RICERCA ITALIANA     

INTERACTION PARTNERS OF AMYLOIDOGENIC PROTEINS TO STUDY MISFOLDING AND AGGREGATION PROCESSES; POSSIBLE APPLICATIONS

Università degli Studi di Udine

Abstract

Some human diseases with a relevant socioeconomic impact are caused by the formation of insoluble protein aggregates of fibrillar nature, referred to as amyloid deposits.
The formation of these aggregates requires conformational modifications of the precursor proteins, which can be considered as a deviation from the normal protein folding process. Amyloid deposits, however, can also be formed by fragments, either structured or not, obtained by proteolytic events from larger precursor proteins. This project aims at studying proteins of either amyloidogenetic pathways. It has been shown that a large proportion of physiologically relevant amyloid deposits in tissues are made up by protein fragments derived from relatively larger protein precursors; on this basis, it is reasonable to propose the use of protein fragments as most suitable experimental systems for exploring the mechanisms of amyloid fibrils formation. On the other hand during the molecular process of fibrillar conversion of other proteins, aggregation requires a molecular destabilization phase and the formation of intermediate states that are stabilized by intramolecular interactions affecting the secondary structure. Fibrillogenesis can be modulated by the modification of chemical and physical conditions affecting protein solvation and the charge of a few key residues, but also by the interaction with natural or synthetic ligands. Metal ions appear important to this purpose because their binding can affect >>>

Principal Investigator

Paolo VIGLINO Università degli Studi di UDINE

Research Objectives

Aim of the project is to investigate three different topics regarding the aberrant assembly of proteins or protein fragments which lead to the formation of fibrils and plaques of amyloidogenic diseases.
Six research units will collaborate to the realization of the project; three research units (Pavia, Firenze, Padova) are mainly involved in the expression or production of biological material (proteins, proteins fragments, antybodies etc), but also contribute to the characterization of thermodinamical, biochemical and kinetic parameters which control oligomerization and fibrillogenesis; the three other research units (Udine, Genova, Napoli) contribution is mainly due to the use of sophisticated techniques such as Atomic Force Microscopy, Multidimensional NMR spectroscopy and limited proteolysis, H/D exchange and chemical crosslinking coupled to Mass Spectroscopy.
- Studies on the aggregation of protein fragments. Amyloid deposits can be formed by fragments, either structured or not, obtained by proteolytic events from larger precursor proteins. Protein fragments can adopt in isolation partly folded states that expose hydrophobic patches that are usually hidden in the interior of a native intact protein. Therefore, fragments are capable of strong intermolecular hydrophobic interactions, thus leading to their association to form soluble oligomers, which constitute a critical nucleus of the overall process of fibrillogenesis towards the final, well-ordered mature >>>

First Results

In phase Ia several structural, thermodynamic and kinetic aspects of the fibrillogenic process of selected amyloidogenic proteins will be established. One of the predicted results is represented by the characterisation of intermediates of the folding/misfolding pathway stabilised by metal ions.
In phase Ib the results are the antibodies that target b2microglobulin, Hypf and sinuclein. For each antibody exaustive characterisation of binding properties, and definition of structural properties of the epitope will be provided.The results will consist in the identification of antibodies and their fragments able to interfere with the fibrillogenic process and in the possible elucidation of mechanism of correction of fibrillogenic propensity of the protein model under investigation. Once the functional (anti-fibrillogenic) antibodies will be availabe and characterised, it will become affordable the definition of favourable modifications induced by the antibodies, regarding the tertiary structure, the surface topology and thermodynamic stability.

Timescale

24 months

National and international background

Some human pathologies with a great socioeconomic impact are caused by the formation of insoluble protein aggregates of fibrillar nature, referred to as amyloid deposits.
The formation of these aggregates requires conformational modifications of the precursor proteins, in analogy to the onset of intermediate states along the normal protein folding process. Formation of such intermediates appears to be critical for the onset of fibril formation, since these species are capable of strong intermolecular interactions given by the exposure of the polypeptide main-chain and hydrophobic side-chains that are otherwise buried in the overall fold of the native protein [Dobson 2003]. This view has led to the proposal that all polypeptide chains in principle can form amyloid aggregates under appropriate experimental conditions [Dobson 1999]. However, since all fibrils derived from different proteins share a common structural motif, the cross beta-structure, it is clear that major conformational rearrangements should take place in order to produce the well-ordered protein aggregates. Nowadays, there is a common belief that partly folded or molten globule states of proteins can be key intermediates in protein aggregation and fibrillogenesis (Fink 1998). Amyloid deposits, however, can also be formed by fragments, either structured or not, obtained by proteolytic events from larger precursor proteins. This project aims at studying proteins of either amyloidogenetic pathways.
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