The
photo taken in Snowdonia, Wales
Curriculum Vitae
Born in
STUDY AND POSITIONS
After the high school at “Gimnazija Bežigrad”, I studied chemistry at the
From 1979 till 1982: master
science studies at the Medical Faculty and the interdisciplinary program of Biochemistry
at the Faculty of Chemistry and Technology,
From 1981 till 1986 worked as a young researcher and assistant with
professor Savo Lapanje at Department of Physical Chemistry at the Faculty of
Chemistry and Technology,
From 1986 till 1989 was a postdoc at Department of Molecular Biology and
Genetics, University of Newcastle upon Tyne, U.K., working with professor Roger
H Pain on a project: “The role of »molten globule« as a general intermediate in
protein folding” (Ptitysn OB et al., 1990).
From 1989 till now, am employed at
the Department of Biochemistry and Molecular Biology (at present Department of
Biochemistry, Molecular and Structural Biology), Jožef Stefan Institute, presently appointed
as “higher scientific co-worker”, i.e., senior researcher.
Member of the program “proteolysis and its regulation”,
led by professor Boris Turk.
Lecturer at International Postgraduate
School (IPS) at JSI, co-lecturing the course: Stability,
folding and aggregation of proteins. And another: Mechanism
and biological implications of protein aggregation (NANO3)
RESEARCH
WORK:
From the year 1990 till 2000: various
stability and protein folding studies were performed.
From the year 2001 we work on
amyloid fibril formation of proteins. Interesting and novel was our finding
that stefin B (cystatin
B gene)
makes fibrils easily, under physiologically relevant conditions, whereas stefin
A is more resistant and can only be transformed into fibrils under extreme
conditions (Žerovnik E et al., 2002,
Jenko S et al 2004). As the the mechanism of amyloid fibrillation of
proteins remains unsolved and clearer answers of the molecular details of the
process would help to understand neurodegenerative disease, we have chosen the
two human stefins as model proteins (Žerovnik E, 2002). In our previous
studies on folding we already had prepared chimeras, which led us to observe propensity
to undergo fibril formation and significant differences were found (Kenig et al., 2006).
Our in vitro studies were increased
to include various mutants, among them the two functional mutants observed in
patients with EPM1
(Kenig et al., 2004, Rabzelj et al, 2005). In studies of
the mechanism of amyloid-fibril formation we searched temperature and protein
concentration dependencies of the rates and proposed a model (Škerget et al., 2009, Smajlović et al.,
2009). We also studied the influence of metal ions, pH and solvent to
these processes (Katja Škerget PhD thesis, Žerovnik
et al., 2006, 2007). As for the structure two studies were done:
one resulted in the 3D structure of the tetramer (Jenko Kokalj et al., 2007) and
another in the model for the structure of the fibril (Morgan et al., 2008). We hope, together with collaborators at
home and abroad (see, below) to arrive at a plausible mechanism and to solve
some more oligomers structures important for fibril formation.
Equally important is the finding
that stefin B, in contrast to stefin A, exerts toxicity to cells and binds to
acidic phospholipid membranes (Anderluh
et al., 2005). The mechanism of toxicity of amyloidogenic proteins is
most likely connected to membrane interaction. In the frame of two doctoral
theses (Sabina Rabzelj and Slavko Čeru) we have isolated well defined
oligomers and measured their interaction with membranes and toxicity to cells (Čeru & Žerovnik 2008, Čeru et
al., 2008). We also performed electrophysiological measurements and
showed that stefin B wt makes well defined pores into acidic phospholipids
membranes (Rabzelj et al., 2008).
We have expressed stefin B in mammalian and yeast cells and have found
aggregates of the “aggresome” type (Kopito, 2000). We have characterised those
in collaboration with
To ressume: stefin B protein
(cystatin B gene) served as a very suitable model protein to study amyloid-fibril
formation and amyloid-induced toxicity (it is easy to prepare and has well
defined oligomers). Over-expression of stefin B but also the endogenous protein
led to intracellular protein “aggresome”- like
aggregates.
