Alzheimer's Amyloid - Helsinki.fi

CONFORMATION -> FOLDINGProtein misfoldingInsightProtein misfoldingADAM SMITHProtein folding and misfoldingCHRISTOPHER M. DOBSONQuality control in the endoplasmic reticulum proteinfactory 891ROBERTO SITIA AND INEKE BRAAKMANVol 426 No 6968 pp739-91118/25 December 2003Protein degradation and protection against misfolded ordamaged proteins 895ALFRED L. GOLDBERGFolding proteins in fatal waysDENNIS J. SELKOETherapeutic approaches to protein-misfoldingdiseases 905FRED E. COHEN AND JEFFERY W. KELLY

Protein non-folding, what’s going on?A)ConformationalchangeFolded functionalnative proteinNon-foldedNon-functional proteinB)ConformationalchangeNon-foldedNon-functional protein(Ubiquitin-Proteasome) degradation Pathway

Protein folding occur in the ERSchematic of typical animal cell, showing subcellularcomponents.Organelles: (1) nucleolus (2) nucleus (3) ribosome (4)vesicle, (5) rough endoplasmic reticulum (ER), (6) Golgiapparatus, (7) Cytoskeleton, (8) smooth ER, (9)mitochondria, (10) vacuole, (11) cytoplasm, (12) lysosome,(13) centrioles

Ubiquitin-Proteasome degradation PathwayThe Nobel Prize in Chemistry 2004"for the discovery of ubiquitin-mediatedprotein degradation"CiechanoverRoseHershkoIn THE CELL AT THE ER...We’reProteins,I feel it!I think that Ihave to get abetter shape!RULES AND REGULATIONS CONTROLTHE PROTEIN NUMBERS AND FOLDINGWell all I wantTo know is,am ICORRECT?E3 makesThe decisionsandfixes thelabelsOhNoooMarked proteins are hacked tobits in the mincerAre you forthe mincertoo?No,Not me!Ups, I do notknow my nativestructureWith a label round your neck,there is no going back...Order can berestored – forthe time being!

Protein misfolding, what can it do?Foldedfunctional proteinConformationalchangeAbnormal proteinNon-foldedNon-functional proteinCan cause a loss of thephysiological functionCan cause aggregationand depositionCan become toxic

Examples of protein misfolding disordersDiseaseAlzheimer's diseaseCJD, FFI , Kuru, BSEProtein involvedAmyloid-ß proteinPrion proteinParkinson disease -synuclein, parkin (?)Huntington diseaseDiabetes type IIAmyotrophic lateral sclerosisSerpin deficiency, emphysema, cirrhosisHaemodialysis amyloidosis, prostatic amyloidCystic fibrosisCADASIL diseaseHuntingtinAmylinSuperoxide dismutaseSerpinsß2-microglobulinCFTR proteinNotch3 receptor proteinEtc, etc, etc...

Some protein misfolding without a disorderProtein involvedE.ColiCurlin proteinP. Falciparum Malarial coat proteinSpiderMammalian melanosomesMammalian brainSpider silkpMELApolipoprotein EEtc, etc, etc...

What is the mechanism of proteinmisfolding and aggregation?Lets take an example:THE AMYLOIDWhy does Amyloid form?

Protein Aggregates in Conformational DisordersAlzheimer’samyloid plaquesPrion plaquesParkinson’sLewy bodiesXAggregatesAmyloid fibers

Ultrastructure of AmyloidFoldedfunctional proteinAmyloid

Ultrastructure of aggregatesVery often these aggregates resembleAmyloid fibers although they do notfulfil the criteria to be Amyloid

The Alzheimer’s Amyloid Precursor Protein

The critical amino acids40 – 46 (47)amino acids

Physico-chemical properties of the critical amino acids:Alzheimer’s AmyloidHighAverageLow

Random database search...Amphoterin (HMG-1)….MSSYAFFVQT….Alzheimer’s Amyloid….HQKLVFFAED….HMG-1, Aggrecan, Misshapen kinase isoform MINK, MMP-2, TRP3 cation channel…

Physico-chemical properties of the critical amino acids:AmphoterinHighAverageLow

Amphoterin (HMG-1)EPEPGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEDDEEDEEDEEEEEEEEDEDEEEDDDDE

