition or reverse fibril 

formation. To date, no effective treatment has been developed for SCA3 disease 

and no compounds were tested for their effect on AT3 aggregation process. For 

this reason, we sought to evaluate the effects of tetracycline and epigallocatechin- 

3-gallate (EGCG), two well-known inhibitors of amyloid aggregation, on AT3 

fibrillogenesis and cytotoxicity. We observed that tetracycline does not apparently 

change the aggregation mode, as supported by Fourier Transform Infrared 

spectroscopy and Atomic Force Microscopy data, but increase the solubility of the 

different aggregated species. Otherwise EGCG interferes in the early steps of 

aggregation, accelerating the misfolding of the Josephin Domain, and leads to the 

formation of off-pathway, non-amyloid, final aggregates. In both cases, coincubation 

of AT3 with these compounds reduces the toxicity of protein aggregates. 

76 77 

REFERENCES 

[1] H. Y. Zoghbi, H. T. Orr Annu. Rev. Neurosci., 2000, 23, 217–247. 

[2] J. R. Gatchel, H. Y. Zoghbi Nat. Rev. Genet., 2005, 6, 743–755. 

[3] B. Burnett, F. Li, R. N. Pittman Hum. Mol. Genet., 2003, 12, 195–205. 

[4] L. Masino, V. Musi, R. P. Menon, P. Fusi, G. Kelly, A. Frenkiel, Y. Trottier, A. Pastore FEBS Lett., 

2003, 549, 21–25. 

[5] E. W. Doss-Pepe, E. S. Stenroos, W. G. Johnson, K. Madura Mol. Cell. Biol., 2003, 23, 6469–6483. 

[6] H. Ouyang, Y. O. Ali, M. Ravichandran, A. Dong, W. Qiu, F. Mackenzie F J. Biol. Chem., 2012, 287, 

2317-2327. 

P-27







P-28 

INTERACTIONS OF THE GLIAL FIBRILLARY ACIDIC PROTEIN 

WITH CEFTRIAXONE REVEALED THROUGH SRCD. 

P. Ruzza 1 , G. Siligardi 2 , R. Hussain 2 , B. Biondi 1 , A. Calderan 1 , G. P. Sechi 3 

1 Institute of Biomolecular Chemistry of CNR, Padua Unit, Padua, Italy. 

2 Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, 

United Kingdom. 

3 Department of Clinical and Experimental Medicine, Medical School, University of Sassari, Sassari, 

Italy. 

Early determinants of human prion protein conversion investigated by solutionstate 

NMR, XAFS and nanobody-assisted crystallography 

Gabriele Giachin 1 , Giulia Salzano 1* , Gregor Ilc 2 , Ivana Biljan 2,3 , Romany N. N. Abskharon 4 , 

Alexandre Wohlkonig 4 , Sameh H. Soror 4 , Els Pardon 4 , Jan Steyaert 4 , Federico Benetti 1 , Paola 

D’angelo 5 , Janez Plavec 2 and Giuseppe Legname 1,6 

1 Department of Neuroscience, Laboratory of Prion Biology, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy. 

2 Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia. 

3 Department of Chemistry, University of Zagreb, Zagreb, Croatia. 

4 Vrije Universiteit Brussel, Structural Biology Brussels, Brussels, Belgium. 

5 Department of Chemistry, University of Rome “La Sapienza”, Rome, Italy. 

6 ELETTRA Laboratory, Sincrotrone Trieste S.C.p.A., Basovizza, Trieste, Italy. 

*Presenting author: gsalzano@sissa.it 

P-29 

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) type III protein 

that, along with microtubules and microfilaments, makes up the cytoskeleton of 

most eukaryotic cells. [1] GFAP has been extensively investigated because it is 

involved in the reactive changes in astrocytes (gliosis) associated with ageing [2], 

following brain injury, in neurodegenerative disorders such as Alzheimer’s and 

The conversion of the cellular prion protein (PrP C ) into its misfolded and amyloidogenic isoform, denoted 

as prion or PrP Sc , is the central event in prion diseases. In prion biology unraveling the molecular 

mechanism leading the conversion process whereby α-helical motifs are replaced by β-sheet secondary 

structures is of utmost importance. The structure of human PrP C consists of a disordered N-terminal part 

(residue 23-127) and a structured C-terminal domain (residue 128-228). Genetic disease-linked mutations 

in the human prion protein (HuPrP) cause spontaneous conversion of PrP C to PrP Sc . We investigated the 

78 

structural consequences of mutations (V210I, Q212P and the polymorphism E219K) clustered on the 

79 

Alexander’s [3] diseases, and multiple sclerosis. 

