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Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90

ABSTRACT: The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.

SUBMITTER: Schmauder L

PROVIDER: S-EPMC8560915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Small cell lung cancer (SCLC) at advanced stage is considered an incurable disease. Despite good response to initial chemotherapy, the responses in SCLC patients with metastatic disease are of short duration and resistance inevitably occurs. Although several target-specific drugs have altered the paradigm of treatment for many other cancers, we have yet to witness a revolution of the same magnitude in SCLC treatment. Anthracyclines, such as doxorubicin, have definite activity in this disease, and ganetespib has shown promising activity in preclinical models but underwhelming activity as a single agent in SCLC patients. Using SCLC cell lines, we demonstrated that ganetespib (IC50: 31 nM) was much more potent than 17-allylamino-17-demethoxygeldanamycin (17-AAG), a geldanamycin derivative (IC50: 16 μM). Ganetespib inhibited SCLC cell growth via induction of persistent G2/M arrest and Caspase 3-dependent cell death. MTS assay revealed that ganetespib synergized with both doxorubicin and etoposide, two topoisomerase II inhibitors commonly used in SCLC chemotherapy. Expression of receptor-interacting serine/threonine-protein kinase 1 (RIP1), a protein that may function as a pro-survival scaffold protein or a pro-death kinase in TNFR1-activated cells, was induced by doxorubicin and downregulated by ganetespib. Depletion of RIP1 by either RIP1 small interfering RNA (siRNA) or ganetespib sensitized doxorubicin-induced cell death, suggesting that RIP1 may promote survival in doxorubicin-treated cells and that ganetespib may synergize with doxorubicin in part through the downregulation of RIP1. In comparison to ganetespib or doxorubicin alone, the ganetespib+doxorubicin combination caused significantly more growth regression and death of human SCLC xenografts in immunocompromised mice. We conclude that ganetespib and doxorubicin combination exhibits significant synergy and is efficacious in inhibiting SCLC growth in vitro and in mouse xenograft models. Our preclinical study suggests that ganetespib and doxorubicin combination therapy may be an effective strategy for SCLC treatment, which warrants clinical testing.

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Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90

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