Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding.

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

The Role of HSP70 in Cancer and its Exploitation as a Therapeutic Target.

BACKGROUND: Sustained proliferation and genetic instability of cancer cells are associated with enhanced production of mutated and conformationally unstable proteins. Excessive proteosynthesis along with increased metabolic turnover generates stress conditions that cancer cells must permanently compensate for. Tumor cells thus become dependent on the maintenance of protein homeostasis, which involves protein quality control, folding, transport and stabilization. These tasks are provided by molecular chaperones, predominantly the stress proteins HSP70 and HSP90. Their expression and activity is increased in all malignant tumors, where they associate with their cochaperones to form large multiprotein complexes. HSP70 and HSP90 maintain the malignant phenotype because they facilitate the folding of numerous oncogenic proteins, maintain proliferative potential, and inhibit apoptosis. In this regard, heat-shock proteins represent an important target for cancer therapy because their inactivation results in the simultaneous blockade of multiple signaling pathways. Although several specific HSP90 inhibitors have been developed in the past decade, their antitumor activity as single agents is limited due to the induction of HSP70, which enables cell survival. Inhibitors of HSP70 thus present new possibilities for targeting proteostatic mechanisms in cancer cells. AIM: The aim of this article is to summarize information on the structure of HSP70 and its role in maintaining protein homeostasis in normal and cancer cells. The mechanisms of HSP70 inhibition by low-molecular weight compounds and their application in targeted antitumor therapy are also described. Key words: HSP70 - stress proteins - molecular chaperons - cellular stress - tumours - protein folding This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 08. 2018.