Recently, reports from our group showed that ceftriaxone, a safe and multi-potent 

β-lactam antibiotic able to pass freely the blood-brain barrier, successfully 

eliminated the cellular toxic effects of misfolded GFAP in a cellular model of 

Alexander’s disease [4]. Here we used synchrotron radiation circular dichroism 

(SRCD) spectroscopy to obtain structural information about GFAP-ceftriaxone 

molecular interactions. The stability of GFAP with and without ceftriaxone was 

studied by UV-denaturation using the high-photon flux of Diamond B23 beamline. 

The B23 UV-induced denaturation, unattainable with bench-top CD instruments, 

provided a novel type of assay for biopolymer stability and binding interaction 

assessments, permitting to identify GFAP ligands and evaluate their ability to 

increase the protein stability in a short period of time, and with very small amount 

of sample material. 


We thank Diamond Light Source for access to beamline B23 (SM8034-1) that contributed to the results 

presented here. The research leading to these results has received funding from the European 

Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 226716. 

REFERENCES 

[1] D. Dahl, D.C. Rueger, A. Bignami, K. Weber, M. Osborn Eur. J. Cell Biol. 1981, 24, 191-196. 

[2] J. Middeldorp, E.M. Hol Prog. Neurobiol. 2011, 93 421-443. 

[3] T. Yoshida, M. Nakagawa Neuropathology 2012, 32, 440-446. 

[4] G. Sechi, I. Ceccherini, T. Bachetti, G.A. Deiana, E. Sechi, P. Balbi JIMD Rep. 2013, 9, 67-71. 

HuPrP structured C-terminal domain by NMR. [1,2,3,4] We found that amino acidic substitutions affect the 

hydrophobic interactions between residues clustered at the interface of the β2-α2 loop and the α3 helix. 

These studies led us to conclude that the structural disorders of the β2-α2 loop, together with the 

increased spacing between this loop and the C-terminal part of α3 helix are key pathological features. 

The disruption of these interactions and the consequent exposure to the solvent of the hydrophobic core 

led to the suggestion that the early stage of prion conversion possibly involves the critical epitope formed 

by the β2-α2 loop and the α3 helix. We then evaluated the effect of the Q212P mutation on the copper 

coordination in one of the copper binding sites located in the unstructured N-terminal domain using X-ray 

absorption fine structure techniques and we compared these findings with the wild-type HuPrP. We found 

that this mutant causes a dramatic modification on the copper coordination in this binding site. [5] Finally, 

we investigated if the N-terminal domain may acquire a structured fold upon binding to an antibody 

targeting a critical epitope involved in the conversion process (the palindromic motif AGAAAAGA, 

residues 113-120). We crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits 

prion propagation. While the segment from residue 128 to 225 shares a fold that is very similar to the 

corresponding NMR HuPrP structures, the binding of Nb484 to a region adjacent to the first β-sheet (β1) 

unveils key structural features of the hydrophobic segment from residue 117 to 128, which had remained 

unresolved in all the PrP structures published so far. In our X-ray structures, the palindromic motif 

arranges in a novel β-strand, denoted as β0 (residues 118–122), which folds into a three-stranded 

antiparallel β-sheet with β1 and β2. The implications of these findings are remarkable, as we provide a 

first atomic structural view of the palindromic region adopting a well-defined β-sheet conformation. [6] 

REFERENCES 

[1] I. Biljan, G. Ilc, G. Giachin, J. Plavec, G. Legname Biochemistry, 2012, 51, 7465-7474 

[2] I. Biljan, G. Ilc, G. Giachin, A. Raspadori, I. Zhukov, J. Plavec, G. Legname J Mol Biol, 2011, 412(4), 660-673 