The fragment of Amphoterin (HMG-1) makes amyloid –like fibrils

Physico-chemical properties of the critical amino acids:Of the known Amyloid Core Sequences1.51.4Beta-sheet1.3Beta-sheet1.2Alpha-helix1.10.9Alpha-helix1Beta-turnAmphoterin fragment0.70.5Gelsolin fragmentBeta-turn0.80.6M S S Y A F F V Q T CN G N C F I L D1.41.6Beta-sheet1.2Beta-sheet1.41.2Alpha-helix1Alpha-helix1Islet amyloid fragmentBeta-turn0.80.6Alzheimer’s amyloid fragmentBeta-turn0.80.60.4S N N F G A IL S SH Q K L V F F A EKallijärvi et al (2001) Biochemistry 40: 10032-10037

Also other non-disease related proteins make amyloid –like fibrilsGammaD crystallin, SH domain phosphoglycerate kinase (PGK), SH3domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase(PI3-SH3)..............AND share the neurotoxic properties of amyloidsAll those which we have so far tested sharea common ”amyloid core sequence”which is responsible for their misfolding.

Why do misfolded proteins kill??The “stick to all” theory

STDApolipoprotein E4 isA risk factor for ADA-betaNACA-betaACApoE

apoE4(34kDa)apoE4(34kDa)1,91,9intenzity1,4ApoE4 +Ab(38kDa)intensity1,40,930000 35000 40000 m/z0,930000 35000 40000 m/zintensity2076With ApoE51543210103800 5800 7800 9800505500 10500 15500 20500 25500m/zMS analysisof the effectof ApoE onAlzheimer’s Amyloidpolymerization20715Ab dimerWithout ApoE6543intensity105Ab trimerAb tetramer2103800 5800 7800 980005500 10500 15500 20500 25500m/z

Physico-chemical properties of some of the ApoE4Sequence epitopesApolipoprotein E4HighAverageLow

Sequences in ApoE predicted to be able to form amyloid-like structuresnum. sequence from-to1. WKYRRPVTT 21-332. KKVFFSTQ 36-483. ALTPGVVL 61-784. VQLGRDTSV 89-1115. AASVFTRKLPYT 121-1486. EAWWTRVFLRE 183-221intensity9876542633020100Analysis of the parts onApoE which are protectedby Alzheimer’s amyloidpeptide (limited enzymaticcleavage assay)4000 6000 8000321000 6000 11000 16000 21000 26000 31000 m/zintensity987654330201004000 6000 80002m/z1000 6000 11000 16000 21000 26000 31000

ApoE sequences protected by Alzheimer’s amyloid fragment1 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNHintensity141210864AbC-termN-termN-termN-term N-termProtected areas highlightedin red20m/z4000 5000 6000 7000 8000 9000 100001412Predicted sequencesable to form amyloid-likestructures are inside of themeasured areasintensity1086420m/z4000 5000 6000 7000 8000 9000 10000

ApoE sequences protected by Alzheimer’s amyloid fragmentintensity1412MS1086420m/z4000 5000 6000 7000 8000 9000 10000Inhibition of the binding ofAlzheimer’s amyloidfragment to ApoE by asynthetic beta-sheetbreaker peptide madetowards ApoEintensity141210864MS(GRFEQWARAVQ)20m/z4000 5000 6000 7000 8000 9000 100001 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

The answer to the question...1 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNHApoE interacts with Alzheimer’s amyloid fragment by its ownamyloidogenic sequence motifs! What about all the others. If theyalso posess their own amyloidogenic motifs, why would they notuse the same way to act??

Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.Wisniewski, T. et alA carboxyl-terminal fragment (residues 216-299)of apolipoprotein E is present in Alzheimer'sdisease lesionsIn vitro this fragment from recombinant apolipoprotein Ecould form amyloid-like fibrils, which were Congo-redpositiveThus senile plaques contain at least both amyloid beta andapolipoprotein E amyloid fibrils.

Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.Wisniewski, T. et alA carboxyl-terminal fragment (residues 216-299) ofapolipoprotein E is present in Alzheimer's disease lesionsIn vitro this fragment from recombinantapolipoprotein E could form amyloid-like fibrils,which were Congo-red positiveThus senile plaques may contain both amyloid beta andapolipoprotein E amyloid fibrils.

Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.Wisniewski, T. et alA carboxyl-terminal fragment (residues 216-299) ofapolipoprotein E is present in Alzheimer's disease lesionsIn vitro this fragment from recombinant apolipoprotein Ecould form amyloid-like fibrils, which were Congo-redpositiveThus senile plaques contain at least both amyloidbeta and apolipoprotein E amyloid fibrils

MISFOLDING DISORDERS – MIGHT THEY BE PROTEINCONFORMATIONAL DISORDERSWHERE MORE THAN ONE PROTEIN PARTICIPATE IN THECASCADE OF MISFOLDING EVENTS?

MYSTERIOUS FACTOR-X IN PRION DISORDERS?IS IT JUST ANOTHER MISFOLDING PROTEIN?AENMR structures of three single-residue variants of the human prion proteinCalzolai et al. PNAS | July 18, 2000 | vol. 97 | no. 15 | 8340-8345

CADASILa Notch3 mutation causingmisfolding

CADASIL• Name given and linkage established: CADASIL fromcerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathy. Linkedto chromosome 19 (q12) by Tournier-Lasserve et al.Nature Genetics 1993; 3:256-59• Gene defect and defective protein identified: Notch3 at19p13, by Joutel et al. Nature 383:707-710, 1996

MRI FLAIR: lacunar infarcts** Frontal horn of left lateral ventricle

CADASIL: A vascular dementia the diagnosis of which can bemade on a skin biopsyVSMCLumen* EEVSMC>VSMCVSMC*E**VSMC>Electron micrograph of a small dermal artery: widened sub-endothelial space(*), irregularity of vascular smooth muscle cells (VSMCs) and granularosmiophilic material (GOM >). E = endothelium

0.4 m1 mElectron microscopy of a dermal artery: deposition of granular osmio-philicmaterial (GOM *) in indentations (notches) of degenerating smooth musclecells and between these cells. (N = nucleus)

Skin biopsy: N3ECDimmunostaining***nerve*confocalPathognomonic finding is deposition of Notch3 extracellulardomain (N3ECD) in the walls of arteries (*)

Notch3 extracellular domain (N3ECD) is a maincomponent of GOMIshiko et al: Acta Neuropathol 2006; 112: 333-9(immunoelectronmicroscopy)

CADASIL WM: arterioles(N3ECD)CADASIL WM: capillary(N3ECD)CADASIL cortex: small arteriolesand a capillary (N3ECD)CADASIL cortex: capillary(N3ECD)In CADASIL Notch3 extracellular domain(N3ECD/GOM) accumulates not only onWM arterioles but also on WM capillaries(pericytes) as well as on these vessels incerebral cortex, although corticalarterioles are not equally thickened.CADASIL subarachnoidal space:arteries (N3ECD)

Pathogenesis• Haploinsufficiency/hypomorphic effect unlikely• Gain or loss of a cysteine molecule affects formation ofsulphur bridges and causes conformational change inthe Notch3 molecule (protein misfolding andaggregation). Thus, gain of function of the mutatedprotein most likely.• Most likely mechanism gain of function:– Dominant negative effect (reduced function of thewild-type allele): does not appear to occur– Hypermorphic effect (increased function of themutated allele): does not appear to occur– Neomorphic effect (mutant protein has newadditional (toxic?) functions(Opherk et al. CADASIL mutations enhance spontaneous multimerizationof NOTCH3. Hum Mol Genet. 2009;18:2761-7)

Signal sending cellDelta/JaggedligandA. Mutation ( ) in theligand binding area ( ) no ligand bindingor signalingB. Mutation outsidethe ligand binding area ligand binds, S3cleavage and signalingoccur, but…ABC. Mutated misfolded Notch3 does notundergo S2 cleavage and sop up Delta/Jagged ligands, block other ligandsor dimerize with other receptorsCAccumulationD. Even after S2 andS3 cleavages misfoldingpreventsinternalization of thecomplex (with orwithout the ligand)DAccumulationAccumulationNo internalizationsignalNo internalizationsignalS2S3S2S3NICD to nucleus signallingS3Vascular smooth muscle cellNotch3

Mol Med 2007; 13: 305-314

Mol Med 2007; 13: 305-31411 differentially expressed proteins discovered• Proteins related to protein degradation and folding andfree radical scavenging– Proteasome components, HSP27 and free radical scavengingenzymes: Expected consequence due to the unpaired cysteinerelated misfolding -> unfolderd protein response -> ER stress ;depletion of glutathione and production of reactive oxygen species(ROS)• Proteins related to vascular smooth muscle cell (VSMC)contraction– accentuated angiotensin II response

