Progress 10/01/09 to 09/30/14
Outputs
Target Audience: - Departments and department heads - OSU administrators - Other faculty and other scientific researchers in DASNR, at OSU & the scientific community - Students and post-docs - Federal, state, and private funding agencies - Scientific journal editors, readers & the scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students Maurie Balch- Trained in: preparation of cell culture sample for stable isotope labeling of cell in culture (SILAC); Mass spectroscopy and analysis of MS data; expression and purification of recombinant proteins; and setting up protein crystallization arrays. Wei Xia- Trained in 2- and 3-dimensional cell culture techniques. Undergraduates Dakota Cryer- Ectopic expression of proteins in cultured cells by transfection Alexander Brown: culturing 3-dimensional spheroids in vitro. How have the results been disseminated to communities of interest? Through publication of manuscripts in the primary scientific literature. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: The fact that eating your fruits and vegetables leads to a healthy life should be of conventional knowledge, as it is now supported by four decades of research that have demonstrated that that bioactive compounds are part of the food chain and have effects on human health. It is noteworthy that the majority of drugs approved for use by the FDA during the past 30 to 50 years are natural products or derivatives thereof. With the growth of scientific enquiry into the field of bioactive compounds there has been an explosion in the amount of novel small molecules being discovered whose health benefits have been previously overlooked, but whose mechanism of action remain uncharacterized. The challenge of the USDA, to have the general public accept new recommendations in regards to their diet or use of dietary supplements, is to demonstrate to them that the recommended changes have proven health benefits. The argument in support of adoption of dietary changes becomes far more persuasive when the bioactive principles in the food, herbals or dietary supplement have been identified and their mechanism of action characterized. The molecular chaperone Hsp90 and its associated co-chaperones exist at the hub of a cell’s protostasis network balancing protein functional activity with protein breakdown. Hsp90 inhibitors have been demonstrated to have beneficial chemo-preventative and chemotherapeutic properties with regards to cancer, and in the prevention and progression of neurodegenerative diseases. Numerous natural product inhibitors of Hsp90 have been identified, and promotion of the consumption of functional foods and herbals that contain Hsp90 inhibitors among their bioactive compounds would like benefit the health of the US population. We have screen natural compound libraries for inhibitors of Hsp90, and have identified over a 100 new compounds, with a literature search indicating that 44 of these new compounds having known beneficial pharmacological properties, but unknown mechanisms of action. Twelve of the compounds come from foods and herbal remedies utilized in folk medicines from various world cultures. Characterization of the biological and chemical properties of the compounds confirms their Hsp90 inhibitory activity. In addition, other studies have expanded our understanding of Hsp90/co-chaperone interaction with target protein, advancing our knowledge into the mechanism through which these compounds have beneficial effect on human physiology. Thus, new inhibitors of Hsp90 chaperone machine have discovered that have the potential to be beneficial for treatment of cancer and neurodegenerative diseases, in addition to have a better understanding of there mechanism of action on cells. Accomplishments: 1. To identify new inhibitors of Hsp90 chaperone machine and determine how these new pharmacological agents that target the Hsp90 chaperone machine and disrupt its function, and how the effects of disrupting the function of the Hsp90 chaperone machine on the growth, differentiation and viability of cells. Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent a six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we utilized a high throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to down regulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized Hsp90 inhibitors. Four compounds, anthothecol (Khaya anthotheca), garcinol (Mango), piplartine (long peppers), and rottlerin (fruit of kamala tree) were further characterized. The compounds had anti-proliferactive effects reducing the rate of growth of MCF7 breast cancer cells by 50% at 0.5 to 10 µM concentrations which correlated with there ability inhibit luciferase refolding. In addition the compounds suppressed the Hsp90-dependent maturation of the heme-regulated eIF2a kinase, confirming their identity as Hsp90 inhibitors. Thus, an additional 44 compounds with known beneficial pharmacological properties, but unknown mechanisms of action as possibly new Hsp90 inhibitors. A search of the literature indicates twelve of the compounds have been consumed as food and herbal remedies in folk medicine with little adverse affect. 2. To determine the mechanism through which the heat shock protein (Hsp)90 chaperone machine functions to regulate the biogenesis of proteins in vivo, and identify novel chaperone components and new protein clients of the Hsp90 chaperone machine. We investigate the activator of heat shock protein 90 (Hsp90) ATPase’s (Aha1) protein-protein interaction (PPI) network to gain critical insights into the relationship of Aha1 with multi-molecular complexes and shred light onto Aha1’s interconnections with Hsp90-regulated biological functions. Flag-tagged Aha1 was over-expressed in HeLa cells and isolated by anti-Flag affinity pull downs, followed by trypsin digestion and identification co-adsorbing proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). A probability-based identification of Aha1 PPIs was generated from the LC-MS/MS analysis by using a relative quantification strategy, spectral counting (SC). By comparing the SC-based protein levels between Aha1 pull-down samples and negative controls, 147 Aha1-interacting proteins were identified that were quantitatively enriched in the pull-down samples over the controls. The identified Aha1-interacting proteins are involved in a wide number of intracellular bioprocesses, including DNA maintenance, chromatin structure, RNA processing, translation, nucleocytoplasmic and vesicle transport, among others. The interactions of 33 of the identified proteins with Aha1 were further confirmed by Western blotting, demonstrating the reliability of our affinity-purification-coupled quantitative SC-MS strategy. It is noteworthy that greater than 80% of the proteins that were identified have not been previously reported to be in the Hsp90-protein interacting network. This proteomic data indicates that Hsp90Aha1 participates in diverse biological pathways to facilitate Hsp90 chaperone functions in response to stress. These interaction networks have previously been underappreciated, and the work expands our knowledge on how Hsp90 inhibitors work, as it enhances the number of cellular biological pathways that are impacted by inhibition of Hsp90.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Davenport, J., Balch, M., Galam, L., Antwan Girgis, A., Jessica Hall, J., Blagg, B.S.J., and Matts, R.L. (2014) High-Throughput Screen of Natural Product Libraries for Hsp90 Inhibitors. Biology 3, 101-138.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Manjarrez, J.R., Sun, L., Prince, T., and Matts, R.L. (2014) Hsp90-dependent assembly of the DBC2/RhoBTB2-Cullin3 E3-ligase complex. PloS One 9(3):e90054.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Sun, L. and Matts, R.L. (2014) Biochim. Biophys. Acta Identification of Aha1 Interacting Proteins in HeLa Cells by Quantitative Proteomics. submitted
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: OSU Departments and department heads OSU administrators Other faculty and other scientific researchers in DASNR, at OSU & the scientific community Students and post-docsFederal, state, and private funding agencies Scientific journal editors, readers & the scientific community Agricultural, environmental, life, and human science industries General public and elected officials Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Version:1.0 StartHTML:0000000225 EndHTML:0000002984 StartFragment:0000002637 EndFragment:0000002948 SourceURL:file://localhost/Users/robertmatts/Documents/%20Grant%20folder/OEAS/CSRS%20report%2013-Matts/CRSR%202013.doc Graduates students have been trained in producing and analyzing advanced proteomic data, generated by the use of a state-of-the-art LC-MS/MS mass spectrometer. This is a skill that will be valued highly as they pursue their future careers. How have the results been disseminated to communities of interest? Version:1.0 StartHTML:0000000225 EndHTML:0000002900 StartFragment:0000002636 EndFragment:0000002864 SourceURL:file://localhost/Users/robertmatts/Documents/%20Grant%20folder/OEAS/CSRS%20report%2013-Matts/CRSR%202013.doc The results have been published in highly regarded scientific journals and presented at national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Version:1.0 StartHTML:0000000225 EndHTML:0000003785 StartFragment:0000002639 EndFragment:0000003749 SourceURL:file://localhost/Users/robertmatts/Documents/%20Grant%20folder/OEAS/CSRS%20report%2013-Matts/CRSR%202013.doc Objectives- To continue to identify new inhibitors of Hsp90 chaperone machine; to determine how these new pharmacological agents that target the Hsp90 chaperone machine and disrupt its function; to determine: the mechanism through which the Hsp90 chaperone machine functions to regulate the biogenesis of proteins in vivo; to extend our LC-MS/MS results on Hsp90 inhibitor impact on cancer cell proteomes by quantifying changes utilizing Stable Isotopic Labeling of Cells in Culture (SILAC); identify novel chaperone components and new protein clients of the Hsp90 chaperone machine; and determine the effects of disrupting the function of the Hsp90 chaperone machine on the growth, differentiation and viability of cells. Expected Outputs: New inhibitors of Hsp90 chaperone machine will be identified and characterized that may represent a new class of drugs for treatment of disease states. New client proteins and co-chaperones of Hsp90 will be identified that will enhance the scientific community’s understanding of Hsp90 function.