[3] G. Ilc, G. Giachin, M. Jaremko, L. Jaremko,F. Benetti, J. Plavec, I. Zhukov, G. Legname PLoSOne, 2010, 5, e11715 

[4] I. Biljan, G. Giachin, G.Ilc, I. Zhukov, J. Plavec, G. Legname Biochem J, 2012, 446, 243-251 

[5] P. D'Angelo, S. Della Longa, A. Arcovito, G. Mancini, A. Zitolo, G. Chillemi, G. Gachin, G. Legname Biochemistry, 2012, 6068- 

6079 

[6] RN. Abskharon, G. Giachin, A. Wohlkonig, SH. Soror, E. Pardon, G. Legname, J. Steyaert J Am Chem Soc, 2014, 136(3), 937- 

944









P-30 

ELONGATION OF MOUSE PRION PROTEIN AMYLOID-LIKE 

FIBRILS: EFFECT OF TEMPERATURE AND DENATURANT 

CONCENTRATION 

Katazyna Milto, Ksenija Michailova, Vytautas Smirnovas 

α-synuclein aggregation: effect of protein charge on fibril 

elongation and membrane interaction 

Alex I.M. van der Wateren 1 , Alexander K. Büll 1 , Céline Galvagion 1 , 

Christopher M. Dobson 1 

P-31 

Department of Biothermodynamics and Drug Design, Vilnius University Institute of 

Biotechnology, Vilnius, Lithuania, vytautas@smirnovas.info 

Prion protein is known to have the ability to adopt a pathogenic conformation, 

which seems to be the basis for protein-only infectivity. The infectivity is based on 

self-replication of this pathogenic prion structure. One of possible mechanisms for 

such replication is the elongation of amyloid-like fibrils. 

We measured elongation kinetics and thermodynamics of mouse prion amyloid-like 

fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show 

that both increases in temperature and GuHCl concentration help unfold 

monomeric protein and thus accelerate elongation. Once the monomers are 

unfolded, further increases in temperature raise the rate of elongation, whereas the 

addition of GuHCl decreases it. 

We demonstrated a possible way to determine different activation energies of 

amyloid-like fibril elongation by using folded and unfolded protein molecules. This 

approach separates thermodynamic data for fibril-assisted monomer unfolding and 

for refolding and formation of amyloid-like structure. 

1 Department of Chemistry, University of Cambridge, Cambridge, UK, imv22@cam.ac.uk 

α-synuclein (AS) is a small protein implicated in synaptic function [1] and is 

strongly implicated in Parkinson’s Disease (PD) where it forms intracellular fibrillar 

aggregates [2]. These aggregates were long thought to result in neuronal cell 

death, but more recently it has been suggested that oligomeric (precursor) 

structures might damage membranes and cause cell death [3]. The majority of PD 

cases is sporadic but some people develop PD much earlier in life due to a 

pointmutation in AS [4]. The protein in vivo is also found to be post-translationally 

modified (such as phosphorylation and truncation) [5]. We are interested in how 

such modifications, in particular truncations of all or part of the negatively charged 

C-terminus, affect the kinetics of aggregation and the ability of the protein to 

interact with lipid membranes, in an effort to study protein aggregation in a more 

physiologically relevant setting. Our preliminary results show that C-terminally 

truncated AS (the first 103 residues: AS 1-103) is more prone to form higher order 

assemblies of aggregates than wild-type (WT) protein. We also show that WT 

aggregates are able to “seed” elongation of AS 1-103, more so than vice versa. 

The behavior of AS 1-103 renders the use of the classical fluorescent dyes, such 

as Thioflavin T to monitor aggregation, challenging and we will discuss strategies 

to curcumvent these difficulties. Both WT and AS 1-103 similarly interact with small 

unilamellar vesicles (SUVs) of DMPS, but we observe large differences between 

WT and AS 1-103 when studying aggregation in the presence of SUVs. 

