Doxorubicin, a highly effective therapeutic agent against several types of cancer, is associated with serious side-effects, particularly cardiotoxicity. In addition, drug resistance leads to unsuccessful outcomes in many patients. There are no current biomarkers to indicate doxorubicin treatment response in patients. To understand the mechanisms of toxicity of doxorubicin, a whole-genome sensitivity screen was performed in the yeast S. cerevisiae. A deletion mutant of YDJ1, a J-domain 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 native conformation. HSP40 binds the misfolded client protein, presents it to HSP70 and stimulates the ATPase activity of HSP70 needed for protein refolding. The HSP40 YDJ1 is comprised of several highly-conserved domains and motifs that are essential in the heat-shock response. The cysteine-rich region has been implicated in protein-protein interaction with client proteins, farnesylation of YDJ1 facilitates attachment of YDJ1 to the ER and perinuclear membranes, and the HPD tripeptide motif present in the J-domain, is responsible for the regulation of the ATPase activity of HSP70s. YDJ1 is also predicted to be post-translationally modified by SUMO proteins, an alteration that has been shown to affect the function and binding substrates of several proteins. The purpose of this study is to determine the relevance of each of the above-mentioned domains/motifs of HSP40 in its protective role from cytotoxic stress. Findings may be able to be exploited to develop specific inhibitors for therapeutical purposes.

Yeast strains harboring mutations in the cysteine-rich region, farnesylation site, HPD motif, and predicted SUMOylation sites of YDJ1 were created using site-directed mutagenesis. Mutants were exposed to heat shock, doxorubicin, and cisplatin. Doxorubicin induces cellular stress by two major mechanisms: 1) DNA damage by generation of DNA double-strand breaks (DSBs) through inhibition of topoisomerase II and DNA intercalation, and 2) the generation of reactive oxygen species (ROS) through its quinone ring. We have dissected these functions by separately exposing the mutant strains to menadione, which contains a quinone ring and generates ROS, and to etoposide, which inhibits topoisomerase II generating DSBs, in an attempt to further characterize the role that conserved domains play in the protection to these agents.

We find that a mutation in the HPD motif abolishes the activity of YDJ1, while a mutation in farnesylation results in a slightly protective effect against cytotoxic agents, and a mutation in the cysteine repeats results in different survival depending on the type of stress. Mutation of predicted SUMOylation sites increased YDJ1 resistance to all types of stress.

Our data indicate that the conserved domains/motif of the YDJ1 play distinct roles in the protection to cellular stresses, which is stress type-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 target sites 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 tissue-specific manner, particularly in cardiomyocytes, this approach may offer a protective effect against cardiotoxicity.