RICERCA ITALIANA     

A multidisciplinary approach to the study of in vivo and vitro aggregation of polyglutamine-containing proteins. Role of molecular and environmental factors.

Università degli Studi di Milano-Bicocca

Abstract

Protein misfolding and aggregation underlie severe and widespread neurodegenerative disorders, like Parkinson, Alzheimer, prion, Huntington, and ataxia diseases, for which no treatment is available. The development of possible therapeutic approaches is hampered by the poor understanding of the molecular mechanisms that cause the onset and the development of these “protein conformational disorders”. Although protein deposition in amyloid fibrils characterizes the pathological state, growing evidence indicates that the toxic agents are the oligomeric and protofibrillar intermediates of the aggregation process. The investigation of these molecular species is hampered by their short life and heterogeneous state, as well as by the high number of factors that affect their structural and biochemical properties in vivo. This project recruits advanced molecular and biophysical technologies to a multidisciplinary investigation of the mechanisms underlying protein aggregation in vitro and in vivo. The focus is on a particular class of amyloidogenic proteins, whose misfolding and aggregation is triggered by expanded polyglutamine (polyQ) sequences. These are the pathogenic agents of a wide group of neurodegerative diseases, among which Huntington disease and spinocerebellar ataxia type 3 are the most widespread. Thus, the proteins huntingtin (htt) and ataxin-3 (AT3) will be employed, along with chimeric constructs in which polyQ tails will be joint to reporter protein domains.
>>>

Principal Investigator

Paolo Tortora Università degli Studi di MILANO-BICOCCA

Research Objectives

This project is aimed at the investigation of the molecular mechanisms underlying 1) aggregation of polyglutamine (polyQ) proteins and 2) their toxicity for eukaryotic cells. In order to shed light on the aggregation mechanism, it is essential to understand the nature of the protein-protein interactions that mediate aggregation and to characterize the molecular species at final and intermediate aggregation states. The latter are thought to act as nucleation factors inducing protein misfolding and eliciting fibrillation. Correspondingly, goal 1 consists of two major tasks: 1.1) investigation of the regions involved in aggregation and 1.2) structural characterization of different aggregation states. Tackling the issue of toxicity of polyQ proteins in vivo, we propose on one side to use yeast as an eukaryotic model system convenient to manipulate and to engineer and, on the other side, human cell cultures approaching as close as possible physiological conditions. Thus, goal 2 implies two tasks: 2.1) investigation of toxicity in Pichia pastoris and 2.2) investigation of toxicity in human neuroblastoma cell. The specific aims that compose these tasks are illustrated below. For each of these issues, we plan to investigate the effect of metal ions, since they seem to play a critical role modulating aggregation and toxicity of amyloidogenic proteins in general. Thus, studying the effect of metals is not listed as a distinct aim of the project, but it is indeed a central focus of the >>>

First Results

RESULTS OF THEORETICAL RELEVANCE

1) Identification of protein contexts favoring or disfavoring protein aggregation, with special regard to the effect towards polyQ protein aggregation.

2) Characterization of the structure of early and intermediate aggregates, and of the kinetics of their appearance.

3) High resolution structural characterization of amyloid fibrils by AFM and STM.

4) Characterization of the interaction surfaces within fibrils by MS and related techniques.

5) Insights into the mechanisms of cellular toxicity of different polyQ proteins. Both naturally occurring proteins and artificial constructs carrying polyQ stretches will be assayed in E. coli, yeast and neuroblastoma cells.

6) Identification of metals enhancing the aggregation and their mode of action at the molecular and cellular level.

RESULTS OF BIOMEDICAL RELEVANCE

1) A deep understanding of the aggregation mechanisms might help identify the step(s) leading to the appearance of the most toxic species which, based on the current knowledge, are likely to occur at the early stages of the overall process. In turn, these achievements should pave the way to the development of screening assays for the selection of compounds capable of preventing the accumulation of these species.

2) Our studies at the cellular level, in particular in neuroblastoma cells, should provide >>>

Timescale

24 months

National and international background

Several neurodegenerative diseases are associated with protein misfolding and aggregation, which results in formation of insoluble amyloid-like deposits in different regions of the brain. They display fibrillar appearance, Congo red staining and birefringence, and cross-beta-structure. It is believed that such “protein conformational disorders” share common mechanisms at the molecular level, which also are paralleled by similar clinical and histological features [Chiti and Dobson (2006) Annu Rev Biochem 75, 333-366].
This diverse grouping include Alzheimer, Parkinson, prion diseases, Huntington and related polyQ diseases. In the pathway of amyloid fibril formation, protofibrils arise first from misfolded proteins. They are roughly spherical or tubular assemblies, 2.5–5.0 nm in diameter with high beta-sheet content. Such assemblies are generally precursors of longer protofilaments and mature fibers. The species most highly toxic to cells are likely to be the prefibrillar aggregates (or protofibrils) rather than the mature fibrils [Dobson and Stefani (2003) Mol Med 81, 678-699].
PolyQ diseases share a number of clinical features, yet each affects different areas of the brain. Each of them is associated with a protein harboring, in its pathological form, an uninterrupted stretch of polyQ of length in excess of a threshold, typically of 40 consecutive residues. Misfolding of these proteins has long been known to be associated with the formation of insoluble >>>