80 81 

REFERENCES 

[1] Bellani et al. 2010, The regulation of synaptic function by a-synuclein, Communicative & Integrative 

Biology 3:2, 106-109 

[2] Baba et al. 1998, Aggregation of a-synuclein in Lewy Bodies of sporadic Parkinson’s disease and 

dementia with Lewy Bodies, American Journal of Pathology 153:4, 879-884 

[3] Kalia et al. 2012, a-synuclein oligomers and clinical implications for Parkinson Disease, Annals of 

Neurology 73:2, 155-169 

[4] Klein and Westenberger 2012, Genetics of Parkinson’s Disease, Cold Spring Harbor Perspectives in 

Medicine 

[5] Schmid et al. 2013, a-synuclein post-translational modifications as potential biomarkers for 

Parkinson’s Disease and other Synucleinopathies, Molecular & Cellular Proteomics 12:12, 3543-3558









P-32 

Chaperon-like activity of intrinsically disordered caseins in A 

fibrillogenesis 

S Vilasi 1 , R Carrotta 1 , GC Rappa 1 , MG Ortore 2 , Canale C 3 , PL San Biagio 1 , 

D Bulone 1 

1 Institute of Biophysics, National Research Council, Palermo, Italy. silvia.vilasi@pa.ibf.cnr.it 

2 Department of Life and Environmental Sciences and CNISM, Marche Polytechnic University, Ancona, 

Italy 

3 Nanophysics, Italian Institute of Technology (IIT), Genova, Italy. 

Alzheimer's disease (AD) is a chronic and progressive syndrome, which affects 

about 5% of the population over age 65 and is characterized by the accumulation 

of a 40-42 aminoacids peptide, the amyloid-beta peptide (Aß), in insoluble plaques, 

known as amyloid fibrils. Recent studies have attributed to small Hsp, and other 

little molecules able to exert an chaperon-like activity, an important role in amyloid 

neurodegenerative diseases and their stabilizying function is often correlated to 

their intrinsically disorder structure [1]. An example is provided by the intrinsically 

disordered proteins, β-, and κ-caseins that are able to inhibiting protein 

aggregation and amyloid fibrils formation and this chaperon-like activity could be 

largely due to their open and flexible conformations. [2,3]. In the present study we 

compare the effect of the three caseins on 1-40 β-amyloid peptide fibrillogenesis by 

Thioflavin T fluorescence and Circular dichroism. Results show that k-casein, that 

in its native state is in a multimeric oligomer composition [4], is able to significantly 

increase lag-time and reduce fibril amount in Aβ amyloid formation. Therefore, we 

explored the action mechanisms of k-casein on Aβ aggregation process by using 

protein intrinsic fluorescence, AFM measurements, light and Small Angle X Ray 

Scattering. Data demonstrate that the k-native casein in its oligomeric state 

incorporates Aβ on its surface, by means of a holding action mainly due to its 

intrinsic disorder [1], thus reducing the amount of peptide available for subsequent 

aggregation. Our results contribute to clear the role of intrinsically disordered 

proteins and their mechanism of action, and offer insight in the field of prevention 

and therapy in Alzheimer diseases, and, in general, of amyloid pathologies. 

Insights into structural features of intermediate states along the 

fibrillogenesis pathway of amyloid-like systems 

L. Vitagliano 1 , A. De Simone 2 , L. Esposito 1 

1 Institute of Biostructures and Bioimaging . CNR, Napoli (Italy), luigi.vitagliano@unina.it. 

2 Division of Molecular Biosciences, Imperial College South Kensington Campus London SW7 2AZ, UK 

The definition of the molecular basis of human diseases is one of the most 

important goals of structural biology. Computational methodologies represent 

valuable tools for coping with these challenging projects. In this scenario, we have 

undertaken molecular dynamics simulations on several amyloid-like systems to 

gain insights into the structural basis of the fibrillogenesis process, which is a key 

step in the insurgence of widespread neurodegenerative diseases. These studies 

have provided some interesting clues on the structural features of small soluble 

precursors of amyloid-like fibers [1-4], which are the actual toxic species. On this 

basis, we have related the toxicity of these oligomers with some specific structural 

features such as the exposure of sticky β-strands [1-2]. 

