One of the most challenging problems in Cell Biology is to understand the mechanisms by which Molecular Chaperones assist protein folding in nature. These mediators have been implicated in a wide range of fundamental biological events including; preventing formation of proteinaceous aggregates, promoting assembly and/or disassembly of oligomeric enzymes, and aiding in the protein translocation process. The emerging evidence suggests that Chaperones are ubiquitous and there is intensive interest in unraveling the precise molecular events by which this class of proteins functions at the cellular level. Moreover, recent studies conclusively demonstrate that the etiology of a variety of pathological conditions could be explained based in alterations in the expression and function of Molecular Chaperones. For instance, alterations in the expression and activation of these mediators have been convincingly demonstrated in the development of autoimmune diseases, viral and bacterial infections, cancer, and muscular dystrophy. During the last few years, we have been able to identify and biochemically characterize three Chaperones, Cpn60, Cpn10 and Hsp70 from the bacterium C. vinosum, and more. Recently, we been have successful in identifying other Chaperone systems, from the same organism, which appear to be homologs of the DnaJ, GrpE and ClpB families. Some of these mediators suffer phosphorylation and, in the present application we intend to obtain information in regards to the biological significance of this finding. The hypothesis to be tested in this proposal is that the ability of Chromatium vinosum Cpn60 in modulating protein folding events is controlled by protein phosphorylation and by its direct physical interaction with other Molecular Chaperones. This hypothesis will be tested by pursuing the following specific aims: (1) to evaluate the influence of various Molecular Chaperone Systems; DnaK, GrpE, DnaJ, Cpn10 and CIpB, in modulating the autophosphorylation of Cpn60 from C. vinosum; (2) to study the ability of Cpn60, in combination with other Chaperone systems, to favor disaggregation and refolding of denatured proteins; (3) to study the intracellular location of various Chaperone systems from C. vinosum, namely DnaK, DnaJ, GrpE, ClpB, and Cpn60/Cpn10, under heat shock and other stressful conditions; (4) to study the conditions that favor the Cpn60 binding to the cytoplasmic membranes of C. vinosum; (5) to study the properties of phosphorylated Cpn60 from C. vinosum under heat shock conditions and; (6) to study the impact of the Cpn60 catalyzed phosphorylation on the functional properties of RuBisCO. Results from these experiments promise to advance our understanding on the mechanisms by which Chaperones modulate protein folding.
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