Role of HSP40 Domains/Motifs in Protection from Cytotoxic Stress

Purpose Doxorubicin, a highly effective therapeutic agent against several types of cancer, is associated with serious side‐effects, particularly cardiotoxicity. To understand the mechanisms of toxicity of doxorubicin, a whole‐genome sensitivity screen was performed in the yeast S. cerevisiae . YDJ1, a heat‐shock protein 40 (HSP40) was among the most sensitive strains. Heat‐shock proteins (HSPs) are molecular chaperones that are upregulated as a response to various types of stresses to aid in the refolding of denatured proteins. HSP40 is a co‐chaperone to HSP70, forming a complex along with HSP104 to break up aggregation of misfolded proteins and refold them into correct conformation. HSP40 stimulates ATPase activity needed for protein refolding by HSP70. The HSP40 YDJ1 is comprised of several highly conserved domains and motifs that are essential in the heat‐shock response. The cysteine‐glycine‐rich region has been implicated in protein‐protein interaction with unfolded, non‐native proteins, the farnesylation site facilitates attachment of YDJ1 to the ER and perinuclear membranes, and the HPD tripeptide motif is responsible for the regulation of the ATPase activity of HSP70s. The purpose of this study is to determine the relevance of each of the above mentioned domains/motifs in the protection of HSP40 from cytotoxic stress. Findings may be able to be exploited to develop specific inhibitors for therapeutical purposes. Methods Yeast strains harboring mutations in the cysteine‐glycine‐rich region, farnesylation site, and HPD motif of YDJ1 were created using site‐directed mutagenesis. Mutants were exposed to heat shock, doxorubicin, and cisplatin. Considering that Doxorubicin induces cellular stress by two major mechanisms (1. DNA damage by generation of DNA double‐strand breaks through inhibition of topoisomerase II and DNA intercalation, and 2. The generation of free oxygen radicals through its quinone ring), we have dissected these functions by separately exposing the mutant strains to menadione, which contains a quinone ring, and etoposide, which only inhibits topoisomerase II, in an attempt to determine if the conserved domains play a distinct roles in the protection to these agents. Results We find that a mutation in the HPD motif abolishes the activity of YDJ1, while a mutation in farnesylation results in a slighty protective effect against cytotoxic agents, and a mutation on the cys/gly domain results in different survival depending on the type of stress. Conclusion Our data indicate that the conserved domains/motif of the YDJ1 play distinct roles in the protection to cellular stresses, which is stress‐specific. We believe that characterizing the role of the conserved domains of YDJ1 in the response to cytotoxic chemotherapy will lead to the identification of targets with potential for drug development that can be used to enhance anthracycline chemotherapy. Since the heat shock response is upregulated in many cancer cells, the inhibition of HSP40s in these cancer cells may sensitize them to chemotherapy. In addition, if the heat shock response proteins such as HSP40 can be upregulated in a site‐specific manner, particularly in cardiomyocytes, this approach may offer a protective effect against cardiotoxicity. Support or Funding Information Research supported by National Institute of Health (NIH) under Award Number G12MD007582 (previous award NIH/NCRR/RCMI G12 RR03020).