Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.
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