Preservation of organ function after trauma or operative stress is critical for patient recovery. Natural mechanisms of cellular protection could be harnessed to improve the preservation of organ function after injury. However, these protective mechanisms need to be well understood to unlock their clinical potential. One natural mechanism of protection is the expression of heat shock proteins (hsp). Hsp are involved in cellular protection from harmful environmental conditions, termed acquired stress tolerance, which is the result of the stabilization of various cellular structures and processes, including protein synthesis. The specific focus of this application is to elucidate the mechanism of hsp mediated protein synthesis protection during stress. Investigations performed during recent years have shown that Hsp70 binds with strong affinity to 40S ribosomal subunits and that the presence of Hsp70 is necessary for the protection of protein synthesis during heat shock. Thus, the central hypothesis to be tested is whether the interaction between Hsp70 and the ribosome subunits is responsible for the stabilization of protein synthesis during heat shock. To elucidate the possible mechanism of protein synthesis protection, the ribosomal binding domain on Hsp70, as well as the ribosomal compounds that interact with Hsp70, need to be identified.
Specific Aim 1. Identify the ribosomal binding domain on Hsp70. The methodology to be used is based on observation that Hsp70 binds to ribosomal subunits whereas a highly homologous protein, Hsc70, interacts with nascent polypeptide chains. The exchange of domains between them will modify their binding pattern resulting in the possible identification of the ribosomal binding site. The ribosomal binding domain will be further characterized by site directed mutagenesis. An important part of this aim is to obtain a Hsp70 mutant that does not bind ribosome subunits. This mutant should not rescue protein synthesis during heat shock. A reverted mutant should restore the protective ability.
Specific Aim 2. Detect the ribosomal component that interacts with Hsp70. Binding of Hsp70 with 18S rRNA will be investigated by RNA gel mobility shift. The polypeptide target for Hsp70 will be identified by selective extraction of ribosomal proteins with high concentrations of salt, immunoprecipitation and indirect Western blotting.
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