RICERCA ITALIANA     

Identification of folding and misfolding determinants by site-directed mutagenesis.

Università degli Studi di Firenze

Abstract

Dialysis-related amyloidosis represents an inevitable and severe complication of long-term hemodialysis and is characterised by the deposition in essential tissues, such as the skeletal muscle, of fibrillar aggregates, termed amyloid aggregates, formed by beta-2-microglobulin (b2-m). The research project presented here aims at extending the present knowledge on the involvement of both specific residues of b2-m sequence, and its different structural regions in the folding and misfolding events. Thus, this project will focus on several topics that require the involvement and the cooperation of different research units (RU). The dealed topics are:
1) Production of b2-m variants. We will design a number of mutations that are useful to study both the processes of folding and aggregation.
2) Characterisation of the solution structure of the variants by standard 1D and heteronuclear 2D and 3D NMR spectroscopy. Gross structural comparisons between different mutants will be obtained by analyisis of the paramagnetic attenuation pattern measured in the presence and absence of a spin label or even from the differential water accessibilty pattern. NMR spectroscopy will be also used to obtain the profiles of segmental mobilities between different mutants and information on the stability of the different secondary structure elements. Real time NMR determinations will then enable us to measure the kinetics of the slow refolding intermediates of several b2-m variants and infer >>>

Principal Investigator

Fabrizio Chiti Università degli Studi di FIRENZE

Research Objectives

Dialysis-related amyloidosis represents an inevitable and severe complication of long-term haemodialysis. Under this pathological condition, protein aggregates known as amyloid fibrils, accumulate in essential tissues, such as the skeletal muscle, interfering with their normal functions. A major constituent of the amyloid fibrils related to this pathological condition is beta-2-microglobulin (b2-m). The research program described here aims at characterising the underlying events of folding and misfolding of such protein at a molecular level.
The first objective of the research program is to gain insight, at a residue level, of the process by which b2-m folds, i.e. convert from its unfolded state to its fully folded, functional state. The proposed research will use previous models of folding, previously characterised by the research units participating to this PRIN as well as other internationally recognised groups, to investigate the folding process with deep molecular insight. We propose to identify the residues that participate to the formation and stabilisation of the native state and of the various partially folded states that accumulate during folding.
The second objective of the program is to determine, with similar molecular insight, the residues or regions of the sequence that promote the process of amyloid aggregation of b2-m. The process of aggregation will be studied not just for free b2-m, but also for the protein in the presence of collagen >>>

Timescale

24 months

National and international background

Amyloidosis is an heterogeneous disease category in which peculiar proteins undergo a pathological self aggregation into insoluble fibrils (1). These fibrils accumulate in target tissues causing irreversible damage. In the last few years the RU of Pavia has gathered a significant collection of natural amyloidogenic proteins isolated from different biological sources, in particular the different isoforms of b2-microglobulin (b2-m), present in amyloid deposits of different patients affected by dialysis–related amyloidosis (DRA), have been characterised. Native b2-m is 99 residues in length and has a seven stranded beta-sandwich fold typical of the immunoglobulin superfamily. The protein contains two beta-sheets (one comprising beta-strands A, B, D, E and the other beta-strands C,F and G) that are held togheter by a single disulphide bond linking Cys25 and Cys80. In vivo, b2-m is present as the non-polymorphic light chain of the class I major histocompatibility complex (MHCI). As part of its normal catabolic cycle, b2-m dissociates from the MHCI complex and is trasported in the serum to the kidneys where the majority is degraded (2). In haemodialised patients (>20000 people in Italy, and >250000 in the EU) the b2-m concentration increases by 20–50 fold and by a mechanism that is currently not well understood, b2-m self aggregate into amyloid fibrils that tipically accumulate in the musculoskeletal system (3). b2-m is mainly present in amyloid fibrils as full >>>