In more recent years, these computational approaches have been extended to the 

analysis of the intrinsic stability of the β-sheet of the prion protein, a region that is 

believed to play a crucial role in the protein aggregation [5]. Extensive replica 

exchange molecular dynamics simulations clearly indicate that the native 

antiparallel β-structure of the prion is endowed with a remarkable stability. 

Therefore, upon unfolding, the persistence of a structured β-region may seed 

molecular association and influence the subsequent phases of the aggregation 

process. The analysis of the four-stranded β-sheet detected in the dimeric 

assemblies of PrP shows a tendency of this region to form dynamical structured 

states. We also evaluated the impact on the β-sheet structure and dynamics of 

disease associated point mutations. 

82 83 

P-33 

REFERENCES 

[1] V. Uversky, Protein chaperones and protection from neurodegenerative diseases, 2011, Wiley. 

[2] R. Carrotta, C. Canale, A. Diaspro, A. Trapani , PL Biagio, D Bulone Biochim Biophys Acta., 2012, 

1820(2),124. 

[3] D.C. Thorn, S. Meehan, M. Sunde, A. Rekas, S.L. Gras, C.E. MacPhee, C.M. Dobson, M.R. Wilson 

MR, JA. I. Carver, Biochemistry, 2005,44(51),17027-36. 

[4] L.K. Rasmussen. P. Højrup, T.E. Petersen, Eur. J. Biochem., 1992, 207, 215-222. 


This work has been supported by FIRB RBFR12SIPT MIND: “Indagine multidisciplinare per lo sviluppo 

di farmaci neuro-protettori.” 

REFERENCES 

[1] L. Esposito, A. Paladino, C. Pedone, L. Vitagliano Biophysical J., 2008, 94, 4031-4040. 

[2] A. De Simone, L. Esposito, C. Pedone, L. Vitagliano Biophysical J., 2008 95, 1965-1973. 

[3] A. De Simone, C. Pedone, L. Vitagliano BBRC, 2008, 366, 800-806. 

[4] L. Vitagliano, L. Esposito, C. Pedone, A. De Simone BBRC, 2008, 377, 1036-1041. 

[5] A. De Simone, F. Stanzione, D. Marasco, L. Vitagliano, L. Esposito J Biomol Struct Dyn. 2013, 31, 

441-52.

P-34 




Assembly mechanism of hereditary amyloid-β variants 

promoted on their specific gangliosides 

M. Yagi-Utsumi 1,2 , K. Kato 2 , C. M. Dobson 1 

1 Dept. of Chemistry, University of Cambridge, Cambridge (United Kingdom), my322@cam.ac.uk 

2 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki (Japan) 

Lipid membranes provide active platform for dynamic interactions of a variety of 

biomolecules on cell surfaces, where glycolipids are involved in physiological and 

pathological molecular recognition events. Growing evidences have demonstrated 

that gangliosides on neuronal cell membranes can be targets for various 

amyloidogenic proteins that are associated with neurodegenerative disorders (e.g. 

Aβ in Alzheimer’s disease and α-synuclein in Parkinson’s disease). To provide a 

structural basis for this pathogenic interaction associated with AD, we conducted 

NMR analyses of the interactions of Aβ with ganglisoides. Our findings suggest 

that (1) the ganglioside clusters offer a unique platform at their 

hydrophobic/hydrophilic interface for binding coupled with α-helix formation of Aβ 

molecules restricting their spatial rearrangements to promote specific 

intermolecular interactions, and (2) the Aβ density on the gangliosidic clusters can 

be a determining factor of an occurrence of the Aβ-Aβ interactions and their 

consequent amyloid formation [1,2,3]. 

Interestingly, it has been reported that assembly of hereditary variant Arctic-type 

(E22G), Dutch-type (E22Q) and Flemish-type (A21G) Aβs is specifically 

accelerated by GM1, GM3 and GD3 ganglioside, respectively, which are 

predominantly expressed at the respective sites of preferential deposition of these 

Aβ variants in the brain. To provide a more detailed understanding of the role of 

gangliosides in the Aβ-species-dependent deposition, we are attempting to 

elucidate the biophysical basis for the assembly mechanisms of hereditary Aβ 

variants promoted on their specific gangliosides. 