SOME USEFUL RELATED WEB SITES (LINKS)
The Dobson Group ,
P Lansbury’s group, Amyloid
Pore Section
P Muchowski’s group, ULSA
Hilal A. Lashuel
group, EPFL
MAIN
COLLABORATIONS
Structural Biology Group - Dusan Turk's
lab
Department of Condensed Matter Physics (Igor Muševič,
Miha Škarabot and Andrej Vilfan)
Department of Biology, Biotechnical Faculty,
Department of Biochemistry and Molecular Biology, Toxinology
group (Uroš Petrovič and Igor
Križaj,)
Department of Plant Physiology and Biotechnology, National Institute of Biology (Magda
Tušek-Žnidarič, Maruša
Pompe-Novak, Maja Ravnikar)
Department of Biomolecular NMR, NIC,
In U.K
Department of Biomolecular NMR,
School of Biomedical Sciences,
University of Nottingham,
Nottingham, U.K. (Robert Layfield
and Lynn Bedford)
In
NeuroBioGen Lab. Centro S.Giovanni di Dio-Fatebenefratelli,
Brescia, Italy (Giuliano
Binetti and Luisa Benussi).
In Estonia
Peep Palumaa, Department of Gene Technology, Tallin
University of Technology
SELECTED
PUBLICATIONS from 1999 to 2009
1. Difference in the effects of TFE on
the folding pathways of human stefins A and B.
(1999) Proteins 36, 205-216.
2.
Accessing the global minimum conformation of stefin A dimer by annealing under
partially denaturing conditions. (1999) J. mol. biol. 291, 1079-1089.
3.
The major transition state in folding need not involve the immobilization of
side chains. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5790-5795.
4.
Human stefin B readily forms amyoid
fibrils in vitro. (2002) Biochim. biophys. acta, Prot. struct. mol. enzymol. 1594, 1-5.
5.
Conformational changes preceding amyloid-fibril
formational of amyloid-beta and stefin
B; parallels in pH dependence. (2002) Curr.
med. chem. 9, 1717-1724.
6.
Amyloid-fibril formation :
proposed mechanisms and relevance to conformational disease. (2002) Eur. j.
biochem. 269, 3362-3371.
7.
Different propensity to form amyloid-fibrils by two
homologous proteins - human stefins A and B : searching for an explanation. (2004) Proteins 55,
417-425.
8.
Interaction of human stefin B in the prefibrillar oligomeric form with
membranes : correlation with cellular toxicity. (2005) Eur. j. biochem. 272, 3042-3051.
9.
In vitro study of stability and amyloid-fibril
formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1. (2005) Protein sci., 14,
2713-2722.
10. Folding and amyloid-fibril formation for a series of human stefins' chimeras: any correlation? (2006) Proteins; Structure, Function and Bioinformatics 62, 918-927.
Some recent publications (more can be found on Pubmed under Zerovnik E)
Essential role of proline isomerization in stefin B tetramer formation. J Mol Biol. (2007) 366, 1569-1579. Epub 2006 Dec 16.
The mechanism of amyloid-fibril formation by stefin B: temperature and protein concentration dependence of the rates. Proteins (2009) 74, 425-436.
Essential role of Pro 74 in stefin B amyloid-fibril formation: dual action of cyclophilin A on the process. FEBS Lett. (2009) 583, 1114-1120. Epub 2009 Mar 3.
The emerging role of cystatins in Alzheimer's disease. Bioessays (2009) 31, 597-599.
The research group over the last 3 years have done some work in close connection to molecular bases of neurodegeneration and molecular pathology of myoclonus epilepsy of type 1 (EPM1). In their studies they are using stefin B as a good molecular model of protein aggregation, in vitro and ex vivo - in cell culture. The department also possesses KO mice of stefin B (cystatin B) gene, which represent a relatively good model of EPM1.
A. molecular bases of neurodegeneration: in connection to Alzheimer’s disease and other neurodegenerative pathologies the following papers appeared:
1. review: Zerovnik E. Protein conformational pathology in Alzheimer's and other neurodegenerative diseases; new targets for therapy. Curr Alzheimer Res. 2010 Feb;7(1):74-83.
Abstract – as shortenned
from Pubmed: The whole set of so-called “conformational”
disorders, among them systemic amyloidoses, various
dementias and other neurodegenerative diseases such as Parkinson's, Alzheimer's
and amyotropic lateral sclerosis, may have similar
molecular backgrounds: changes in protein conformation and aggregation lead to
toxic amyloid oligomers and
fibrils. The so called aggresomes in eukaryotes
(equivalent to inclusion bodies in prokaryotes), located at the centriole by the nucleus and composed of aggregated
proteins, are believed to sequester the toxic material. They eventually get
cleared from the cell by autophagy. The advances in molecular and cellular
studies will hopefully lead to novel therapies and eventually to a cure.
