The heat stress induced or enhanced synthesis of a small number of specific proteins is a fundamental phenomenon of widespread application and relevance in cellular biology. The goal of this proposal is unchanged and is to study the role of heat stress proteins (hsp) in survival and adaptation following hyperthermic stress. The overall hypothesis is that the hsp act at multiple throughout the cell by mediating the interaction of cellular molecules. Some of these mediated interactions are seen as associated with protein translation and translocation at or near membrane interfaces. Others are seen as associated with cellulaR regulatory and signaling systems involving Ca++ homeostasis, calmodulin, protein kinases and hormones at or near membrane interfaces.
The specific aims for this project period are designed to test selected aspects of that hypothesis by characterizing the hyperthermic response of cells and the interactions of their hsp in macromolecular associations at or near the plasma membrane.
The aims are: (1) To more completely define the conditions and extent of the locations of a subfraction of the hsp and their cognates (hsc) occurring at or near the plasma membrane in apparent associated with cytoskeletal proteins, transmembrane proteins, and other less characterized proteins. (2) To characterized the nature and role of the apparent associations of hsc/hsp with cytoskeletal, transmembrane and other proteins in submembranous assemblages. (3) to modulate the amount of calmodulin (CAM) in cells and ask how this affects the macromolecular associations of hsc/hsp with other cellular proteins as defined above. Further, to ask now the amount of CaM affects synthesis of hsc/hsp, thermotolerance and heat killing. (4) To modulate the amount of protein kinase C (PKC) in cells and ask how this affects the macromolecular association of hsc/hsp with other cellular proteins as defined above. Further, to ask how the amounts of PKC affects synthesis of hsc/hsp, thermotolerance and heat killing.
These aims will be achieved using a number of biochemical techniques including metabolic labeling, one- and tow-dimensional, native- and SDS-gel electrophoresis, chemical crosslinking, affinity isolation, protein blotting, and antibody staining techniques. Studies also involved cell culture techniques and clonogenic survival studies and cell lines containing expression vectors to overexpress calmodulin and certain protein kinases. The last studies will be previously constructed vectors and some to be made during this research.
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