84 85 


Financial support from the Naito Foundation is acknowledged. 

REFERENCES 

[1] M. Utsumi, Y. Yamaguchi, H. Sasakawa, N. Yamamoto, K. Yanagisawa, K. Kato, Glycoconjugate J. 

2009, 26, 999-1006. 

[2] M. Yagi-Utsumi, T. Kameda, Y. Yamaguchi, K. Kato, FEBS Lett. 2010, 584, 831-836. 

[3] M. Yagi-Utsumi, K. Matsuo, K. Yanagisawa, K. Gekko, K. Kato, Int. J. Alzheimer’s Dis. 2011, 2010, 

e92507. 

AUTHOR INDEX 

Abedini A. PL-4 

Abskharon R. N. N. P-29 

Altieri F. P-1 

Ami D. P-20 

Andreasen M. OC-10 

Arcari P. P-1 

Arciello A. P-19 

Baldassarre M. P-2 

Barbet-Massin E. OC-1 

Barbiroli A. OC-1 

Barth A. OC-5, P-2 

Beekes M. P-7 

Belgacem O. ET 

Bellotti V. 

OC-1 

Benetti F. P-29 

Bernardini G. P-18 

Bieschke J. OC-14 

Biljan I. P-29 

Biondi B. P-28 

Bitler C. P-25 

Blinov N. OC-13, P-3 

Bobba F. P-8 

Bolognesi M. OC-1, P-20 

Bonanomi M. P-27 

Borg C.B. P-23 

Botyriute A. P-14 

Braconi D. P-18 

Brown J. P-4 

Buell A. P-4, P-24, P-31 

Bulone D. P-32 

Buxbaum J.N. P-5 

Calamai M. P-11 

Calderan A. P-28 

Canale C. P-32 

Cao P. 

PL-4 

Carrotta R. P-32 

Cascella R. P-5, P-11 

Cashman N. OC-13, P-3 

Castangia R. ET 

Cavaliere P. P-22 

Cebey Zas L. OC-2 

Cecchi C. P-5, P-11 

Chino M. P-6 

Chiti F. PL-2, P-5 , P-11 

Choo M. S. F. ET 

Colby D. 

OC-11 

Colombo G. P-27 

Crea R. SN, P-25 

Cucolo A. P-8 

D’Angelo P. P-29 

D’Errico G. P-9 

D’Ursi A. M. P-8, P-12 

Dargužis D. P-14 

Daus M.L. P-7 

de Chiara C. OC-12 

De Simone A. OC-3, OC-4, 

P-10, P-33 

DeGrado W. F. P-6, P-21





Del Giudice R. P-19 

Dell A. 

ET 

Di Marino S. P-12 

Di Natale C. P-15 

Di Pierro P. P-16 

Di Stadio C.S. P-1 

Dobson C. M. 

OC-3, P-4, P-5, 

P-31, P-34 

Doglia S.M. P-20, P-27 

Emendato A. P-9 

Esposito L. P-10, P-33 

Evangelisti E. P-5, P-11 

Falanga A. P-9 

86 87 

Fernández J.-J. 

OC-2 

Foderà V. P-26 

Fusco G. 

OC-3 

D’Alessio G. P-24 

Galdiero S. P-9 

Galvagnion C. P-4, P-31 

Gambassi S. P-18 

Garcia G.A. P-17 

Geminiani M. P-18 

Ghezzi L. P-18 

Giachin G. P-29 

Giancola C. P-22 

Giorgetti S. 

OC-1 

Giosafatto C. V. L. P-16 

Granata V. P-22 

Grandori R. 

Gräslund A. 

OC-1 

PL-3 

Grimaldi M. P-8, P-12 

Halabelian L. OC-1, P-20 

Hemmingsen L. P-23 

Horrocks M. P-13 

Hussain R. P-28 

Iannuzzi C. 

OC-7 

Ilc G. P-29 

Iljina M. P-13 

Irace G. 

OC-7 

Itri F. P-19 

Iuliano A. P-12 

Kanthasamy A. 

Karjalainen E.-L. 

Kaspersen J. D. 

OC-8 

OC-5 

OC-10 

Kato K. P-34 

Kelly G. 