Impacts
What was accomplished under these goals?
Version:1.0 StartHTML:0000000225 EndHTML:0000005345 StartFragment:0000002641 EndFragment:0000005309 SourceURL:file://localhost/Users/robertmatts/Documents/%20Grant%20folder/OEAS/CSRS%20report%2013-Matts/CRSR%202013.doc The 90 kDa heat-shock protein (Hsp90) and other co-chaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy since the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. AUY922 is a potent synthetic Hsp90 inhibitor that is moving steadily through clinical trials against a small range of cancers. To identify protein markers that might measure the drug’s effects, and to gain understanding of mechanisms by which AUY922 might inhibit the proliferation of leukemia, we characterized AUY922’s impacts on the proteomes of cultured Jurkat leukemia cells. A robust and readily assayed proteomics fingerprint of was identified that AUY922 shares with the flagship Hsp90 inhibitors 17-DMAG and radicicol. These findings were extended from proteomics assays, demonstrating that an unrelated antagonist of protein folding potentiates the anti-proliferative effects of AUY922. Results provide a set of candidate biomarkers for responses to AUY922 in leukemia cells, and suggest new modalities for enhancing AUY922’s anti-cancer activities. Early clinical trials have been disappointing with Hsp90 inhibitors. An approach to overcome these pitfalls is to identify compounds with improved Hsp90 inhibitory activity in the cancer cell milieu. To accomplish this objective a panel of cancer cell lines that express luciferase were constructed and an in-cell Hsp90-dependent luciferase refolding assay was developed. The assay was optimized using previously identified Hsp90 inhibitors against prostate, colon and lung cancer cell lines. This assay exhibits good interplate precision, a signal to noise ratio and an approximate Z-factor ranging from 0.5 – 0.7 indicating that the results from the assay are statistically reliable. Subsequently, a pilot screen was conducted, and two compounds, biperiden and ethoxyquin, revealed significant Hsp90 inhibitory activity. Thus, this in-cell Hsp90-dependent luciferase refolding assay is amenable across cancer cell lines for screening of inhibitors in their specific milieu.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sadikot,T., Swink, M., Eskew, J.D., Brown, D., Zhao, H., Kusuma, B.R., Rajewski, R.A., Blagg, B.S.J. Matts, R.L., Holzbeierlein, J.M., and Vielhauer, G.A. (2013) Development of a high-throughput screening cancer cell-based luciferase refolding assay for identifying Hsp90 inhibitors. Assay and Drug Development Technologies 11, 478-488.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Voruganti, S., Lacroix, J.C., Rogers, C.N., Rogers, J., Matts, R.L., Hartson, S.D. (2013) The anti-cancer drug AUY922 generates a proteomics fingerprint that is highly conserved among structurally diverse Hsp90 inhibitors. J. Proteome Res. 12, 3697-706.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). MCF7 breast cancer cells were transfected to ectopically over-express DBC2 to investigate the mechanism through which DBC2 induced cell death. Cell extracts were Western blotted for ubiquitin and PARP1 to test the hypothesis that DBC2-induced polyubiquitination of cellular proteins was followed by apoptosis. A series of site specific mutations were constructed that created phospho-mimetic and non-phosphorylatable tyrosine mutants of Hsp90 and Cdc37. These mutant constructs were ectopically expressed in COS7 cells and the effect of the mutations on Hsp90's and Cdc37's interactions with protein kinases and co-chaperones was assessed by Western blotting. The interaction of Hsp90 and Aha1 with client proteins and co-chaperone partners was assessed in vitro expression of putative clients by coupled transcription/ translation in reticulocyte lysate, and in cultured cells using specific antibodies, pull-down assays, autoradiography, and Western blotting. The Aha1-client complexes formed in transfected cells expressing epitope tagged-Aha1 were analyzed by LC/MS/MS tandem mass spectroscopy and verified by Western blotting. The work will be disseminated by publication in the general scientific literature. PARTICIPANTS: Graduate Students, Jacob Manjarrez Liang Sun; Collaborators, Steve Hartson, Director of OSU DNA/Protein Core Facility trained students in Mass Spec and data analysis, Len Neckers and Wanping Xu, National Cancer Institute, N.I.H. TARGET AUDIENCES: General Scientific Community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
: Ectopic overexpression of DBC2 in MCF7 breast cancer cells induced accumulation of polyubiquitinated proteins. Western blotting indicated that DBC2 over expression was accompanied by cleavage of PARP1 at 36 hours, consistent with the induction of apoptosis. Hsp90 acts to facilitate the folding of protein kinases through a reaction cycle that is driven by the binding and hydrolysis of ATP. This cycle requires the binding and release of Cdc37, as well as Aha1, which stimulates the ATPase activity of Hsp90. How these interactions are regulated is not well understood. Our results are consistent with a model that directionality of the folding process is driven by a series of events involving the phosphorylation of specific tyrosine residues on Hsp90 and Cdc37. Phosphorylation of Cdc37 on Y4 and Y298 disrupts the interaction of Cdc37 with client kinases, while the phosphorylation of Hsp90 on Y197 appears to cause Cdc37 to dissociate from Hsp90. The phosphorylation of Hsp90 on Y313 promotes the recruitment of Aha1, which is known to stimulate Hsp90 ATPase activity, and thus Hsp90's progression through its reaction cycle. Subsequently, phosphorylation of Hsp90 on Y627 phosphorylation induces dissociation of client kinase and remaining co-chaperones completing its reaction cycle. The interaction of Aha1 with Hsp90 during its reaction cycle was characterized. Complexes formed by Aha1 with Hsp90 were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in rabbit reticulocyte lysate. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. The proteins identified to interact with Aha1 suggest that Aha1 plays roles in modulating RNA splicing, DNA repair and nuclear transport, in addition to other cellular processes. These results: 1) increase our understanding of how the DBC2 tumor suppressor gene functions and potentially how its expression or activity can be manipulated to induce apoptosis in cancer cells, 2) expands our knowledge of how Hsp90's ATP-dependent reaction cycle is regulated and how it may be manipulated by small molecule inhibitors; and 3) furthers our understanding of how the Hsp90 co-chaperone Aha1 functions and expands our knowledge of the protein clientele with which Hsp90/Aha1 interact.
Publications
- Sun, L., Prince, T., Manjarrez, J.R., Scroggins, B.T., and Matts, R.L. (2012) Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins. Biochim. Biophys. Acta 1823, 1092-101.
