RICERCA ITALIANA     

ROLE OF INTRACELLULAR PROTEOLYSIS IN THE REGULATION OF INNATE AND ACQUIRED IMMUNE RESPONSE.

Università degli Studi di Genova

Abstract

This research project is addressed to study by means of new inter-disciplinary approaches the role of the calcium dependent proteolytic system (the calpain/calpastatin system) in the regulation of the immune response. The involvement of the proteolytic system in such a process is suggested by the increase in intracellular free Ca2+ occurring in stimulated leukocytes, the presence in these cells of specific calpain isoforms, and the cytolytic effect exerted by some of these immunocompetent cells following activation.
The project will study the following points:
-Identification and characterization of the calpains expressed in homogenous leukocyte populations or in cell lines showing functional properties similar to those of cells involved in the immune response. This will be followed by the characterization of the regulation system, identifying the molecular and inhibitory properties of calpastatin.
-These studies will be completed by experiments in intact cells by using of the FRET technology. With this procedure, it will be obtained information on the expression, localization, activation, target degradation and regulation of the calpain/calpastatin system.
-Correlation between changes in intracellular calcium homeostasis and activation of the calcium dependent proteolytic system in cells involved in immune response and stimulated with natural external signals.
-Extracellular role of calpains and interaction of the calcium dependent >>>

Principal Investigator

Edon MELLONI Università degli Studi di GENOVA

Research Objectives

The aims of this research project is to find answers to still unclear aspects of the calpain/calpastatin system concerning its function and its involvement in the onset and regulation of the immune response.
A role of the calcium dependent proteolysis in immune response can be hypothesized on the basis of an observed increase in intracellular free calcium accompanying the stimulation of leukocyte cells, the presence in such cells of specific calpain isoforms and the cytolytic effect exerted by some immunocompetent cell members. On the basis of these considerations, the project aims are addressed to the understanding of such arguments using, new experimental approaches ranging from the classical biochemical techniques to more appropriate methodologies of molecular biology and biophysics. Many of the problems limiting the experimental studies on the characterization and function of this system concern the difficulties in the finding of appropriate experimental cell or animal models, in the identification of the calpain isoforms expressed in a cell and their specific action, the lack of direct in vivo observations on the activation and regulation mechanism of the system, and finally the high number of involved protease members and of targets to be digested. We will try to clarify some of these uncertainties using appropriate multi-disciplinary technologies in order to identify the occurrences of these processes directly into the cells in the basal conditions and >>>

Timescale

24 months

National and international background

The calpain/calpastatin proteolytic system (1-3) which is present in the cytosol of all mammalian cells is composed by one or more proteases belonging to calpain family and one or more forms of the natural inhibitor calpastatin. In calpain family are present about 15 members sharing some catalytic and regulatory properties. One of the features common to all calpain forms is the identification in the catalytic domains of two subdomains. This condition keeps the amino acids of the catalytic triad (Cys, His, and Asn) separated and calpain is inactive (3, 4). Thus, a conformational modification is required to achieve the setting of the catalytic triad and the activation of the protease. Another characteristic shared by all calpains is the presence of a calmodulin-like domain which contains five EF-hand structures able to bind calcium ions. This property and the calcium dependence of the protease is even enhanced by other calcium binding sites localized also near calpain catalytic site. Many calpain forms have a dimeric structure where the catalytic heavy subunit is associated to a light subunit also containing five EF-hand structures. At basal calcium concentration calpain is not active and becomes active following a modification of calcium homeostasis.
Almost all mammalian cells contain two calpain forms, named µ- and m-calpain that are both eterodimers and show a strict calcium dependence (3, 5).Their tridimensional structure has been determined both in the absence >>>