The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.

Humans contain many HSP (heat-shock protein) and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of protected cells from heat-induced cell death, overexpression of Hduanyu18426 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of Hduanyu18426 did not impair the development of heat-induced thermotolerance. Yet, Hduanyu18426 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical Hduanyu18421A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified Hduanyu18426 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved family, functional differentiation is larger than expected, with Hduanyu18426 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.

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