OC-12 

Klenerman D. P-13 

Knowles T.P.J. PL-5, P-17, P-24 

Kovalenko A. OC-13, P-3 

Lasch P. P-7 

Laschi M. P-18 

Legname G. P-29 

Lemmin T. P-6 

Leone M. P-15, P-26 

Lombardi A. P-6 

Lorenzen N. 

OC-10 

Mališauskas R. P-14 

Mannini B. P-5 

Manno M. 

OC-6 

Marasco D. P-15 

March Z. 

OC-11 

Mariniello L. P-16 

Maritato R. 

Marrone A. 

Martorana V. 

OC-7 

OC-9 

OC-6 

Mazzarella L. P-24 

Menon R. P. 

Meredith S. 

OC-12 

PL-7 

Merlino A. P- 24 

Michaels T.C.T. P-17 

Michailova K. P-30 

Middleton C. T. 

PL-4 

Militello V. P-26 

Millucci L. P-18 

Milto K. P-30 

Miselli G. P-1 

Monti D. M. P-19 

Morelli G. P-15 

Morozova O. 

OC-11 

Natalello A. P-20, P-27 

Nick M. C. P-21 

Nielsen S. B. 

OC-10 

Nori S. L. P-8 

Novellino E. P-22 

Ortore M.G. P-32 

Otzen D. E. 

Paciotti R. 

OC-10 

OC-9 

Pagano B. P-22 

Pardon E. P-29 

Paslawski W. 

Pastore A. 

Pedersen J. S. 

OC-10 

OC-12 

OC-10 

Pedersen J. T. P-23 

Penco A. P-27 

Peters P. J. 

OC-2 

Pica A. P- 24 

Piccoli R. P-19 

Picone D. P-9 

Pintacuda G. 

OC-1 

Pizzo E. P-24 

Plavec J. P-29 

Polverino A. P-12 

Pontoniere P. P-25 

Porta R. P-16 

Prigent S. P-22 

Prusiner S.B. 

PL-1 

Punzo V. P-15 

Raccosta S. 

Raleigh D.P. 

OC-6 

PL-4 

Rao E. P-26 

Rappa G.C. P-32 

Regonesi M.E. P-27 

Relini A. P-5, P-27 

Requena J. R. 

OC-2 

Rezaei H. P-22 

Ricagno S. OC-1, P-20 

Riek R. 

PL-6 

Rippa E. P-1 

Ruzza P. P-28 

Salzano G. P-29 

San Biagio P.L. P-32 

Santambrogio C. 

OC-1 

Santucci A. P-18 

Schmidt A. M. 

PL-4


88 

Scognamiglio P. L. P-15 

Scrima M. P-8 P-12 

Sechi G. P. P-28 

Shorter J. 

PL-8 

Sica F. P-24 

Siligardi G. P-28 

Sirangelo I. OC-7, P-16 

Sivasankar S. OC-8 

Smirnovas V. P-14, P-30 

Soror S. H. P-29 

Sorrentino A. P-16 

Sorrentino G. P-12 

Spadaccini R. P-9 

Stefani M. P-11 

Steyaert J. P-29 

Stöhr J. P-21 

Storchi L. 

OC-9 

Sublimi Saponetti M. P-8 

Tata G. 

OC-3 

Teilum K. P-23 

Thomsen K. OC-10 

Tiribilli B. P-5 

Tu L.-H. 

PL-4 

van der Wateren A.I.M. P-31 

Vázquez-Fernández OC-2 

Veglia G. 

OC-3 

Vendruscolo M. OC-3 

Vetri V. P-26 

Vilasi S. P-32 

Visentin C. P-27 

Vitagliano L. P-10, P-15, P-33 

Vos M. 

OC-2 

Vostrikov V. OC-3 

Wang H. 

PL-4 

Wille H. 

OC-2 

Wilson M.R. P-5 

Wohlkonig A. P-29 

Yagi-Utsumi M. P-34 

Yen C.-F. 

OC-8 

Young H. 

OC-2 

Zagari A. P-22

2 University by the Bay 2014: Biophysics of Amyloids and Prions

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