The Role of HSP40 Conserved Motifs in the Response to Cytotoxic Stress

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 the yeast DNAJ (YDJ1), a J-domain heat-shock protein 40 (HSP40) was among the most sensitive strains. HSP40 is a co-chaperone to HSP70 and together refold denatured proteins into native conformation. 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 histidine-proline-aspartic acid (HPD) tripeptide motif present in the J-domain, is responsible for the regulation of the ATPase activity of HSP70s. We have investigated the role of these motifs in the protection cytotoxic stress. We find that mutations in the HPD motif and cysteine-rich region of YDJ1 sensitize cells to doxorubicin and cisplatin, while a mutation in farnesylation results in a slightly protective effect. The sensitivity of the HPD and cysteine mutants is specific to oxidative stress and not to DNA double-strand breaks.