Projects / Programmes
Oligomers of amyloidogenic proteins from a to z: biophysical properties, structure, function and mutual interactions
| Code |
Science |
Field |
Subfield |
| 7.00.00 |
Interdisciplinary research |
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| Code |
Science |
Field |
| B000 |
Biomedical sciences |
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| Code |
Science |
Field |
| 1.07 |
Natural Sciences |
Other natural sciences |
three-dimensional structure, domain-swapping, amyloid fibril, amyloid toxicity, neurodegeneration, stefins, cystatins, alpha-synuclein, protein folding, interaction with membranes, chaperone effect
Researchers (13)
Organisations (3)
Abstract
Oligomers of amyloid forming proteins will be the connecting theme of this project, which will encompass cross-disciplinary aspects stemming from higher oligomers structure, their role in the kinetics and thermodynamics of amyloid fibril formation, novel physico-chemical properties of the oligomers, molecular interactions of the oligomers with membranes and among themselves (protein: peptide interactions) and cellular phenomena which occur as a consequense of protein oligomerization and aggregation, such as autophagy and oxidative stress. In more detail, we will try to determine stefin B higher oligomer (higher than the tetramer) 3D structure, by using crystallography and supplemented by heteronuclear NMR. Further we will determine stability of the oligomers, dimers, tetramers and higher in comparison to the monomer, using spectroscopic and calorimeric (DSC) techniques. Role of the oligomers in the kinetics of fibril formation and role of proline residues to oligomerization and amyloid formation will be determined by the usual kinetic approaches and mutant studies. Oligomers conformational changes at membranes and permeability studies will be made. Protein protein interaction between the oligomers of stefin B (or ß2-microglobulin) and other amyloid peptides, such as a-synuclein (or amylin) will be measured by using surface plasmon resonance (SPR). We will study oligomers of a globular protein human stefin B– as a good model (protocols exist how to isolate them) but not exclusively. As comparison we will study a-synuclein from the class or natively unfolded proteins, which build Lewy bodies in Parkinson's disease and ataxin – which is representative of the polyglutamine repeats disorders and possibly another member of globular proteins - ß2-microglobulin, which is found deposited in joists and bones of patients undergoing long-term hemodialysis. The mechanistic and structural studies of the prefibrillar oligomers in vitro will be supplemented by cell culture studies, with an aim to reveal cellular processes accompanying protein aggregation. Cystatins, including stefin B, are involved in important physiological processes and their gene alleles may represent a risk for Alzheimer’s disease (AD). Cystatin C allele-B undergoes impaired secretion and represents a risk factor for AD. As recent data show, cystatin C also has a role in autophagy, which is the primary means of clearance protein aggregates from the cell. Stefin B, whose mutations cause progressive epilepsy of type 1 (EPM1), on one side prevents oxidative stress and cell death (apoptosis), which are increased in stefin B KO mice. However, stefin B on the other side reduces autophagy, which means that authophagy proceeds to completion in stefin B KO cells, which helped to clear amyloid pathology in a mouse AD model. Stefin B also is involved, by an unknown mechanism, in innate immunity. Many of these effects are cathepsin independent and cystatins alternative functions to protease inhibition are suggested. Studying cellular and membrane phenomena caused by oligomerization and amyloid fibril formation of stefin B and its EPM1 mutants as well as a-synuclein and its mutants involved in PD, is of direct medical relevance; contributes to Molecular Medicine (paper in press by Polajnar & Žerovnik in Trends Molecular Medicine).
The project will connect different disciplines and is thus interdisciplinary: it spans the fields from structure, molecular and cellular properties to eventual function and pathophysiology of the prefibrillar oligomers. The project entitled: ‘Oligomers of amyloidogenic proteins from a to z: biophysical properties, structure, function and mutual interactions’ falls under 5th priority of national (and EU) research program: health and science about life, interdisciplinary studies in the field of natural, biotechnical and medical sciences.