- Xu, W., Mollapour, M., Prodromou, C., Wang, S., Scroggins, B.T., Palchick, Z., Beebe, K., Siderius, M., Lee, M.-J., Couvillon, A., Trepel, J., Miyata, Y., Matts, R., and Neckers, L. (2012) Dynamic tyrosine phosphorylation modulates cycling of the Hsp90-p50cdc37-Aha1 chaperone machine. Molecular Cell 47, 434-43.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). Mutation of critical residues required for the GTP binding activity of DBC2 were created to verify its GTP binding activity and determine the role of GTP binding on its ubiquitin ligase activity in reticulocyte lysate and upon over-expression of DBC2 in HeLa and MCF-7 breast cancer cells was investigation. Identification of DBC2 binding partners was studied using DBC2 and trypsin finger printing of associated proteins by LC/MS/MS. We have screened chemical and natural compound libraries using a high throughput screen (HTS) based on the Hsp90-dependence of the renaturation of heat denatured firefly luciferase in rabbit reticulocyte lysate. Hits were analyzed for their effects on the structure and function of the complexes formed between Hsp90, and its co-chaperones, and client proteins in vitro by coupled transcription/ translation of nascent protein, and in cultured cells using specific antibodies, pull-down assays, and Western blotting. Hits were analyzed for how they altered chaperone activity and the physiology of cultured cells. The structure and function of chaperone-client complexes formed in transfected cells expressing known and novel Hsp90 co-chaperones that were epitope tagged was analyzed by LC/MS/MS tandem mass spectroscopy. A surface plasmon resonance (SPR) assay for binding of novel Hsp90-inhibitors to full length Hsp90 and the C-terminal domain of Hsp90 was developed. The binding site of C-terminal Hsp90 inhibitors was localized by photo-crosslinking of a novobiocin derivative to Hsp90' C-terminal domain, followed by identification of the crosslinked peptide by LC-MS/MS. The mechanism of action of a new generation of C-terminal Hsp90 inhibitors was investigated by LC-MS/MS of cell extracts of inhibitor-treated MCF7 breast cancer cells. The work will be disseminated by publication in the general scientific literature. PARTICIPANTS: Collaborators: Brian S.J. Blagg, Department of Medicinal Chemistry, The University of Kansas Steven, D. Hartson, Department of Biochemistry and Molecular Biology, Oklahoma State University TARGET AUDIENCES: General scientific community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The high throughput screen for natural product inhibitors Hsp90 identified gambogic acid, anthothecol, rottlerin and piperlongumine as potential Hsp90 inhibitors. These natural products inhibitors were demonstrated to block the interaction of Hsp90 and its kinase specific cochaperone Cdc37 with the heme-regulated eIF2alpha kinase (HRI), and block the maturation and activation of HRI in rabbit reticulocyte lysate. Gambogic acid inhibited the proliferation of MCF7 and SKBr3 cells by 50% (IC50) at a concentration of 1 microM, which correlated with its concentration dependence for depletion of cells of Hsp0 dependent clients. SPR analysis indicated the gambogic acid bound to the N-terminal domain of Hsp90 with low microM affinity and at a site distinct from geldanamycin. The effect of Hsp90 inhibitors that bind to its N-terminal ATP binding domain (17-diaminoallyl-geldanamycin/ 17-DMAG) or its C-terminal nucleotide bind domain (KU174 and KU412) on the proteome of MCF7 and SK-Br3 breast cancer cell lines are being analyzed by LC-MS/MS. SPR analysis demonstrated that the C-terminal Hsp90 inhibitor, KU175, bound to Hsp90 with a Kd of 100 microM. LC-MS/MS analysis of tryptic digests of Hsp90 crosslinked to a photo-activatable novobiocin derivative indicated a single polypeptide (KKQEEK) located in Hsp90's C-terminal domain was derivatized. The results from these screens may lead to the identification of better Hsp90-inhibitors for the treatment cancers that are caused by dysregulation of proto-oncogenic protein kinases. These drugs may also have efficacy in the treatment of neurodegenerative diseases and diseases of the cardiovascular system. The identification of the binding site of C-terminal inhibitors of Hsp90 will facilitate the development of more potent drugs that bind to this inhibitory site, in lieu of N-terminal inhibitors that have been shown to have issues with hepato- cardio- and ocular toxicity in clinical trials. The binding of a V191I mutant in the Rho domain of DBC2 to GTP-agarose was competed by excess GTP demonstrating the specificity of GTP binding to this domain. LC-MS/MS of DBC2 complexes reconstituted in vitro demonstrated Hsp90 dependent assembly of DBC2 into a complex containing the Cullin3 E3 ubiquitin ligase and the COP9 signalosome. HeLa cells are resistant to the effects of over-expression of DBC2, while MCF7 are sensitive to its over-expression and undergo apoptosis. LC-MS/MS analysis of DBC2-interacting proteins from HeLa cell extracts indicated that it was associated with stress granules and targeted for degradation by the proteosome. In MCF7 cells DBC2 was associated with components of the Cul3 ubiquitin ligase complex, proteins that modulate transcription and chromatin remodeling, structural components of the cytoskeleton and proteins involved in vesicular transport. These results: 1) expand our knowledge of Hsp90-dependent client proteins; 2) clarify the mechanism of new, potentially clinically useful drugs; and 3) further our understanding of how a tumor suppressor gene functions and potentially how its expression or activity can be manipulated to induce apoptosis in cancer cells.
Publications
- Davenport, J., Manjarrez, J. R., Peterson, L., Krumm, B., Blagg, B. S. J., and Matts, R. L. (2011) Gambogic Acid, a Natural Product Inhibitor of Hsp90 Journal of Natural Products, 74, 1085-1092.
- Matts, R.L., Dixit, A., Peterson, L.B., Sun, L., Voruganti, S., Kalyanaraman, P., Steve D. Hartson, S.D., Verkhivker, G.M., and Blagg, B.S.J. (2011) Elucidation of the Hsp90 C-terminal Inhibitor Binding Site. ACS Chemical Biology 6, 800-807.
- Hartson, S.D. and Matts, R.L. (2011) Approaches for Defining the Hsp90-dependent Proteome. Biochim. Biophys. Acta in press
- Eskew, J.D., Sadikot, T., Morales, P., Duren, A., Dunwiddie, I., Swink, M., Zhang, X., Hembruff, S., Donnelly, A., Rajewski, R.A., Blagg, B.S., Manjarrez, J.R., Matts, R.L., Holzbeierlein, J.M., Vielhauer, G.A. (2011) Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells. BMC Cancer 11, 468.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). DBC2 (aka, RhoBTB2) is a multi-domain protein consisting of a Rho-like GTP binding domain, a poly-proline domain, 2 BTB domains, and a putative ring finger followed by a ser-rich C-terminal tail. DBC2 functions as part of an ubiquitin ligase complex that regulates the breakdown of as yet unidentified proteins. Expression of DBC2 induces apoptosis suggesting that it is a tumor suppressor protein. To better understand the function of this protein, DBC2 was dissected into single and multiple domain constructs containing or lacking interdomain linkers. Mutation of critical residues required for the GTP binding activity of DBC2 were created to verify its GTP binding activity and determine the role of GTP binding on its ubiquitin ligase activity in reticulocyte lysate and upon over-expression of DBC2 in HeLa and MCF-7 breast cancer cells was investigation. Identification of DBC2 binding partners was studied using DBC2 and trypsin finger printing of associated proteins by LC/MS/MS. We have screened chemical and natural compound libraries using a high throughput screen (HTS) based on the Hsp90-dependence of the renaturation of heat denatured firefly luciferase in rabbit reticulocyte lysate. Hits were analyzed for their effects on the structure and function of the complexes formed between Hsp90, and its co-chaperones, and client proteins in vitro by coupled transcription/ translation of nascent protein, and in cultured cells using specific antibodies, pull-down assays, and Western blotting. Hits were analyzed for how they altered chaperone activity and the physiology of cultured cells. The structure and function of chaperone-client complexes formed in transfected cells expressing known and novel Hsp90 co-chaperones that were epitope tagged was analyzed by MALDI-TOF and LC/MS/MS tandem mass spectroscopy. A surface plasmon resonance assay for binding of novel Hsp90-inhibitors to full length Hsp90 and the C-terminal domain of Hsp90 was developed. The work will be disseminated by publication in the general scientific literature. PARTICIPANTS: Collaborators: Brian S.J. Blagg, Department of Medicinal Chemistry, The University of Kansas Steven, D. Hartson, Department of Biochemistry and Molecular Biology, Oklahoma State University TARGET AUDIENCES: General Scientific Community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Hsp90 and accelerated the turnover of Hsp90-dependent proto-oncogenic proteins and inhibit the proliferation of cells were characterized using a systematic protocol. Among the compounds characterized were celastrol, gedunin and sylibin. The best compound identified to date, gambogic acids has an IC50 of 1 microM depleted cells of Hsp0 dependent clients and bound to a site in Hsp90's N-terminal domain distinct from geldanamycin. The effect of Hsp90 inhibitors that bind to its N-terminal ATP binding domain (geldanamycin and radicicol) or its C-terminal nucleotide bind domain (novobiocin and 2 novobiocin analogues) on the differential expression of genes is currently being analyzed MCF-7 cells and compared to the natural product inhibitors curcumin and derrubone. Concentration and time-dependent effects of the drugs were determine and common up- and down-regulated genes were identified. The surface plasmon resonance spectroscopy assay has shown that coumerimycin A, chorobiocin and novobiocin bind to the C-terminal domain of Hsp90 with Kds similar to which they bind full length Hsp90: 0.05, 0.10 and 1.3 mM, respectively, and did not bind to the N-terminal domain of Hsp90. A Novel C-terminal inhibitor of Hsp90 (KU175) was found to bind Hsp90 with a Kd of 100 microM. The results from these screens may lead to the identification of better Hsp90-inhibitors for the treatment cancers that are caused by dysregulation of proto-oncogenic protein kinases. These drugs may also have efficacy in the treatment of neurodegenerative diseases and diseases of the cardiovascular system. The domain of DBC2 that binds to the Hsp90 chaperone machine was localized to the Rho domain, which was also demonstrated to bind GTP. The ubiquitin ligase activity localized to the C-terminal construct containing the second BTB domain, the ring finger domain and the C-terminal Ser rich domain. LC-MS/MS of DBC2 complexes reconstituted in vitro demonstrated Hsp90 dependent assembly of DBC2 into a complex containing the Cullin3 E3 ubiquitin ligase and the COP9 signalosome. Over 150 proteins that specifically interact with Aha1, a co-chaperone of Hsp90, were identified by LC-MS/MS markedly expanding the list of potential Hsp90-dependent clients. These results: 1) expand our knowledge of Hsp90-dependent client proteins; 2) clarify the mechanism new, potentially clinically useful drugs; and 3) further our understanding of how a tumor suppressor gene functions and potentially how its expression or activity can be manipulated to induce apoptosis in cancer cells.