2. critical
literature review Zerovnik
E. The
emerging role of cystatins in Alzheimer's disease.
Bioessays. 2009 Jun;31(6):597-9.
Recently opposing effects of cysteine protease inhibitors, the human cystatins, on neurodegeneration have been reported. Human cystatin C is a risk factor for late-onset Alzheimer's disease (AD), whereas human stefin B (cystatin B) has no direct involvement in AD. Conflicting data show that their target protease, cathepsin B, might be anti-amyloidogenic, helping in amyloid-beta (Abeta) clearance or, instead, might be involved in Abeta production. Some reports claim that cystatin C binds soluble Abeta, making transgenic animals healthier, others, in contrast, that deleting cystatins genes may contribute to- or in some cases ameliorate amyloid pathology in animal models of AD.
3. Skerget K, Taler-Vercic A, et al. Zerovnik E. Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells. J Biol Chem. 2010 Jan 29;285(5):3201-10. Epub 2009 Dec 2.
Abstract – as shortened from Pubmed: To contribute to the question of the putative role
of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction
between human stefin B (cystatin
B) and amyloid-beta-(1-40) peptide (Abeta). The dimers and tetramers
of stefin B, which bind Abeta,
are domain-swapped as judged from structural studies. Consistent with the binding
results, the same oligomers of stefin
B inhibit Abeta fibril formation. When expressed in
cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is
another APP/Abeta-binding protein in vitro and likely
in cells.
4. Taler-Verčič A, Zerovnik
E. Bioessays. 2010 Dec;32(12):1020-4.
doi: 10.1002/bies.201000079.
Epub 2010 Oct 22. Binding of amyloid peptides to domain-swapped dimers of other amyloid-forming
proteins may prevent their neurotoxicity.
At first, based on their previous study (Škerget et al., 2010, J.Biol.Chem.) the authors suggest that binding of amyloid peptides to domain-swapped dimers of other amyloid-forming proteins may be a general feature. In such a way these oligomers would serve an “amateur” chaperone (Wilhelmus, 2007) [1] function and prevent amyloid neurotoxicity. The authors then show that generalization of findings from stefin B to other cystatins, stefin A and cystatin C, are not straightforward. Amyloid-beta interacts with cystatin C dimers but not so with stefin A dimers. Pressumably the dimers prepared are of domain-swapped type. This shows that binding of amyloid-beta to stefin B oligomers (Škerget et al., 2010, J.Biol.Chem) and cystatin C (Sastre, 2004) [2] is more specific, dependent also on protein sequence and amino-acid chemical properties, not only on the structural features of binding.
B. molecular pathology of myoclonus epilepsy of type 1 (EPM1): protein aggregation as an additional cause of pathology in EPM1
Zerovnik and co-workers already in 2005 came with a hypothesis that protein aggregation may have a role in pathology of a subset of EPM1 patients [3]. They have studied in vitro properties, membrane interaction and toxicity of some of the exonic EPM1 mutants [4-7]. Recently, they confirmed and extended the observation that stefin B forms oligomers and aggregates in the cell [8-9].
1. Ceru
S, Layfield
R, Zavasnik-Bergant T, Repnik U, Kopitar-Jerala
N, Turk
V, Zerovnik
E. Intracellular aggregation of human stefin B: confocal and electron
microscopy study. Biol Cell. 2010 Mar 17;102(6):319-34.
Pubmed abstract: BACKGROUND:
Protein aggregation is a major contributor to
the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the
CSTB (cystatin B) gene [StB
(stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with
features of neurodegeneration and increased oxidative
stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has
also been observed that StB is overexpressed
after seizures and in certain neurodegenerative conditions, which could
potentially lead to its aggregation. Human StB proved
to be a good model system to study amyloid fibril
formation in vitro and, as we show here, to study protein aggregation in cells.
RESULTS: Endogenous
human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin-proteasome system) led to an increase in the
amount of the endogenous protein and also increased its aggregation. Further,
we characterized both the untagged and T-Sapphire-tagged StB
on overexpression in mammalian cells. Compared with
wild-type StB, the EPM1 missense
mutant (G4R), the aggregate-prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear
aggregates accompanied by cytoskeletal
reorganization. Non-homogeneous morphology of these large aggregates was
revealed using TEM (transmission electron microscopy) with StB
detected by immunogold labelling.