SMAWe have found in 2D-gel electrophoresis of genuine human vascular smooth muscle cellsfrom a patient with CADASIL and controls, that the expression of the following proteinsinvolved in actin metabolism were different: Rho protein dissociation inhibitor (RhoGDI):upregulated; Profilin: upregulated; HSP27 upregulated; Cysteine and glysine rich protein(CRP): upregulated. (Ihalainen et al. Molec Med 2007; 13:305-14)Hence the actin organization was analysed in VSMCs from different vascular beds in CADASILpatients (pre- and post-mortem). The actin network was altered suggesting that Notch3 isinvolved in the regulation of actin, the major the contractile protein in VSMCs. (Tikka et al.submitted)

In ConclusionMisfolded proteins are quite naturally occuringrisk factors for the life…which the nature can just sometimesnot deal with.They form spontaneously by mutationswhich are only controlled by the evolution.BAD LUCK…or ???

The Yeast story...Yeast uses a prion-like protein for to control its lifein various environmental conditions.It keeps this protein in an amyloid-like form if it is notneeded…When needed, it can produce the same protein in asoluble form.

Protein misfoldingHelsinki-BiomedicumMarc BaumannGibril Fadika (Gelsolin AA)Candace Laverette (SH3)Christina Pompey (Cystatin)Vonda Meeks (Cystatin, SH3)Can Hekim (Cystatin)Jukka Kallijärvi (Alzheimers AA, Amphoterin)Riikka Nissinen (Alzheimers AA, Melatonin)Petra Gromova (Alzheimers AA and ApoE)Bronislaw Clod (Polish Acad. of Sci.)(Alzheimers AA, Melatonin)Maciej Lalowski (Polish Acad. of Sci.)(Alzheimers AA)Katri Niemi (Secondary AA)Kari Eklund (Secondary AA)Saara Tikka (CADASIL)Eeva Kauppi (Technical Assistance)Rabah Soliymani (Technical Assistance)

Protein misfoldingHelsinki-HaartmanInstituteMatti HaltiaHannu KalimoUniv of HelsinkiCentral HospitalSari Kiuru-EnariNYU Medical CenterNew York, U.S.A.Blas FrangioneThomas WisniewskiJorge GhisoAsok KumarFrances PrelliMaciej LalowskiTexas Univ. MedicalBranch, Galveston, USAClaudio SotoStatens Serum InstitutCopenhagen, DenmarkNiels H.H. HeegaardThe Scripps Research InstituteLa Jolla, USAJeffery KellyLaura Morelli (Univ. Buenos Aires)

Nordic CADASIL research groupClinical studies– Matti Viitanen U of T, Karol Inst– Susanna Roine U of T– Auli Verkkoniemi U of HPathology– Hannu Kalimo U of H– Qing Miao* U of TGenetics– Minna Pöyhönen U of H– Kati Mykkänen* U of T– Maija Junna * U of TProteomics and molecular biology– Marc Baumann U of H– Saara Tikka (nee Ihalainen)* U of H– Yan Peng Ng U of H– Urban Lendahl and team Karol Inst* PhD or DMedSci studentImaging and PET studies– Juha Rinne U of T– Susanna Roine U of TOphthalmology– Tero Kivelä U of H– Paula Summanen U of H– Mika Harju U of HNeuropsychology– Kaarina Amberla* Karol InstVasoregulation– Anna Stenborg U of Uppsala– Andreas Terent U of Uppsala– Robert Bergholm U of HU of H = Univ of Helsinki, FinlandU of T = Univ of Turku, FinlandKarol Inst = Karolinska Institutet, Sweden

Amyloids are always found i twodistinct conformations

AmyloidogenicproteinsPathogenicformNon-pathogenicform

AmyloidogenicconformationNon-amyloidogenicconformation

Sequences of Alzheimer’s -sheet breaker peptides1 695N A C APPA1-42DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGVVIA1 42-sheet breaker peptidesiA11iA9iA7iA6iA5iA4iA3RDLPFFPVPIDRDLPFFPVDLPFFPVDLPFFVDLPFFDLPFFPFFSoto and Baumann (1996) Biochem. Biophys. Res.Commun. 226: 672-680

Activity of a -sheet breaker peptideIn vitro activityActivity in cellsControlTreatmentControlTreatmentActivity in rat modelControlTreatment