Publications
- Matts, R.L., Brandt, G.E., Lu, Y., Dixit, A., Mollapour, M., Wang, S., Donnelly, A.C., Neckers, L., Verkhivker, G., Blagg. B.S. (2010) A systematic protocol for the characterization of Hsp90 modulators. Bioorg. Med. Chem. In press.
- Zhao, H., Brandt, G.E., Galam, L., Matts, R.L. and Brian S.J. Blagg (2011) Identification and Initial SAR of Silybin: an Hsp90 Inhibitor Bioorg. Med. Chem. Let In press.
- Matthews SB, Vielhauer GA, Manthe CA, Chaguturu VK, Szabla K, Matts RL, Donnelly AC, Blagg BS, Holzbeierlein JM. (2010) Characterization of a novel novobiocin analogue as a putative C-terminal inhibitor of heat shock protein 90 in prostate cancer cells. Prostate 70, 27-36.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). DBC2 (aka, RhoBTB2) is a multi-domain protein consisting of a Rho-like GTP binding domain, a poly-proline domain, 2 BTB domains, and a putative ring finger followed by a ser-rich C-terminal tail. DBC2 functions as part of an ubiquitin ligase complex that regulates the breakdown of as yet unidentified proteins. Expression of DBC2 induces apoptosis suggesting that it is a tumor suppressor protein. To better understand the function of this protein, DBC2 was dissected into single and multiple domain constructs containing or lacking interdomain linkers. The role of Hsp90 and Cdc37 in regulating the GTP binding activity of DBC2 and its ubiquitin ligase activity in reticulocyte lysate and upon over-expression of DBC2 in HeLa and MCF-7 breast cancer cells was investigation. Identification of DBC2 binding partners was studied using DBC2 and trypsin finger printing of associated proteins by LC/MS/MS. We have screened chemical and natural compound libraries using a high throughput screen (HTS) based on the Hsp90-dependence of the renaturation of heat denatured firefly luciferase in rabbit reticulocyte lysate. Hits were analyzed for their effects on the structure and function of the complexes formed between Hsp90, and its co-chaperones, and client proteins in vitro by coupled transcription/ translation of nascent protein, and in cultured cells using specific antibodies, pull-down assays, and Western blotting. Hits were analyzed for how they altered chaperone activity and the physiology of cultured cells. The structure and function of chaperone-client complexes formed in transfected cells expressing known and novel Hsp90 co-chaperones that were epitope tagged was analyzed by MALDI-TOF and LC/MS/MS tandem mass spectroscopy. A surface plasmon resonance assay for binding of novel Hsp90-inhibitors to full length Hsp90 and the C-terminal domain of Hsp90 was developed. The work will be disseminated by publication in the general scientific literature. PARTICIPANTS: P.I. Robert L. Matts Collaborator: Steve Harston, Oklahoma State University- MS analysis Collaborator: Brian Blagg, Department of Medicinal Chemistry, Kansas University- synthesis of Drug derivatives for SAR analysis TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A natural product library from Analyticon of 2,400 compunds has been screened with approximately 50 new inhibitors being identified. The HTS for Hsp90 inhibitors was implemented at the NIH funded Scripps Research Institute Molecular Screening Center and the 300,000 compounds in the NIH Molecular Libraries Small Molecule Repository were screened, and 10 new high affinity scaffolds were identified and are currently being analyzed. The ability of the most promising of these compounds identified in previous years to inhibit Hsp90, accelerated the turnover of Hsp90-dependent proto-oncogenic proteins and inhibit the proliferation of cells were characterized. The best compound identified to date, gambogic acid has an IC50 of 1 microM. The effect of Hsp90 inhibitors that bind to its N-terminal ATP binding domain (geldanamycin and radicicol) or its C-terminal nucleotide bind domain (novobiocin and 2 novobiocin analogues) on the differential expression of genes is currently being analyzed MCF-7 cells. Concentration and time-dependent effects of the drugs were determine and common up- and down-regulated genes were identified. The surface plasmon resonance spectroscopy assay has shown that coumerimycin A, chorobiocin and novobiocin bind to the C-terminal domain of Hsp90 with Kds similar to which they bind full length Hsp90: .05, .10 and 1.3 mM, respectively. Novel C-terminal inhibitors KU135 and KU36 bound with Kds of approximately 2 and 100 microM. The results from these screens may lead to the identification of better Hsp90-inhibitors for the treatment cancers that are caused by dysregulation of proto-oncogenic protein kinases. These drugs may also have efficacy in the treatment of neurodegenerative diseases and diseases of the cardiovascular system. The domain of DBC2 that binds to the Hsp90 chaperone machine was localized to the Rho domain, which was also demonstrated to bind GTP. The ubiquitin ligase activity localized to the C-terminal construct containing the second BTB domain, the ring finger domain and the C-terminal Ser rich domain. Dbc2 was ubiquitinated by multiple ubiquitin mutants, so the type of ubiquitin chain linkage was not identified. Narg1, a subunit of an N-alpha-acetyltransferase complex, was identified as a new DBC2 interacting partner. Suppression of Narg1 activity causes cells to go apoptotic and Narg1 has been reported to be over-expressed in various cancers. These results further our understanding of how a tumor suppressor gene functions and potential how its expression or activity can be manipulated to induce apoptosis in cancer cells.
Publications
- Matts, R.L. and Manjarrez, J.R. (2009) Assays for identification of Hsp90 inhibitors and biochemical methods for discriminating their mechanism of action. Current Topics in Medicinal Chemistry in press
- Hadden, M.K., Hill SA, Davenport J, Matts RL, Blagg BS (2009) Synthesis and evaluation of Hsp90 inhibitors that contain the 1,4-naphthoquinone scaffold. Bioorg. Med. Chem. 17, 634-40.
- Shawna B. Comer, S.B.,Vielhauer, G.A., Manthe, C.A., Chaguturu, V.K., Szabla, K., Matts, R.L., Donnelly,A.C., Blagg, B.S.J., and Holzbeierlein, J.M. (2009) Characterization of a novel novobiocin analogue as a putative C-terminal inhibitor of heat shock protein 90 in prostate cancer cells. Prostate in press
- Shelton, S.N., Shawgo, M.E., Comer, S.B., Lu, Y., Donnelly, A.C., Szabla, K., Tanol, M., Vielhauer, G.A., Rajewski, R.A., Matts, R.L., Blagg, B.S.J., and Robertson, J.D. (2009) KU135, a Novel Novobiocin-derived C-terminal Inhibitor of Hsp90, Exerts Potent Antiproliferative Effects in Human Leukemic Cells. Molecular Pharmacology in press
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). DBC2 (aka, RhoBTB2) is a multi-domain protein consisting of a Rho-like GTP binding domain, a poly-proline domain, 2 BTB domains, and a putative ring finger followed by a ser-rich C-terminal tail. DBC2 functions as part of an ubiquitin ligase complex that regulates the breakdown of as yet unidentified proteins, and the genetics of DBC2 expression suggest that it is a tumor suppressor protein. To better understand the function of this protein, DBC2 has been dissected into single and multiple domain constructs containing or lacking interdomain linkers, and the interaction of each construct with Hsp90 and Cdc37 was studied after generating the protein construct de novo by coupled transcription/ translation in reticulocyte lysate. The role of Hsp90 and Cdc37 in regulating the GTP binding activity of DBC2 and its ubiquitin ligase activity upon over-expression of DBC2 in MCF-7 breast cancer cells is currently under investigation. Renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. We have developed a high throughput screen (HTS) assay based upon this observation to identify Hsp90-inhibitors that obstruct the chaperone activity of Hsp90. Three natural product libraries from TimTec (800 compounds), BioMol (540 compounds) and BioFocus (240 compounds) have been screened with approximately 80 inhibitors being identified. The ability of the most promising of these compounds to inhibit Hsp90, accelerated the turnover of Hsp90-dependent proto-oncogenic proteins and inhibit the proliferation of cells is being characterized. The effect of Hsp90 inhibitors that bind to its N-terminal ATP binding domain (geldanamycin and radicicol) or its C-terminal nucleotide bind domain (novobiocin and 2 novobiocin analogues) on the differential expression of genes were studied in MCF-7 cells. Concentration and time-dependent effects of the drugs were determine and common up- and down-regulated genes were identified. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have identified a new tumor suppressor protein that is recognized by Hsp90 and Cdc37, which may allow us to manipulate the function of the protein and give us a better understanding of how tumor cells transition to highly aggressive metastatic states. While the DBC2 Rho domain has been postulated to not bind GTP, we find that DBC2 and its Rho domain specifically bind to GTP-agarose. The DBC2 Rho domain was found to interact strongly with Hsp90 and Cdc37. Deletion of the Rho domain is accompanied by polyubiquitination of the DBC2 constructs, which required the presence of at least one of the BTB domains, suggesting that the Rho domain regulates its putative ubiquitin ligase activity. We have developed a HTS to identify Hsp90 inhibitors present in chemical scaffold and natural product libraries. The results from these screens may lead to the identification of better Hsp90-inhibitors for the treatment cancers that are caused by dysregulation of proto-oncogenic protein kinases. These drugs may also have efficacy in the treatment of neurodegenerative diseases and diseases of the cardiovascular system. We have identified changes in the differential expression of genes in MCF-7 cells that are specific to the mechanism by which pharmacological agents bind and inhibit Hsp90. Genes common to Hsp90 inhibition were also identified. These findings will allow us to identify more readily the mechanism of action of Hsp90 inhibitory compounds discovered in our HTS screens.