StB-positive cytoplasmic
aggregates were partially co-localized with ubiquitin,
proteasome subunits S20 and S26 and components of
microfilament and microtubular cytoskeleton using confocal microscopy. StB
aggregates also co-localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed
that protein aggregation was associated with reduced cell viability.
CONCLUSIONS: We
have shown that endogenous StB aggregates within
cells, and that aggregation is increased upon protein overexpression
or proteasome inhibition. From confocal
and TEM analyses, we conclude that aggregates of StB
show some of the molecular characteristics of aggresomes
and may be eliminated from the cell by autophagy.
Intracellular StB aggregation shows a negative
correlation with cell survival.
2. http://www.cell.com/trends/molecular-medicine/newarticles
In the paper entitled: Impaired autophagy:
a link between neurodegenerative diseases and progressive myoclonus
epilepsies, Trends in Molecular Medicine - in press, Polajnar M. & Žerovnik
E. from Dept. Biochemistry Molecular & Struct.
Biology, J.Stefan Institute,
Grants supporting the work: Currently, E.Ž. leads a research project J7-4050: Oligomers of amyloidogenic proteins from a to z: biophysical properties, structure, function and mutual interactions. Her work is partially supported by the program P1-0140 (proteolysis and its regulation - led by B. Turk; till 2009 by V. Turk) and the project J3-2258 (V. Stoka), both financed by the Slovenian Research Agency (ARRS).
REFERENCES:
1. Wilhelmus,
M. M., de Waal, R. M. & Verbeek, M. M. (2007) Heat shock proteins and
amateur chaperones in amyloid-Beta accumulation and clearance in Alzheimer's
disease, Mol Neurobiol. 35, 203-16.
2. Sastre, M.,
Calero, M., Pawlik, M., Mathews, P. M., Kumar, A., Danilov, V., Schmidt, S. D.,
Nixon, R. A., Frangione, B. & Levy, E. (2004) Binding of cystatin C to
Alzheimer's amyloid beta inhibits in vitro amyloid fibril formation, Neurobiol Aging. 25, 1033-43.
3. Ceru, S.,
Rabzelj, S., Kopitar-Jerala, N., Turk, V. & Zerovnik, E. (2005) Protein
aggregation as a possible cause for pathology in a subset of familial
Unverricht-Lundborg disease, Med
Hypotheses. 64, 955-9.
4. Anderluh,
G., Gutierrez-Aguirre, I., Rabzelj, S., Ceru, S., Kopitar-Jerala, N., Macek,
P., Turk, V. & Zerovnik, E. (2005) Interaction of human stefin B in the
prefibrillar oligomeric form with membranes. Correlation with cellular
toxicity, Febs J. 272, 3042-51.
5. Rabzelj, S.,
Turk, V. & Zerovnik, E. (2005) In vitro study of stability and
amyloid-fibril formation of two mutants of human stefin B (cystatin B)
occurring in patients with EPM1, Protein
Sci. 14, 2713-22.
6. Rabzelj, S.,
Viero, G., Gutierrez-Aguirre, I., Turk, V., Dalla Serra, M., Anderluh, G. &
Zerovnik, E. (2008) Interaction with model membranes and pore formation by
human stefin B: studying the native and prefibrillar states, Febs J. 275, 2455-66.
7. Ceru, S.,
Kokalj, S. J., Rabzelj, S., Skarabot, M., Gutierrez-Aguirre, I.,
Kopitar-Jerala, N., Anderluh, G., Turk, D., Turk, V. & Zerovnik, E. (2008)
Size and morphology of toxic oligomers of amyloidogenic proteins: a case study
of human stefin B, Amyloid. 15, 147-59.
8. Ceru, S.,
Layfield, R., Zavasnik-Bergant, T., Repnik, U., Kopitar-Jerala, N., Turk, V.
& Zerovnik, E. (2010) Intracellular aggregation of human stefin B; confocal
and electron microscopy study, Biol Cell.
9. Cipollini,
E., Riccio, M., Di Giaimo, R., Dal Piaz, F., Pulice, G., Catania, S.,
Caldarelli, I., Dembic, M., Santi, S. & Melli, M. (2008) Cystatin B and its
EPM1 mutants are polymeric and aggregate prone in vivo, Biochim Biophys Acta. 1783,
312-22.