Significance for science
Our studies are of basic nature, however, they are oriented to gain understanding of molecular backgrounds of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), etc. Studies of EPM1 mutants of stefin B contribute to understanding at least some part of pathology of this progressive myoclonus epilepsy, which also can be regarded as neurodegenerative process. More knowledge of the properties of oligomers will help in search for therapies for this kind of diseases. Oligomers of amyloid forming proteins proved cytotoxic and are main players in the pathological cascade in AD and other neurodegenerative conditions. Interaction with membranes (even making pores into membranes) is likely the key mechanism of their cytotoxic action. As the structure of the oligomers is not known, as well conformation at membranes is not clear, these remain open and hot questions. Original is our goal to study as yet unknown, novel biophysical properties of the prefibrillar oligomers (and amyloid fibrils), such as optical and electrical properties. One could wander, why using stefins as a primary model? One good reason is that 3D structures of stefins A and B have been determined and lately the structure of its tetramer (1) as well as the model for the fibrils (2), which renders them especially suitable for such studies. Stefins have been studied thoroughly by our group from stability, folding to amyloid fibril formation, which in contrast to stefin A or cystatin C occurs under mild, physiologically relevant conditions. Sequential influences (by studying chimeras) and role of the conserved prolines have been studied, resulting in many general findings (as reviewed Biochimie) (3). 1. New knowledge with potential application to translational medicine. Model studies which explain basic molecular mechanisms taking place in neurodegenerative conditions (9,10). The mechanism of protein aggregation to amyloid fibrils and toxicity exerted by the soluble oligomers and prefibrillar aggregates, are two most challenging questions of the today’s neuroscience. It is believed that cellular inclusions - aggresomes or extracellular plaques are less toxic than the soluble oligomers. 2. Other important contributions C.06: Editorial board of Amyloid. Žerovnik, Eva (member of editorial board 2005-). Carnforth: Parthenon. ISSN 1350-6129. [COBISS.SI-ID 512143641] Amyloid, the international journal of experimental and clinical investigation. Official journal of the “International Society of Amyloidosis” G Applications of research G.01 / G.01.02 Contribution to postgraduate Education 1. Introduction of a new research field on postgraduate education at J.Stefan's International Postgraduate School (IPS). PI holds two courses at IPS. http://www.mps.si/splet/studij.asp?lang=slo&main=1&left=4&id=457&m=4 2. PI has ERASMUS exchange grant with Technical University of Tallinn, Estonia. G.09 Basic research for Health in search of new therapies: „molecular medicine”, A review paper entitled: Protein Conformational Pathology in Alzheimer's and Other Neurodegenerative Diseases; New Targets for Therapy, was published in Current Alzheimer Research, 2010, 7, 74-83. IF= 4.1) (11). Connection to private health and pharma sector: The IRCCS Institute in Brescia will study the role of stefin B as a risk factor for neurodegenerative diseases (i.e. Alzheimer disease and Frontotemporal dementia). As well, LEK Pharmaceuticals d.d., (Novartis, Sandoz group) part of multi-national pharmaceutical company is interested in the results and future co-operation.
Significance for the country
Stefin B is not only an excellent model to study mechanisms of amyloid fibril formation and toxicity, which can be generalised to other proteins, mostly the globular ones – which form domain-swapped oligomers but it also is physiologically important protein. In 2010 we have published our study of the interaction of stefin B with amyloid-beta in vitro and co-localization in cells, which brings stefin B in direct connection to AD (4). Apart from this, Lehtinen et al., 2009 have reported that in KO stefin B cells oxidative stress gets increased (5) and that cystatin C induces autophagy (6). This latter is a process which clears protein aggregates from the cell and has a major role in neurodegenerative diseases. Recently, another study was published, where they have shown that absence of stefin B ameliorates pathology in a mouse AD model (7-8). Altogether, this demonstrates that new functions of this protein have to be studied. Function of stefin B is far from clear. Its function as a protease inhibitor is not the only one – as clearly shown by recent studies. How does it prevent oxidative stress and what role it may have in innate immunity? One possible function, which we have proposed is acting chaperone-like, as the oligomers might bind other amyloid peptides and thus prevent their aggregation. If it would be proven that EPM1 mutants loose such a protective function, this would certainly have implications for EPM1 disease. Similarly, binding of ?-synuclein to stefin B would have important implication to PD. Cellular studies are directly relevant to pathology of AD and EPM1. Markers of protein aggregation, which we think stefin B influences and especially so some EPM1 mutants (we measured markers of oxidative stress and autophagy) could be in common between EPM1 and neurodegenerative diseases, which would prove our opinion (POLAJNAR, Mira, ŽEROVNIK, Eva. Impaired autophagy : in common to neurodegenerative diseases and progressive myoclonal epilepsis. Trends mol. med., IF (2009) = 11.049) (9). Similar conclusion might apply to neurodegenerative disease (10). 1. Jenko Kokalj, S., et al. Essential role of proline isomerization in stefin B tetramer formation. J Mol Biol 366, 1569-1579 (2007). 2. Morgan, G.J., et al. Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein. J Mol Biol 375, 487-498 (2008). 3. Zerovnik, E., Staniforth, R.A. & Turk, D. Amyloid fibril formation by human stefins: Structure, mechanism & putative functions. Biochimie 92, 1597-1607 (2010). 4. Skerget, K., et al. Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells. J Biol Chem 285, 3201-3210 (2010). 5. Lehtinen, M.K., et al. Cystatin B deficiency sensitizes neurons to oxidative stress in progressive myoclonus epilepsy, EPM1. J Neurosci 29, 5910-5915 (2009). 6. Tizon, B., et al. Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines. Plos One 5, e9819. 7. Kaur, G. & Levy, E. Cystatin C in Alzheimer's disease. Front Mol Neurosci 5, 79 (2012). 8. Kaur, G., et al. Cystatin C rescues degenerating neurons in a cystatin B-knockout mouse model of progressive myoclonus epilepsy. Am J Pathol 177, 2256-2267. 9. Polajnar, M. & Zerovnik, E. Impaired autophagy: a link between neurodegenerative diseases and progressive myoclonus epilepsies. Trends Mol Med 17, 293-300 (2011). 10. Polajnar, M. & Zerovnik, E. Impaired autophagy: a link between neurodegenerative and neuropsychiatric diseases. J Cell Mol Med (2014). 11. Zerovnik, E. Protein conformational pathology in Alzheimer's and other neurodegenerative diseases; new targets for therapy. Curr Alzheimer Res 7, 74-83 (2010).
Most important scientific results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