Publications
- Hadden, M.K., Galam, L., Gestwick, J. E., Matts, R.L., and Blagg, B.S.J. (2007) Derrubone, an Inhibitor of the Hsp90 Protein Folding Machinery Journal of Natural Products 70, 2014-8.
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Progress 10/01/06 to 09/30/07
Outputs
The molecular chaperone Hsp90 and its kinase-specific co-chaperone Cdc37 are responsible for facilitating the folding of numerous proto-oncogenic and mutated protein kinases that drive the uncontrolled growth of cancer cells. Drugs that inhibit Hsp90 function are currently in phase II clinical trials for the treatment of various forms of cancer. We have identified a new interacting partner of the Hsp90/Cdc37 chaperone complex, the Deleted-in-Breast Cancer-2 gene product (DBC2). DBC2 functions as part of an ubiquitin ligase complex that regulates the breakdown of as yet unidentified proteins, and the genetics of DBC2 expression suggest that it is a tumor suppressor protein. We have verified that Hsp90/Cdc37 bind DBC2, and the DBC2 interacts with cortactin, a protein that has been implicated in the pathogenesis of human neoplasia. In addition the p21-activated protein kinase PAK1, whose over-expression is associated with aggressive tumors, appears to cause the turnover
of DBC2. DBC2 remains associated with Hsp90 in the presence of the Hsp90-inhibitor geldanamycin, a property consistent with the hypothesis that DBC2 plays a role in regulating the turnover of Hsp90-dependent proto-oncogenic clients. Renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. We have developed a high throughput screen (HTS) assay based upon this observation to identify Hsp90-inhibitors that obstruct the chaperone activity of Hsp90. Approximately 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (avg. Z-factor = 0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microG/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <
10 M and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors. A novel natural product inhibitor of Hsp90, derrubone, was identified during the screen. Derrubone was subsequently demonstrated to inhibit Hsp90 with an IC50 of 0.2 microM, and accelerated the turnover of Hsp90-dependent proto-oncogenic proteins, which correlated with its anti-proliferative effects on cell growth.
Impacts
We have identified a new tumor suppressor protein that is recognized by Hsp90 and Cdc37, which may allow us to manipulate the function of the protein and give us a better understanding of how tumor cells transition to highly aggressive metastatic states. We have developed a HTS to identify Hsp90 inhibitors present in chemical scaffold and natural product libraries. The results from these screens may lead to the identification of better Hsp90-inhibitors for the treatment cancers that are caused by dysregulation of proto-oncogenic protein kinases. These drugs may also have efficacy in the treatment of neurodegenerative diseases and diseases of the cardiovascular system.
Publications
- 1.Galam, L., Hadden, M. K., Ma, Z., Ye, Q.-Z., Yun, B.-G., Brian S. J. Blagg, B.S.J., and Matts, R.L. (2007) High-Throughput Assay for the Identification of Hsp90 Inhibitors Based on Hsp90-Dependent Refolding of Firefly Luciferase. Bioorganic and Medicinal Chemistry 15, 1939-46.
- 2.Matts, R.L. and Caplan, A.J. (2007) Cdc37 and protein kinase folding, in "HEAT SHOCK PROTEINS IN CANCER" (Calderwood, S.K., Ciocca, D. and Michael Sherman, M., eds.) Chapt. 16, pp. 331-350.
- 3.Hadden, M.K., Galam, L., Gestwick, J. E., Matts, R.L., and Blagg, B.S.J. (2007) Derrubone, an Inhibitor of the Hsp90 Protein Folding Machinery Journal of Natural Products in press.
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Progress 10/01/05 to 09/30/06
Outputs
The function of Hsp90 is essential for regulation of a myriad of cellular signal transduction. Protein kinases represent the largest family of Hsp90 clientele, and require the assistance of the Hsp90 co-chaperone Cdc37 to fold into a functional conformation. The interactions of Hsp90 and Cdc37 with the cyclin-dependent kinase, Cdk2 (a kinase previously thought to be Hsp90-independent) were studied to determine structural features of this kinase that interact with these chaperones. Cdk2 was shown to be a genuine client of the Hsp90 chaperone complex, and antibodies directed against the C-helix of Cdk2 disrupted Hsp90 and Cdc37 binding. Using mutants containing deletions of secondary structural elements from Cdk2, we demonstrated that the presence of the G-box motif of Cdk2 was critical for Cdc37 binding, and the stabilization of the E-helix were shown to be needed for Hsp90 binding. c-Jun N-terminal kinases (JNKs) are Hsp90-independent kinases. To investigated what
structural motifs within JNKs that confer or defer Hsp90 and Cdc37, mutants were constructed and utilized to examine the roles that JNK's activation loop, N-terminal non-catalytic structural motif (NSM) and C-terminal non-catalytic structural motif (CSM) play in its ability to function independent of Hsp90 and Cdc37. Both Hsp90 and Cdc37 bound JNK that had its NSM deleted, while only Hsp90 bound JNK that had its CSM deleted. Mutations in JNK's activation loop that are known to constitutively activate or inactivate its kinase activity had no effect on JNK's lack of interaction with Hsp90 and Cdc37. Our findings suggest a novel model by which Cdc37 recognizes features within the NL of kinases, which possibly induces the recruitment of Hsp90. Endothelial nitric oxide synthase (eNOS), via the production of nitric oxide (NO) in the endothelium, plays a key role in cardiovascular biology and is tightly regulated by co- and posttranslational mechanisms, phosphorylation, and protein-protein
interactions. Akt/Hsp90 interaction is dependent on Cdc37. Since both Hsp90 and Akt are key eNOS regulatory proteins, we determined whether Cdc37 interacts with eNOS as part of the Hsp90 regulatory complex. Our results suggest that Cdc37 has a direct regulatory interaction with eNOS and may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation. During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation. Reaper, a key apoptotic regulator, inhibited protein synthesis by binding directly to the 40S ribosomal subunit. This interaction did not affect early initiation events, but instead it interfered with late events in initiation of cap-dependent translation upstream of 60S subunit joining. Reaper's interaction with the ribosome appears to alter start codon recognition during scanning, and these effects on translation are selective for
particular classes of mRNA.
Impacts
Our findings define structural motifs present in kinases are recognized by Hsp90 and Cdc37, and additional structures that hide these motifs, which may allow us to design better inhibitors to treat cancers that are caused by dysregulation of proto-oncogenic protein kinases. The discovery of a Hsp90/Cdc37/Akt/eNOS regulatory complex may allow the development of better drugs to treat diseases of the cardiovascular system. The discovery of new mode of translational inhibition via the virally encoded Reaper protein gives us new insight into a mechanism by which viral infections can cause cell death, and may lead to novel anti-viral therapies.
Publications
- Prince, T., Sun, L., and Matts, R.L. 2005. Cdk2: a genuine protein kinase client of Hsp90 and Cdc37. Biochemistry 44:15287-95.
- Prince,T., and Matts, R.L. 2005. Exposure of Protein Kinase Motifs that Trigger Binding of Hsp90 and Cdc37. Biochemical and Biophysical Research Communications 338:1447-1454.
- Colon-Ramos, D.A., Shenvi, C. L., Weitzel, D. H., Gan, E. C., Matts, R. L., Cate, J. and Kornbluth, S. 2006. Nature Structure and Molecular Biology, Direct ribosomal binding by a cellular inhibitor of translation 13:103-11.
- Harris, M.B., Bartoli, M., Sood, S.G., Matts, R.L., and Venema, R. C. 2006.Direct interaction of the cell division 37 homolog (Cdc37) inhibits endothelial nitric oxide synthase (eNOS) activity. Circulation Research 98:335-41.
