Cytolytic lymphocytes (CTLs) are characterized by their inclusion of secretory granules containing putative effector molecules such as perforin and the serine proteases. We recently described the cDNA cloning and functional characterization of a cytotoxic granule-associated RNA-binding protein designated TIA-1. Purified, recombinant TIA-1 was found to induce DNA fragmentation in permeabilized target cells, suggesting that it may be the granule component responsible for inducing programmed cell death, or apoptosis, in CTL targets. The overall aim of this proposal is to determine the role of TIA-1 in the cytolytic effector function of CTLs.
The specific aims are: i) to perform structure- function analysis of relevant TIA-1 domains, ii) to demonstrate the physiologic role of TIA-1 in cytolytic lymphocyte-mediated killing, iii) to identify TIA-1-binding proteins and nucleic acids in target cells, and iv) to determine how cytotoxic granules deliver TIA-1 to target cells.
These aims will be accomplished by constructing a series of deletion and point mutations in TIA-1 structural domains, and determining the effect of these mutations on nucleolytic activity. Once we have identified the nucleolytic active site, we will transfect wild-type and mutant TIA-1 into rat basophilic leukemia cells and determine whether TIA-1 confers the ability to induce DNA fragmentation in target cells. We will then identify target cell proteins and RNAs that interact with TIA-1, as a first step in unraveling the molecular mechanisms by which TIA-1 induces DNA fragmentation. Finally, we will determine how cytotoxic granules deliver TIA-1 to target cells by studying isolated light and dense-cored granules isolated using immunoaffinity purification. Because cytolytic lymphocytes may contribute to the pathogenesis of autoimmune disease and to the process of transplant rejection, an improved understanding of the molecular toxins employed by these cells may lead to the development of new therapies for these common conditions.
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