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C-terminal amino acids are essential for human heat shock protein 70 dimerization.

ABSTRACT: The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.

SUBMITTER: Marcion G

PROVIDER: S-EPMC4255253 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation.

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Project description:Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

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