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Progress 10/01/04 to 09/30/05
Outputs
The function of the 90 kDa heat shock protein (Hsp90) is essential for the regulation of a myriad of signal transduction cascades that control all facets of a cell's physiology. As derivatives of the Hsp90-inhibitor and tumoricidal agent geldanamycin (GA) move into phase II clinical trials, its potential for triggering adverse effects in non-tumor cell populations requires closer examination. Treatment of differentiating C2C12 myoblasts with GA blocked myogenin expression, inhibited myotubule formation, and led to the depletion of ErbB2, Fyn, and Akt, and induction of apoptosis. GA caused newly synthesized Akt and Fyn to be degraded rapidly, but GA had little effect on the turnover rate of mature Fyn and Akt. Hsp90 maintained an interaction with mature Akt, while Cdc37, Hsp90's kinase-specific co-chaperone, was lost from the chaperone complex upon Akt maturation. GA partially disrupted the interaction of Cdc37 with Akt, but had a much less significant effect on the
interaction of Hsp90 with Akt. Short-term treatment of differentiating C2C12 with GA, novobiocin or okadaic acid increased the phosphorylation of Akt on Ser473. Furthermore, Akt was found to interact directly with catalytic subunit of protein phosphatase 2A (PP2Ac) in C2C12 cells, and this interaction was not disrupted by GA. Total cellular Src (c-Src) protein levels and the turnover rate of newly synthesized c-Src were unaffected by GA. GA also disrupted the interaction of Cdc37 with MyoD. Our findings indicate that Hsp90 functions to balance the phosphorylation state of Akt by modulating the ability of Akt to be dephosphorylated by PP2Ac during C2C12 myoblast differentiation, and that inhibition of Hsp90 caused C2C12 cells to become depleted of multiple signal transduction proteins whose functions are essential for myoblast differentiation, and muscle cell survival, suggesting that GA-derivatives may have the prospective of adversely affecting the physiology of certain sensitive
muscle cell populations in vivo. Pull-down assays and MALDI-TOF analysis were used to identify new Hsp90 co-chaperone partner proteins. A new partnership between Hsp90 chaperone machinery and another chaperone, the 97-kDa Valosin-Containing Protein VCP. Coimmunoadsorption assays indicate that VCP occurs in one or more native heterocomplexes containing Hsp90 and the Hsp90 partner proteins Cdc37, FKBP52, and p23. Functional characterizations indicate that VCP is not an Hsp90 substrate, but rather demonstrates the biochemical hallmarks of an Hsp90 co-chaperone. The activity of the heme-regulated inhibitor of protein synthesis (HRI), an Hsp90 client protein, is activated and suppressed, respectively, by the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD). The effect of hemin, NO and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/deltaHBD) was examined. Hemin stabilized the interaction of the NT-HBD with HRI/deltaHBD, and NO and CO
disrupted, and stabilized this interaction, respectively. Our work demonstrates that HRI's activity is regulated through modulation of the interaction between its NT-HBD and catalytic domain.
Impacts
Our findings indicate that inhibition of Hsp90 caused depletion of multiple signal transduction proteins whose functions are essential for muscle cell differentiation, and muscle cell survival, suggesting that geldanamycin-derivatives that are currently in Phase II clinical trials may have the prospective of adversely affecting the physiology of certain sensitive muscle cell populations in vivo.
Publications
- Yun, B.-G. and Matts R.L. (2005). Hsp90 functions to balance the phosphorylation state of Akt during C2C12 myoblast differentiation. Cellular Signalling 17, 1477-1485.
- Yun, B.-G. and Matts R.L. (2005). Differential effects of Hsp90 inhibition on protein kinases regulating signal transduction pathways required for myoblast differentiation. Exp. Cell Res. 307, 212-223.
- Prince, T., Shao, S., Matts, R.L., and Steven D. Hartson, S.D. (2005). Evidence for chaperone heterocomplexes containing both Hsp90 and VCP Biochem. Biophys. Res. Commun. 331, 1331-7.
- Yun, B.-G., Matts, J.A.B., and Matts, R.L. (2005). Interdomain interactions regulate the activation of the heme-regulated eIF2a kinase Biochim. Biophys. Acta 1725, 174-81.
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Progress 10/01/03 to 09/30/04
Outputs
The heme-regulated eIF2a kinase (HRI) regulates the initiation of translation in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI activates its kinase activity. By utilizing absorption, resonance Raman, EPR and CD spectroscopy, two histidines residues were identified that are crucial for the binding of heme to the NTD. Both His78 and His123 were crucial for stable heme binding, however, the effects of their mutations on the structure of the NTD differed. His78 played the primary role in the specific binding of heme to the NTD, acting analogously to the proximal histidine ligand of globins, while His123 acted as the distal heme ligand. Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. The effect of novobiocin on Hsp90 function was examined to identify structural and functional effects of drug binding to Hsp90's second ATP binding site. Novobiocin inhibited the maturation of HRI in a concentration
dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 co-chaperones p23, FKBP52 and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by ten fold. Recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/co-chaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's auto-kinase activity. The unique structure and properties of novobocin-bound
Hsp90 suggests that it represents the client-release conformation of the Hsp90 machine. Kinase mutations were also constructed to determine features recognized by Hsp90 and its kinase-specific co-chaperone Cdc37. While Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the Lck kinase's catalytic domain, the lobes themselves were not sufficient to trigger Hsp90's high-affinity binding. Only constructs containing a complete N- or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained: the a-C-helix, b4- and b5-strands of the NL through to end of the CL; and the NL through to the a-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the a-C-helix, b4- and b5-strands of this lobe of Lck. Results indicate that the Hsp90's high affinity binding activity is triggered through its
interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the a-C-helix and part of its adjoining loop connection to the b4-strand appear to be the primary determinants recognized by Cdc37.
Impacts
Our findings indicate that: 1) NO and CO may exploited pharmacologically to protein synthesis rates in cells and thus, alter the rate of cell growth, cell viability and differentiation, thus opening new avenues to manipulate the characteristics of aberrant cell populations; and 2) structure-function relationships that regulate the binding of Hsp90 to Cdc37 and protein kinases may be exploited as potential pharmacological targets for intervention in treatment of diseases, such as cancer.
Publications
- Inuzuka, T., Yun, B.-G., Ishikawa, H., Takahashi, S., Hori, H., Matts, R.L., Ishimori, K., Morishima, I. (2004)Identification of Crucial Histidines for Heme Binding in the N-terminal Domain of the Heme-regulated eIF2a Kinase. J. Biol. Chem. 279, 6778-6782.
- Yun, B.-G., Huang, W., Leach, N., Hartson, S.D., and Matts, R.L. (2004) Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions. Biochemistry 43, 8217-29.
- Prince, T. and Matts, R.L. (2004) Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37. J. Biol. Chem. 279, 39975-39981.
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Progress 10/01/02 to 09/30/03
Outputs
The heme-regulated eIF2 a kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing multiple spectroscopic approaches, histidines residues have been identified the heme binding ligans of the NTD. The Hsp90 co-chaperone Cdc37 is essential for the biogenesis of numerous protein kinases. Our work demonstrates that Cdc37 is phosphorylated on Ser13by casein kinase II. Mutation of Ser13 to either Ala or Glu compromised the recruitment of Cdc37 to Hsp90-kinase complexes, but only modestly affected its binding to Hsp90. Cdc37 containing the complementing Ser to Glu mutation showed altered interactions with Hsp90-kinase complexes consistent with compromised Cdc37 modulation of Hsp90s ATP-driven reaction cycle. The data indicate that phosphorylation of Cdc37 on Ser13 is critical for its ability to coordinate kinase and Hsp90 binding.
Structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with the Hsp90-dependent kinase HRI were examined. Our data indicate that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37s middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase. Functional assays suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90s conformation and thereby mediate Hsp90s regulation of Cdc37s kinase-binding activity. Scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to HRI. Our results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules. Crystal
structures of protein kinases were used to guide the dissection of two kinases. the Src-family tyrosine kinase Lck and HRI, and to assess the association of Hsp90 and Cdc37 with protein kinases. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated stable salt-resistant chaperone-client complexes. Dissections identified a specific kinase motif that triggers Hsp90s conformational switching to a high-affinity client binding state and kinase structures that interact with Hsp90 co-chaperones. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of
multiple client segments is communicated to generate or stabilize high-affinity chaperone-client complexes.
Impacts
Our findings indicate that: 1) NO and CO may exploited pharmacologically to protein synthesis rates in cells and thus, alter the rate of cell growth, cell viability and differentiation, thus opening new avenues to manipulate the characteristics of aberrant cell populations.; and 2) structure-function relationships that regulate the binding of Hsp90 to Cdc37 and protein kinases may be exploited as potential pharmacological targets for intervention in treatment of diseases, such as cancer.
Publications
- Ishikawa, H., Yun, B.-G., Takahashi, S., Hori, H., Matts, R.L., Ishimori, K. and Morishima, I. (2002) NO-induced activation mechanism of the heme-regulated eIF2alpha kinase. J. Amer. Chem. Soc. 124, 13696-13697.
- Shao, J., Prince, T., Hartson, S.D. and Matts, R.L. (2003) Phosphorylation of Serine-13 is Required for the Proper Function of the Hsp90 Co-chaperone, Cdc37. J. Biol. Chem. 278, 38117-20.
