We will continue investigating how mammalian cells defend themselves against adverse changes in their local environment. Using a combination of cell biology, biochemistry, immunology, and microinjection techniques, considerable effort will be directed toward understanding the properties and intracellular functions of the proteins induced to high levels in cells experiencing heat shock and/or other forms of physiological stress. Toward these ends, it is planned to complete the purification of the major stress-induced proteins, prepare polyclonal and monclonal antibodies specific to each, and finally complete the determination of their intracellular location in both the normal and stressed cell. New efforts will focus on the biochemistry of the two major stress-induced proteins, 72kDa and 28kDa. It is planned to further characterize the ATP-binding properties of 72kDa, examine its putative ATPase activity, and define substrates which drive such ATPase activity. In vitro and in vivo assays will be designed to test the hypothesis tha t 72kDa is involved in the rescue and/or stabilization of the translation machinery in the stressed, recovering, and/or thermotolerant cell. Characterization of 28kDa, including its subcellular distribution, role of phosphorylation, and relevance of its observed co- distribution with 72kDa will be pursued. Finally, a vigorous effort will be directed at introducing the purified stress proteins (initially 72kDa, 90kDa, and 28kDa) or their corresponding antibodies into living cells via microinjection and assess the effects by morphological criteria, indirect immunofluorescene, and protein synthesis. Using this approach, questions relating to the possible role of the stress proteins in cellular protection and survival, acquisition of thermotolerance, regulation of translation, and autoregulation will be addressed.
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