RICERCA ITALIANA     

PROTEASES IN NORMAL AND PATHOLOGICAL CELL RESPONSES. IDENTIFICATION OF ENDOGENOUS SUBSTRATES AND INDUCED MOLECULAR MECHANISMS.

Libera Università "Vita Salute S. Raffaele" - Milano

Abstract

This project will exploit and set up leading edge technologies to study the role of proteases in normal and pathological cell responses at the proteomic level. This has become possible thanks to the completion of the sequenze of the human genome. Moreover, the project will form an electronic network for the partners with a "Virtual Center" allowing the information and on-line analysis of the results and the sharing of the software for analysis, storage and handling of the data. The choice of the partners, in addition to their scientific excellence, has been based on their scientific and technological convergence as well as on the possibility to transfer the results to the industry.
The project focuses on proteases: five of the Units will analyse in detail various aspects of three types of proteases, while three Units will, in addition, also act as technological motors and provide common excellence services.
The Units of this project will identify new substrates of a serine protease (urokinase), of a calcium-dependent protease (calpain) and of the ubiquitination system. Moreover, new components of the ubiquitin-ligase gene family and other proteins interacting with them will be identified. Moreover, the substrates and the types of protease-induced, or protease-conditioning, post-translational modifications will be analysed. The project aims at obtaining more sensitive and reproducible techniques in the areas of purification of multiprotein complexes, sequencing of complex protein mixtures and identification of new phosphorylatable substrates. New methods overcoming the technical difficulties depending on the mass of existing proteins will be proposed and tested, to bring them to a level of real "high-troughputness".
Objectives of this project:
1. Organization of an interactive Web site and of a Workstation for data analysis and handling.
2. Setting up of technologies of mass spectrometry with on-line chromatographic separation of proteins for the identification of new substrates of proteases and for the analysis of their effects on signal transduction pathways.
3. Setting up of technologies for the qualitative and quantitative analysis of the phosphoproteome through both the identification of serine, threonine and tyrosine phosphorylation sites in cell and tissue extracts and elucidation of the relationship(s) between phosphorylation processes and susceptibility to proteolysis.
4. Setting up of technologies for the rapid identification of both function and tridimensional structure of phosphorylated peptides and analysis of their resistance to the various proteases.
5. Setting up of technologies for the identification at the proteomic level of the substrates of uPA, exemplifying a serine protease, and the analysis of both the molecular mechanisms involved and the signaling pathways affected.
6. Identification of the activation of the calpain-calpastatin system in signal transduction pathways and human pathologies by determining the active forms of calpain, the changes in the intracellular distribution of calapastatin, its phosphorylation state and degradation patterns, and the substrates preferentially degraded in the various activation states.
7. Setting up of technologies for the identification at a proteomic level of novel enzymatic ubiquitination systems, and the elucidation of the different roles played by mono- and poly-ubiquitination in the mechanisms of signal transduction. Identification through informatics and proteomics of new substrates of the SIAH ubiquitin ligases and characterization of their role in the p53-dependent regulation of cell growth both in vitro and in human tumors.
8. Identification and characterization of the components of the human signalosome and systematic identification of the specific target proteins which are ubiquitinated before being degraded by the proteasome under the control of the signalosome. <<<

Principal Investigator

FRANCESCO BLASI Libera Universita' "Vita Salute S.Raffaele" MILANO

Research Goal

The present project is aimed at investigating the role of proteases in both normal and pathological cellular responses. The most recent technologies will be exploited to carry out an in-depth high-throuput study. These technologies are based on one side on the presence in data banks of the nucleotide sequence of the entire human genome (and of a large part of the murine one), and consequently of the amino acids sequence of many proteins. On the other side, they are based on the dramatic improvements in sensitivity and reproducibility of the techniques to purify molecular complexes and to sequence their protein components. It is in fact abundantly clear, that proteins within the cells are present in multi-molecular complexes, the formation of which is essential for the function of the cell. Another important goal is to collect a series of highly qualified laboratories into a single "virtual Centre", allowing both a real-time exchange of information about the results obtained by the various Units and the sharing of dedicated softwares for data processing and analysis.
Finally, the partners were chosen not only on the basis of common fields of interest and scientific excellence, but also to generate results that could be transferred to the pharmaceutical industry. In fact one of the Units belongs to an important pharmaceutical industry, and other Companies have already shown their interest in our project. In addition, since six of the eight participating Units belong to Centri di Eccellenza created by MIUR, the creation of the present network represents the first step in the building of a High Qualification Center for the Study of Proteases joining public and private enterprises.

The number of proteases able to influence cellular responses is increasing continuously. In addition to serine proteases, other relevant examples are provided by the matrix metalloproteases and the calcium-dependent proteases whose active site contain either a cysteine or an aspartic residue. Some of these proteases degrade extracellular proteins preferentially, whereas other proteases hydrolyze specifically cell surface receptors or intracellular signaling molecules. A well characterized mode of proteolysis is represented by a complex biochemical system which, through the attachment of multiple copies of a specific protein module, ubiquitin, serves to direct proteins to their degradation catalyzed by the proteasome. However, this type of covalent modification is not limited to protein degradation. Indeed while polyubiquitination is a signal for proteolysis, mono- and oligo-ubiquitination play a role in signal propagation. Therefore, the definition of the role of the various proteases in cellular responses requires the elucidation of a series of key points, like the identification of substrates, interacting proteins and other components of protease families, as well as of the type of post-translational modifications induced by proteases directly or indirectly, or able to affect protease activity. All these issues can be addressed by applying post-genomics technologies, particularly those grouped under the general name of proteomics. To achieve this goal, however, it is necessary not only to improve or adapt the available techniques bringing them to a higher level of sensitivity and reproducibility, but also to develop and test novel methods which has to overcome the difficulty represented by the very high number of proteins to be analyzed before becoming real "high-throughput" applications.
The following list summarizes the experimental objectives of this project:
1. Organization of an interactive Web site and of a Workstation for data analysis and handling.
2. Setting up of technologies of multi-dimensional chromatography coupled on line with mass spectrometry for the identification of new substrates of proteases and for the analysis of their effects on signal transduction pathways.
3. Setting up of technologies for the qualitative and quantitative analysis of the phosphoproteome through both the identification of serine, threonine and tyrosine phosphorylation sites in cell and tissue extracts and elucidating the relationship(s) between phosphorylation processes and susceptibility to proteolysis.
4. Setting up of technologies for the rapid identification of both the function and the tridimensional structure of phosphorylated peptides and the analysis of their resistance to the various proteases.
5. Setting up of technologies for the identification at the proteome level of the substrates of uPA, exemplifying a serine protease, and the analysis of both the molecular mechanisms involved and the signaling pathways affected.
6. Identification of the activation of the calpain-calpastatin system in signal transduction pathways and human pathologies by determining the active forms of calpain, the changes in the intracellular distribution of calpastatin, its phosphorylation state and its degradation patterns, and the substrates preferentially degraded in the various activation states.
7. Setting up of technologies for the identification at a proteomic level of novel enzymatic ubiquitination systems, and the elucidation of the different roles played by mono- and poly-ubiquitination in the mechanisms of signal transduction. Identification through bio-informatics and proteomics of new substrates of the SIAH ubiquitin ligases and characterization of their role in the p53-dependent regulation of cell growth both in vitro and in human tumors.
8. Characterization of the human signalosome and systematic identification of the specific target proteins which are ubiquitinated before being degraded by the proteasome under the control of the signalosome. <<<

Timescale

36 months