- Scroggins, B.T., Shao, J., Prince, T., Uma, S., Huang, W., Guo, Y., Hedman, H., Robert L. Matts, R.L. and Hartson S.D. (2003) High Affinity Binding of Hsp90 is Triggered by Multiple Discrete Segments of Its Kinase Clients Biochemistry 42, 12550-61.
- Shao, J., Irwin, A., Hartson, S.D. and Robert L. Matts (2003) Functional dissection of Cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding. Biochemistry 42, 12577-88.
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Progress 10/01/01 to 09/30/02
Outputs
Layer-by-layer assembly of collagen films of controlled thickness over nano particle films were found to be biocompatible. Layer-by-layer assembled ion-selective and biocompatible films of TiO2 nanoshells were demonstrated to shown promise for neurochemical monitoring. Thus, films made of a number of types of nanoparticles , some of which required coating with collagenfor biocompatibility, were compatible with the growth and viability of cells, indicating that they may have utility in the engineering of biological-based prosthetics and biocompatible implantable monitoring devices . The maturation and activation of the heme-regulated inhibitor of protein synthesis (HRI) in reticulocytes requires its functional interaction with Hsp90. We demonstrated that protein phosphatase 5 (PP5) is associated with HRI maturation intermediates. The interaction of PP5 with HRI was mediated through Hsp90, as mutants of PP5 that do not bind Hsp90 do not interact with HRI. PP5 was
present in Hsp90 complexes containing p50cdc37 and expression of newly synthesized HRI enhanced the amount of p50cdc37 associated with Hsp90/PP5-HRI complexes. The functional significance of the interaction of PP5 with Hsp90-HRI complexes was examined by characterizing the effects of compounds that impact PP5 activity in vitro. The phosphatase inhibitors okadaic acid and nodularin enhanced the activity of HRI when applied during HRI maturation/activation, while the PP5 activators arachidonic and linoleic acid repressed HRI activity when applied during HRI maturation/activation. However, application of these compounds after HRI's 'transformation' to an Hsp90-independent form did not similarly impact HRI's kinase activity. The Hsp90-inhibitor geldanamycin negated the effects of phosphatase inhibitors on HRI maturation/activation. The finding that PP5 down-regulates an Hsp90-dependent process supports models for regulated Hsp90 function and describes a novel potential substrate for PP5
function in vivo. The ability of two high-affinity Hsc70-binding peptides [FYQLALT (pep-_) and NIVRKKK (pep-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both pep-_ and pep-K inhibited chaperone-dependent renaturation of luciferase in RRL. Pep__, but not pep-K, blocked Hsp90/Hsc70-dependent transformation of HRI into an active, heme-regulatable kinase. In contrast, pep-K, but not pep__, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. HDJ2, but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Pep-K inhibited, while pep__ enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. Thus, pep__ acts to inhibit Hsc70 function by binding to the hydrophobic peptide binding cleft of Hsc70, while pep-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. The data indicate that pep__ and pep-K have differential
effects on Hsc70 functions, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.
Impacts
Our findings indicate that: 1) nanoparticles films are biocompatible, indicating that they will utility in the engineering of prosthetics and biocompatible implantable monitoring devices; and 2) pharmacologic manipulation of the function of the Hsp90 "signal-transduction" chaperone complex may represent a novel pharmacological target for intervention in treatment of diseases,such as cancer.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
p50cdc37 facilitates Hsp90-mediated biogenesis of certain protein kinases. We have examined whether p50cdc37 is required for the biogenesis of the heme-regulated eIF2alpha kinase (HRI). p50cdc37 interacted with nascent HRI cotranslationally and this interaction persisted during HRI biogenesis. p50cdc37 stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50cdc37 function was specific to immature and inactive forms of the kinase. Analysis of cdc37 mutants indicated that the N-terminal portion of p50cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50cdc37 with Hsp90. A C-terminally truncated mutant of p50cdc37 bound HRI and inhibited HRI's activation by preventing the interaction of
Hsp90 with HRI. These results suggest that p50cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90. The relationship between the molecular chaperone Hsp90 and the signal transducing capacity of the Src-family kinase Hck was investigated. Inhibition of Hsp90 with geldanamycin suppressed the ability of bacterial lipopolysaccharide to enhance the cell adhesion properties of macrophages, a phenomenon explained by the reduced expression and activity of Hck in macrophages lacking Hsp90 function. The contribution of Hsp90 to signal transduction by Hck was dissected further by examining its role in the de novo folding and maintenance of wildtype (wt)Hck and its constitutively active counterpart Hck499F. The folding of nascent wtHck and Hck499F into catalytically active conformations and their accumulation in cells were dependent on Hsp90 function. Mature Hck499F had a greater requirement for
on-going support from Hsp90 than did mature wtHck. This particular finding might have important implications for our understanding of the evolution of oncogenic protein kinases. Nitric oxide (NO) inhibits protein synthesis in eukaryotic cells by increasing the phosphorylation of the alpha subunit of eukaryotic initiation factor eIF2. However, the mechanism through which this increase occurs has not been characterized. We have examined the effect of the diffusible gases nitric oxide (NO) and carbon monoxide (CO) on the activation of the heme-regulated eIF2alpha kinase (HRI) in rabbit reticulocyte lysate. Spectral analysis indicated that NO and CO bind to the N-terminal heme-binding domain (HBD) of HRI. While NO was a very potent activator of HRI, CO markedly suppressed NO-induced HRI activation. The NO-induced activation of HRI was transduced through the interaction of NO with the HBD of HRI. The regulation of HRI activity by diffusible gases may be of wider physiological significance,
as NO-generators increase eIF2alpha phosphorylation levels in NT2 neuroepithelial and C2C12 myoblast cells and activate HRI immunoadsorbed from extracts of these non-erythroid cells lines.
Impacts
Our findings indicate that: 1) molecular chaperones may facilitate the evolution of oncogenic protein kinases and suggest that chaperones are good targets for the development of new therapeutic agents for treating cancer; and 2) the heme-regulated eIF2alpha kinase is a target for regulation by nitric oxide (NO), and may play a role in disease states caused by chronic elevation of NO levels.
Publications
- Shao, J., Grammatikakis, N., Scroggins, B.T., Uma, S., Huang, W., Chen, J.-J., Hartson, S.D., and Matts, R.L. (2001) Hsp90 Regulates p50cdc37 Function during the Biogenesis of the Active Conformation of the Heme-regulated eIF2alpha Kinase. J. Biol. Chem. 276, 206-214 .
- Uma, S., Yun, B.-G., and Matts, R.L. (2001) The heme-regulated eIF2alpha kinase: A potential regulatory target for control of protein synthesis by diffusible gases. J. Biol. Chem. 276, 14875-14883.
- Scholz, G.M., Hartson, S.D., Cartledge, K., Volk, L., Matts, R.L., and Dunn, A.R. (2001) The molecular chaperone Hsp90 is required for signal transduction by wildtype Hck and maintenance of an oncogenic mutant of Hck. Cell Growth and Differentiation 12, 409-417.
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Progress 10/01/99 to 09/30/00
Outputs
Protein synthesis in heme-deficient reticulocytes is inhibited by activation of heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI). HRI contains two distinct heme-binding sites. The N-terminal domain (NTD) of the HRI has sequence similarity to mammalian alpha-globins. Recombinant NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI. The kinase activity of two N-terminally truncated mutant of HRI was 50% less than wild type HRI and less sensitive to inhibition by heme. Heme binding of the individual HRI domains showed that the kinase insertion also bound hemin, whereas the C terminus and the catalytic domains did not. The cleavage of eIF2alpha in apoptotic HeLa cells was inhibited by the caspase inhibitor Z-VAD-fmk. In vitro analysis, showed efficient cleavage of eIF2alpha by caspase 3. Only caspase 3
was capable of cleaving eIF2alpha in the eIF2/2B complex. The eIF2/GDP binary complex was not cleaved by caspase 3. Cleavage of eIF2alpha resulted in functional alteration of the eIF2 complex: exchange of GDP bound to eIF2 was no longer dependent upon eIF2B; cleaved eIF2 no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site; and cleaved eIF2 no longer stimulated overall translation. The benzoquinoid ansamycins geldanamycin (GA), herbimycin, and their derivatives are emerging as novel therapeutic agents that act by inhibiting the 90-kDa heat shock protein hsp90. GA inhibited the proliferation of mitogenactivated T cells. GA was toxic to both resting and activated T cells; activated T cells were especially vulnerable. GA treatment depleted the levels of the hsp90-depedent kinase lck in cultuted T-cells. Pulse-chase analyses indicated that GA induces rapid degradation of newly synthesized lck molecules, but not mature lck populations. These
results correlate with slow global losses in lck activity and an inability to respond to TCR stimuli. Cdc37 provides a poorly understood biochemical function essential to certain hsp90-dependent protein kinasess. Cdc37 was recovered in complexes with hsp90 cohorts. Maturation intermediates of the lck and HRI increased the amount of Cdc37 and hsp90 in FKPB52 complexes. Complexes between hsp9 and Cdc37 were salt-labile, but their interaction with kinase substrates was salt-resistant. GA did disrupt the association of hsp90 with Cdc37, but caused formation of salt-labile hsp90-kinase complexes which lacked Cdc37. A temperature-sensitive mutant of the Hck kinase (tsHck499F) was created. Overexpression of Cdc37 rescued the catalytic activity of tsHck499F, and partially buffering it against inactivation at high temperatures. Hsp90 was required for tsHck499F activity and its stabilization by Cdc37, but overexpression of Hsp90 was not sufficient to stabilize tsHck499F. Overexpression of Cdc37
promoted the association of tsHck499F with Hsp90. Results indicate that Cdc37 does not simply serve as a passive structural bridge between hsp90 and its kinase substrates.
