Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

• Keywords
• 14-3-3 protein, 70-kDa heat shock protein (Hsp70), HSP70, Hsp70, Tomm34, dimerization, hydrogen-deuterium exchange, molecular chaperone, phosphorylation, protein folding, protein import, protein kinase A (PKA), protein-nucleic acid interaction, protein–protein interaction, translocase of outer mitochondrial membrane 34 (TOMM34),
• MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Phosphorylation physiology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Molecular Chaperones metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• HSP70 Heat-Shock Proteins metabolism   MeSH
• HSP72 Heat-Shock Proteins metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   metabolism   MeSH
• Mitochondrial Membrane Transport Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BCL2-associated athanogene 1 protein MeSH Browser
• DNA-Binding Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Proteins MeSH
• Molecular Chaperones MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• HSP72 Heat-Shock Proteins MeSH
• HSP90 Heat-Shock Proteins MeSH
• TOMM34 protein, human MeSH Browser
• Transcription Factors MeSH
• Mitochondrial Membrane Transport Proteins MeSH

BACKGROUND: Increased activity of the chaperones Hsp70 and Hsp90 is a common feature of solid tumours. Translocase of the outer mitochondrial membrane 34 (Tomm34) is a cochaperone of both Hsp70 and Hsp90 that was found to be overexpressed in colorectal, hepatocellular, lung and breast carcinomas. The expression profile of Tomm34 in ovarian cancer has not been investigated. Therefore, the aim of the current study was to investigate the expression pattern of Tomm34 in ovarian carcinomas and analyse its correlation with clinico-pathological parameters. RESULTS: Epithelial ovarian cancers (140) were histologically classified based on their morphology and graded into two types comprising 5 histologic subgroups. Type I carcinomas comprise low grade serous (LGSC), clear cell (CCOC) and endometrioid (ENOC), type II comprises high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). Tomm34 was more highly expressed in type II than type I carcinomas (p < 0.0001). Comparing tumours based on the mutation in the TP53 gene revealed similar results, where mutant tumours exhibited significantly higher levels of Tomm34 (p < 0.0001). The decreased levels of Tomm34 in type I carcinomas were particularly evident in clear cell and mucinous carcinomas. The expression of Tomm34 was also positively correlated with FIGO stage (r = 0.23; p = 0.007). Tomm34 levels also indicated poor prognosis for patients with mutant p53. CONCLUSIONS: Our data indicate that Tomm34 is commonly expressed at high levels in epithelial ovarian cancers, except for the clear cell and mucinous subtypes. The expression of Tomm34 corresponds with the dualistic model of ovarian cancer pathogenesis where high grade, type II tumours exhibit higher expression of Tomm34 in contrast to type I tumours. These data are also comparable to the previous findings that Tomm34 is a marker of progression and poor prognosis in human cancer.

• Keywords
• Chaperone, Epithelial ovarian cancer, Heat shock protein, Immunohistochemistry, Ovary, Tomm34, Tumour,
• MeSH
• Adult MeSH
• Carcinoma, Ovarian Epithelial metabolism   pathology   MeSH
• Immunohistochemistry MeSH
• Middle Aged MeSH
• Humans MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mutation MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Ovarian Neoplasms metabolism   pathology   MeSH
• Prognosis MeSH
• Disease Progression MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Neoplasm Staging MeSH
• Mitochondrial Membrane Transport Proteins metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Biomarkers, Tumor MeSH
• Tumor Suppressor Protein p53 MeSH
• TOMM34 protein, human MeSH Browser
• TP53 protein, human MeSH Browser
• Mitochondrial Membrane Transport Proteins MeSH

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.

• Keywords
• Allostery, Chaperone, Cochaperone, Mass Spectrometry, Protein Conformation, Protein Structure, Protein-Protein Interactions, Structural Biology,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Stress, Physiological MeSH
• HEK293 Cells MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• HSP70 Heat-Shock Proteins chemistry   metabolism   MeSH
• Mitochondrial Membrane Transport Proteins metabolism   MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• STUB1 protein, human MeSH Browser
• TOMM34 protein, human MeSH Browser
• Mitochondrial Membrane Transport Proteins MeSH
• Ubiquitin-Protein Ligases MeSH

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.

• MeSH
• Epithelial Cell Adhesion Molecule genetics   metabolism   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mucoproteins MeSH
• Oncogene Proteins MeSH
• Peptides metabolism   MeSH
• Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epithelial Cell Adhesion Molecule MeSH
• AGR2 protein, human MeSH Browser
• EPCAM protein, human MeSH Browser
• MDM2 protein, human MeSH Browser
• Mucoproteins MeSH
• Oncogene Proteins MeSH
• Peptides MeSH
• Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Recombinant Proteins MeSH