We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature-and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino-and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature-and concentration-dependent equilibrium involving monomers, dimers, and trimers. Members of the highly conserved 70-kDa heat shock protein family (HSP70) 1 are involved in several cellular processes such as protein folding, assembly and disassembly of multimeric proteins, protein translocation across membranes, protein degradation, and signal transduction (for reviews see Refs. 1-4). They are thought to act as molecular chaperones by transiently binding hydrophobic regions exposed to the solvent in the nonnative conformations of proteins, thereby preventing off-pathway reactions that lead to aggregation (5).A prominent member of this family, the mammalian, constitutively expressed, 70-kDa heat shock cognate protein (HSC70), has been shown to bind peptides and unfolded proteins (6 -8) and to possess refolding activity in the presence of the cochaperone DnaJ (9, 10). HSC70 shows a very weak ATPase activity that can be stimulated 2-5-fold upon binding of peptides, unfolded proteins, clathrin light chains, and cochaperones of the DnaJ family (11-14). HSC70 seems to function through cycles of binding and release of polypeptide substrates coupled to binding and hydrolysis of ATP (15), in a mechanism involving cochaperones of the DnaJ protein family and a newly isolated factor, Hip (16). HSC70 is made of two domains, an NH 2 -terminal domain of 44 kDa (residues 1-384), which binds and hydrolyzes ATP, and a COOH-terminal domain of about 30 kDa (residues 385-646), which contains the peptide binding site (17, 18). The threedimensional structure of the NH 2 -terminal ATPase domain has been solved by crystallography (19), and the secondary structure topology of the peptide binding site (residues 385-543) has been determined by NMR methods (20). Recently, the structure of a complex between a seven-residue peptide and the COOHterminal domain of DnaK, the bacterial HSP70, has been established (21). However, the three-dimensional structure of the entire protein is still unknown.Self-association is a general and well conserved feature of the HSP70 protein family. BiP (22-24), the constitutive HSC70 (6, 25-30), the heat shock-inducible HSP70 (31), plant HSP70 (32), and DnaK (33), all show self-association properties. Nevertheless, the structural basis an...

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a b-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein.To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385±540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.Keywords: analytical ultracentrifugation; heat shock proteins; HSC70; molecular chaperones; self-association.The constitutively expressed 70-kDa heat shock cognate protein (HSC70), a member of the highly conserved 70-kDa heat shock protein family (HSP70), plays an important role in protein folding, assembly and disassembly, transport and signal transduction (for review see [1±3]). HSC70 is composed of an Nterminal ATPase domain (residues 1±384) [4] and a C-terminal domain (residues 385±646) which can be divided into a peptidebinding subdomain (residues 385±540) and a C-terminal subdomain (residues 540±646) [5,6]. The three-dimensional structure of the isolated N-terminal ATPase domain of HSC70 [7] and the C-terminal domain of the bacterial HSP70, DnaK, complexed with peptide [6], have been solved by crystallography. However, structural information for the whole protein is still lacking. At the functional level, HSC70 binds peptides and unfolded proteins [8,9]. It has very weak ATPase activity which can be stimulated two-to fivefold on binding of peptides [10] Proteins of the HSP70 family from bacteria to man oligomerize into several species in vitro [19±21] as well as in vivo [22±24]. Although the role of such a property has not yet been established, mounting evidence suggests the existence of a relationship between oligomerization and chaperone activity. Indeed, like chaperone activity, oligomerization of the HSP70s is regulated by ATP binding, co-chaperones and peptides [25±27], and the C-terminal domain of the protein (residues 385±646), which is necessary for chaperone activity as it contains the peptide-binding site, has recently been implicated in the process [26].The C-terminal domain of HSC70 is composed of ...

BackgroundThe epithelial-to-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal features and contributes to metastasis and resistance to treatment. This process involves epigenetic reprogramming for gene expression. We explored global histone modifications during TGF-β1-induced EMT in two non-small cell lung cancer (NSCLC) cell lines and tested different epigenetic treatment to modulate or partially reverse EMT.ResultsLoss of classical epithelial markers and gain of mesenchymal markers were verified in A549 and H358 cell lines during TGF-β1-induced EMT. In addition, we noticed increased expression of the axonal guidance protein semaphorin 3C (SEMA3C) and PD-L1 (programmed death-ligand 1) involved in the inhibition of the immune system, suggesting that both SEMA3C and PD-L1 could be the new markers of TGF-β1-induced EMT. H3K79me3 and H2BK120me1 were decreased in A549 and H358 cell lines after a 48-h TGF-β1 treatment, as well as H2BK120ac in A549 cells. However, decreased H3K79me3 was not associated with expression of the histone methyltransferase DOT1L. Furthermore, H3K79me3 was decreased in tumors compared in normal tissues and not associated with cell proliferation. Associations of histone deacetylase inhibitor (SAHA) with DOT1L inhibitors (EPZ5676 or SGC0946) or BET bromodomain inhibitor (PFI-1) were efficient to partially reverse TGF-β1 effects by decreasing expression of PD-L1, SEMA3C, and its receptor neuropilin-2 (NRP2) and by increasing epithelial markers such as E-cadherin.ConclusionHistone methylation was modified during EMT, and combination of epigenetic compounds with conventional or targeted chemotherapy might contribute to reduce metastasis and to enhance clinical responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0380-0) contains supplementary material, which is available to authorized users.