The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.In the absence of hormone, steroid receptors are known to exist in heteromeric complexes containing multiple proteins, including heat shock protein 90 (hsp90) (for reviews see references 20 and 33). In order to understand the role of these proteins in regulating receptor assembly and hormone signalling, we and others have used a cell-free rabbit reticulocyte lysate (RL) system to support assembly of functional progesterone (30) and glucocorticoid (24) receptor complexes in vitro. For progesterone receptor (PR) assembly in vitro, at least eight distinct proteins have been identified as taking part in the ordered assembly of PR complexes (26,31). hsp70 can function as a molecular chaperone in promoting protein folding, and hsp90 may be able to promote folding as well (reviewed in reference 9). The other six proteins have not been shown to have individual chaperone activity, but each commonly associates with hsp70 and/or hsp90, and it is possible that each serves at least as an accessory in chaperone-mediated processes. Thus, all eight receptor-associated proteins can be considered components of the overall molecular chaperone machinery in eukaryotic cytoplasm.Importantly, proper assembly with hsp90 is required to establish and maintain PR's high-affinity progesterone binding state (26), as was previously demonstrated for glucocorticoid receptor (24). In vitro assembly of PR complexes competent for hormone binding requires ATP, Mg 2ϩ , K ϩ , and near-physiological temperature in addition to several protein factors (6-8, 12, 24, 26, 30, 31). A working model outlining the complex and dynamic interactions occurring during PR assembly in vitro was proposed earlier (26). In that model, a transient PR compl...

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90-to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661-677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.

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The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription.

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically ‘break’ right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac–Leu–Thr–Phe–Aib–Glu–Tyr–Trp–Gln–Leu–Cba–Aib–Ser–Ala–Ala–NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

Prior to binding hormone, steroid receptor monomers are typically found in a multiprotein complex containing Hsp90 and Hsp90-associated proteins such as p23 and large immunophilins (for an extensive review, see reference 21). Receptors for progesterone (PR) and glucocorticoids (GR) must be assembled in these complexes in order to bind hormone with high affinity and efficiency. Studies of the receptor assembly process, relying primarily on cell-free reactions in rabbit reticulocyte lysate (RL), have revealed that assembly is an ordered process and involves at least eight proteins that are components of the molecular chaperone machinery. Several of these chaperone components appear transiently prior to formation of mature receptor complexes. For example, Hsp70 is the first protein observed to bind PR in a cell-free assembly, but Hsp70 is probably not a component of mature complexes (1,25). Two Hsp70-associated proteins, Hip and Hop, are recovered in PR complexes shortly after the first appearance of Hsp70, but these proteins are also absent from mature complexes. A model in which Hip and Hop function in a coordinated manner to facilitate Hsp70-mediated folding of proteins has been proposed (8), but it is not clear how this relates to steroid receptor assembly.Hop (alternate names in the literature are p60, IEF SSP 3521, mSti1, and RF-hsp70) is required for formation of mature PR (4) and GR (5, 6) complexes. In a yeast model system,

The present study was to evaluate workplace violence and examine its effect on job burnout and turnover attempt among medical staff in China. A total of 2,020 medical employees were selected from Fujian province by using stratified cluster sampling method. The Chinese version of the Workplace Violence Scale and the Maslach Burnout Inventory-General Survey were used to measure the workplace violence and job burnout, respectively. Other potential influencing factors for job burnout and turnover attempt were collected using a structured questionnaire. The incidence of workplace violence among medical staff was 48.0%. Workplace violence had a positive correlation with emotional exhaustion and cynicism and a negative correlation with professional efficacy. Workplace violence, marital status, employment type, working time (≥ 10 h/day), performance recognition, and life satisfaction were significant predictors for turnover attempt among Chinese medical staff.