download report - Istituto Pasteur

P a r t i c i p a n t s :
Anna Ferraro, Fabio Altieri, Margherita Eufemi, professors;
Silvia Chichiarelli, researcher; Caterina Grillo, post-doc fellow;
Manola Maceroni, Elisa Gaucci, Valentina Arcangeli,
PhD students.
Report of activity
ERp57 is a member of the protein disulfide isomerase
family of proteins, whose roles in the endoplasmic
reticulum as chaperon and disulfide-reshuffling protein
are well understood. ERp57 is a stress-responsive
protein, overexpressed in transformed cells. Its
presence in other subcellular locations, i.e. the cell
surface, the cytosol and the nucleus, has been extensively
documented, and also its ability to interact with
nuclear proteins and with DNA. However, its functions
in these extra-ER locations are still obscure.
Data from different laboratories suggest that ERp57
participate in the regulation of transcription factors
activity and in DNA repair. This suggestion is
strengthened by some findings from our laboratory,
such as the interaction of ERp57 with specific DNA
sequences and its association with APE/Ref-1, which
is a DNA-repair endonuclease and an activator of
transcription factors, and with the nuclear STAT3,
which is a transcription factor involved in inflammation,
cell proliferation, cell survival and oncogenesis.
The aim of the proposed research is to explore the
functions of ERp57 in its non-ER locations and to
verify its role when bound to specific DNA sites,
which we have begun to identify. If the binding of
ERp57 to DNA, which is redox-dependent, has a
truly regulative role, then the protein would appear to
be an oxidative stress-responsive factor capable of
acting directly in the nucleus by regulating the
expression of specific genes.
We previously described the DNA-binding properties
of ERp57 and identified its C-terminal region as
being directly involved in the DNA-binding activity.
71
Molecular genetics of eukaryotes - AREA 3
Roles of ERp57 in DNA repair and in the regulation of gene
expression
Principal investigator: Carlo Turano
Professor of Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910598; Fax: (+39) 06 4440062
carlo.turano@uniroma1.it
In this research, we investigated in some detail the
structural features of ERp57 responsible for this
interaction with DNA. Since the fourth domain of the
protein (called a’) is the one directly involved in DNA
binding, we produced a recombinant isolated a’
domain and demonstrated that its DNA-binding
properties are strongly dependent on its redox state.
Site-directed mutagenesis on the first cysteine
residue of the -CGHC-thioredoxin-like active site
leads to a mutant domain (C406S) lacking DNA-binding
activity. Biochemical studies on the recombinant
domain revealed a conformational change associated
with the redox-dependent formation of a homodimer,
having two disulfide bridges between the cysteine
residues of two a’ domain active sites. The formation
of intermolecular disulfide bridges rather than
intramolecular oxidation of active site cysteines is
important to generate species with DNA-binding
properties. Thus, in the absence of any dedicated
motif within the protein sequence, this structural
rearrangement might be responsible for the DNAbinding
properties of the C-terminal domain.
Moreover, NADH-dependent thioredoxin reductase
is active on intermolecular disulfides of the a’
domain, allowing the control of dimeric protein content
as well as its DNA-binding activity. A similar
behavior was also observed for the entire ERp57.
In order to explore the functions of ERp57, we investigated
the binding of various ligands to the protein
and their effects on the interaction with DNA and on
the enzymatic reductase activity. We found that some
antibiotics, which had already been tested on PDI,
bound to ERp57 with an inhibitory action on its
activities and with an affinity constant two to three
orders of magnitude higher than that displayed
towards PDI. Therefore these inhibitors provide a
mean to differentiate the biological activities of these
two protein-disulfide-isomerases, which share homologous
structures and similar sub-cellular locations.
The DNA-ERp57 interaction was then studied in
vivo by means of chromatin immunoprecipitation