RICERCA ITALIANA     

Molecular features of protein conformational diseases. Role of environmental factors on the structural changes of proteins for the design and the synthesis of agents with antiaggregating, antioxidant, antiglycating and chelating activity and for application in diagnostics.

Università degli Studi di Catania

Abstract

Recently, it has been shown that several pathological conditions are related to protein misfolding. These diseases, which have been grouped under the name “protein conformational diseases”( PCD),
include the Alzheimer’s disease , the prion diseeases, the Huntington’s and the Parkinson’s diseases, the diabetes type II and about 15 other not very well-defined illnesses.
Several environmental factors, including pH, metal ions, oxidative stress may contribute to the destabilization of the native protein’s conformation and, probably, increase the population of misfolded states.
Notwithstanding big research effort, the mechanism by which soluble proteins (or related peptide fragments) with distinct primary structures undergo partial unfolding with subsequent incorrect refolding is still unclear. What is worse is that this aberrant process may lead to the production of very stable oligomers or polymers endowed with new properties.
This project aims at the elucidation of the molecular mechanisms involved in the pathogenesis of the PCD and to the development of new compounds for the pharmacological treatment of these pathologies. To test the activity as well as the effectiveness of new designed potential drugs, either chemical and biological experimental models will be used.
The analysis of the molecular mechanisms, that will be aided by the use of the above cited the experimental models, will allow a better >>>

Principal Investigator

Enrico RIZZARELLI Universita' degli Studi di CATANIA

Research Objectives

The main goal of this project is to understand the molecular basis related to the conformational variations connected with the protein misfolding process and to design and develop new molecules for the therapeutical treatment and the diagnosis of the conformational diseases.
At first, the potential active sites for the new drugs, able to stop or delay the neurons death during the developing of conformational diseases, will be determined. In this regard, the chemical-physic mechanism and the properties that lead to the formation of neurotoxic species will be studied by means of different computational techniques. In particular calculations for the conformational and geometry optimization as well as for the determination of pharmacophore maps will be performed. The calculation of hydropathic and electrostatic potential maps and the determination of formation energies will be done as well. The computational results will also permit to set-up a pharmacological approach to inhibit the formation of the neurotoxic species and the proapoptotic transductional systems.
Suitable software able to create hydropaticity and interaction maps will allow the analysis of the frames obtained by means of MD. This in turn will allow the possibility to discriminate between the different protein “forms” and to give useful indications about the conformational variations. The subsequent steps will be focused on: a) the characterization of the structural alterations that >>>

First Results

The main aim of this research activity is the optimization of protocols for the synthesis of fibrillogenic peptides which will be followingly used for the physico-chemical characterization. Because of the high insolubility of these peptides and consequent difficulties in the analysis of the conformational changes, attention will be focused on the functionalization of these peptides with PEG. Several fragments of alpha-synuclein (aS), as well as the entire protein are already available. Shortly after the beginning of the project, it is anticipated that suitably labeled (15N, 13C, 2H) material will be available for NMR structural analysis. In parallel, this Unit will have obtained the expression of human tyrosinase (Ty) in P. pastoris. An important intermediate result in this phase of the project will be the obtainment of the NAC fragment (56-102), possibly labeled with 15N for structural and topological studies. It is possible that the NMR analysis of the longer peptide fragments, i.e., 1-41 and 1-52, may require their expression at least with 15N-labeling. Once this necessity is established by means of preliminary NMR studies, these fragments should be easily obtained. Another significant result will be the production of suitably spin-labeled material for EPR studies. The first pair of mutated aS proteins will bear the Cys around residue 80, within the NAC region. The other pair, mutated around residue 50, will be produced in a subsequent step. Depending on the success of >>>

Timescale

24 months

National and international background

The failure of proteins to fold correctly, or to remain correctly folded, is the origin of a wide variety of pathological conditions. In the last few years, several diseases have been shown to arise from protein misfolding and they are now grouped together under the name of “Protein Conformational Disorders” (PCDs). This group includes Alzheimer’s Disease (AD), Transmissible Spongiform Encephalopaties (TSES), Huntington’s Disease (HD), Parkinson’s Disease (PD), diabetes type II, dialysis related amyloidosis, Amyotrophic Lateral Sclerosis (ALS) and more than 15 other less known disease (Kelly 1996; Soto 1999). The hallmark event in PCD is a change in the secondary and/or tertiary structure of a normal protein (A-beta amyloid, Prion, Huntingtin, alfa-sinuclein, beta-microglobulin, Cu Zn- superoxidismutase) or its polypeptide fragments without alterations of the primary structure. In each case, there is a progressive transition of normal, correctly folded cellular proteins into insoluble, rich in beta sheet conformation amyloid fibrils (Soto 1999). These are straight, unbranching structures of about 70-120 A in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation >>>