RICERCA ITALIANA     

Analysis of cellular survival processes in eukaryotic orgamisms by proteomic approaches

Università degli Studi di Napoli "Federico II"

Abstract

This project will be addressed to the investigation of cell survival mechanisms at the molecular level by exploiting integrated proteomic-based strategies. Two major guidelines of activity can be foreseen within the project based on two alternative and complementary proteomic approaches, functional proteomics and expression proteomics. The expression proteomic methodologies will be applied to the comparative analysis of the variations in protein profiles and in post-translational modifications in different cell lines and in different conditions in order to identify specific proteins involved in cell survival and drug resistance. Detailed studies on the molecular mechanisms involved in cell survival and drug resistance will be carried out in the same cell lines by using functional proteomic strategies aimed to the identification of protein-protein interactions in vivo. Proteomic analyses will essentially be performed on three different cell lines, a simple eukariotic model system as yeast and two complex sistems, tumoral stem cells and peripheral lymphocytes from AD patients.
Investigation of differential protein profilings will be carried out on both normal and apoptotic yeast cells, following treatment with high level of H2O2 or with the Apoptosis-inducing factor (AIF 1). The same approach will be employed in the comparative analyses of protein profiles from normal and tumor stem cells in the presence and in the absence of IL-4 treatment or to investigate proteins >>>

Principal Investigator

Pietro PUCCI Università degli Studi di NAPOLI "Federico II"

Research Objectives

The aims of this project are addressed to the application of integrated proteomic-based approaches to investigate the role of a variety of effectors in the regulation of the apoptotic cascade at the molecular level. These aims will be approached from different angles and using different techniques as the Research Units partecipating in the project have consolidate expertises in complementary scientific fields. Different complex systems as stem cells and lymphocytes as well as Saccaromices cerevisiae as model system will be investigated. We will therefore outlines these objectives:



1.Comparative analysis of Yeast proteome oxidatively stressed and/or during apoptosis induced by H2O2 and by AIF 1 overexpression.
2. Identifying the redox sensitive and carbonylated proteins involved in ageing and calorie restriction
3. Protein-protein interactions between Apoptosis-inducing factor (AIF) and others yeast proteins.
4. Comparative analysis of the proteic pattern of normal and cancer stem cells with normal and cancer primary cells
5. Comparative analysis of the proteic pattern of IL-4 treated and untreated stem cells

6. Study of expression levels of molecules involved in differentiation such as ZBP-89/BREF-1, nestin and p63
7. Understanding the regulation of JAK/STAT pathway in cancer cells overexpressing SOCS-1
8. identification of the protein partners interacting with SOCS-1 and the other >>>

Timescale

24 months

National and international background

In the last years, following the end of the Human Genome Project, the biological challenge has again shifted from gene to the protein side, giving rise to the so-called "proteomic era", with the aims to identify and localise proteins within a given organelle, cell or even organism as well as to unravel protein pathways in vivo (1-5). These new goals, however, cannot be easily achieved as intrinsic difficulties increase by several order of magnitude when moving from genome to proteome research. The static nature of the genome, in fact, cannot be compared to the dynamic properties of the proteome; protein expression profiles change several times during the cell cycle and are heavily affected by a number of intra- and extracellular stimuli (temperature, stress, apoptotic signals, ecc.) (6). Moreover, the occurrence of alternative splicing and post-translational modifications led to a complete re-thinking of the old paradigm "one gene-one protein" that does not reflect anymore the real nature of the cellular proteome.
Current proteome investigations are basically focused on two major areas, the expression proteomics, which aims to measure up-and down-regulation of protein levels, and the functional proteomics aimed at the characterisation of cellular compartments, multiprotein complexes and signaling pathways. Both approaches rely on two consecutive analytical steps consisting in the separation and identifications of proteins. The main technologies involved in a >>>