Impacts
Our findings indicate that: 1) pharmacologic inhibition of the function of the 90-kDa heat shock protein "signal-transduction" super-chaperone complex may represent a novel immunosuppressant strategy; and 2) have implications for our understanding of the evolution of protein kinases and tumor development, and thus, treatment of cancers.
Publications
- Yorgin, P. D., Hartson, S. D., Fellah, A. M., Scroggins, B. T., Huang, W., Katsanis, E., Couchman, J. M., Matts, R. L. and Whitesell, L. (2000) Effects of Geldanamycin, an Hsp90-binding Agent, on T-Cell Function and T-Cell Non-Receptor Protein Tyrosine Kinases. J. Immunology 164, 2915-2923.
- Uma, S., Matts, R. L., Guo, Y., White, S., and Chen, J.-J. (2000) The N-terminal Region of the Heme-regulated eIF-2a Kinase is an Autonomous Heme-binding Regulatory Domain Eur. J. Biochem. 267, 498-506.
- Rafie-Kolpin, M., Chefalo, P. J., Hussain, Z., Hahn, J., Uma, S., Matts, R. L. and Chen, J.-J. (2000) Two Heme-Binding Domains of Heme Regulated eIF-2a Kinase: N-terminus and Kinase Insertion J. Biol. Chem. 275, 5171-5178.
- Marissen, W., Guo, Y., Thomas, A., Matts, R. L. and Lloyd, R. E. (2000) Identification of Caspase 3-Mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2a in Apoptosis. J. Biol. Chem. 275, 9314-9323.
- Hartson, S.D., Irwin, A.D., Shao, J., Scroggins, B.T., Volk, V., Huang, W., and Matts, R.L. (2000) p50cdc37 is a Non-exclusive Hsp90 Cohort Which Participates Intimately in Hsp90-mediated Folding of Immature Kinase Molecules. Biochemistry 39, 7631-7644.
- Scholz, G., Hartson, S.D., Cartedge, K., Hall, N., Shao, J., Dunn, A. R. and Matts, R.L. (2000) p50cdc37 can Buffer the Temperature-sensitive Properties of a Mutant of Hck. Mol. Cell. Biol. 20, 6984-6995.
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Progress 10/01/98 to 09/30/99
Outputs
Pharmacologic inhibition of Hsp90 with the ansamycin antibiotic and anti-tumor agent geldanamycin disrupted somite development in zebrafish. Our data are consistent with there being a temporal and spatial requirement for Hsp90 function within somitic cells which is necessary for the formation of muscle pioneers and possibly other striated muscle fiber types. To examine the biochemical mechanism by which Hsp90 exerts its essential positive function on certain signal transduction proteins, we characterized the effects of molybdate and geldanamycin on Hsp90 function and structure. Molybdate inhibited hsp90-mediated p56lck biogenesis and luciferase renaturation while enforcing salt-stable interactions with these substrates. Molybdate also reduced the amount of free Hsp90 present in cell lysates, inhibited hsp90's ability to bind geldanamycin, and induced resistance to proteolysis at a specific region within the C-terminal domain of hsp90. In contrast, the Hsp90 inhibitor
geldanamycin prevented Hsp90 from assuming natural or molybdate-induced conformations that allow salt-stable interactions with substrates. When these compounds were applied sequentially, the order of addition determined the effects observed, indicating that these agents had opposing effects on Hsp90. The results indicate that a specific region within the C-terminal domain of Hsp90 (near residue 600) determines the mode by which hsp90 interacts with substrates and that the ability of Hsp90 to cycle between alternative modes of interaction is obligatory to Hsp90 function. The heme-regulated eIF-2 kinase (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme-deficiency, heat shock and oxidative stress. Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to
acquire and maintain a conformation that is competent to activate. The role of Hsc70 in the regulation of HRI biogenesis and activation was examined. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation. Transformation of HRI into an active kinase was inhibited by the Hsc70-antagonist clofibric acid. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI, resulting in the hyperactivation of HRI. Activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase, but is subsequently required to negatively attenuate the activation of transformed HRI.
Impacts
(N/A)
Publications
- Lele, Z., Hartson, S. D., Martin, C. C., Whitesell, L., Matts, R. L. and Krone, P. H. (1999) Disruption of zebrafish somite development by pharmacologic inhibition of Hsp90. Development al Biology 210, 56-70.
- Hartson, S. D., Thulasiraman, V., Huang, W., Whitesell,. L. and Matts, R. L. (1999) Molybdate Inhibits Hsp90, Induces Structural Changes in Its C-terminal Domain, and Alters Its Interactions with Substrates Biochemistry 38, 3837-3849.
- Uma, S., Thulasiraman, V., and Matts. R. L. (1999) Dual role for Hsc70 in the biogenesis and regulation of the heme-regulated eIF-2a kinase. Mol. Cell. Biol. 19, 5861-5871.
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Progress 10/01/97 to 09/30/98
Outputs
The de novo folding of the individual domains of the src-family kinase p56lck was examined in rabbit reticulocyte lysate (RRL). Results indicate that during p56lck biogenesis, the SH2 domain rapidly folds independently of hsp90 function, followed by the slower hsp90-dependent folding of the catalytic domain, and suggest the final stabilization of p56lck structure occurs by phosphorylation-mediated interdomain interactions. Changes in the expression of the heme-regulated eIF-2a kinase (HRI), heat shock proteins (Hsps) and several of their associated cohorts were studied in maturing rabbit reticulocytes during recovery from anemia induced by injection of N-acetylphenylhydrazine or by phlebotomy. Northern and western blot analysis indicated that HRI and hsp mRNA and protein content gradually decreased during maturation of reticulocytes into erythrocytes. Low hematocrits correlated with high levels of hsp expression and with increasing hematocrits, hsp expression
decreased. The role of the heat shock cognate protein, Hsc70, in regulating the activity of the HRI in hemin-supplemented RRL in response to heat and oxidative stress was examined and compared to the effect of Hsc70 on HRI activation in response to heme-deficiency. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficient RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress. The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI.
Impacts
(N/A)
Publications
- Hartson, S. D., Ottinger, E. A., Huang, W., Barany, G., Burn, P., and Matts, R. L. (1998) Modular Folding and Evidence for Phosphorylation-induced Stabilization of an Hsp90-dependent Kinase J. Biol. Chem. 273, 8475-8482.
- Uma,S., Barret, D. J. and Matts, R. L. (1998) Changes in the Expression of the Heme-regulated eIF-2a Kinase and Heat Shock Proteins in Rabbit Reticulocytes Maturing during Recovery from Anemia. Exp. Cell Res. 238, 273-282.
- Thulasiraman, V., Xu, Z., Uma, S., Gu, Y., Chen, J.-J., and Matts, R. L. (1998) Evidence that Hsc70 Negatively Modulates the Activation of the Heme-regulated eIF-2a Kinase in Rabbit Reticulocyte Lysate. Eur. J. Biochem. 255, 552-562.
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Progress 10/01/96 to 09/30/97
Outputs
We examined the interaction of Hsp90 and its cohorts with the heme-regulated eIF-2a kinase (HRI) in rabbit reticulocyte lysate (RRL). The activation of HRI in response to heat and oxidative stress is not mediated by dissociation of Hsp90 from HRI, and inhibition of HRI activity by hemin is not mediated by its reassociation with Hsp90. The role of hsp90 in the maturation of newly synthesized HRI was examined. Hsp90 interacted with nascent HRI cotranslationally.Incubation of HRI in heme-deficient RRL resulted in the transformation of a portion of the HRI into an active heme-regulatable kinase. Transformation of HRI was dependent on autophosphorylation, transformed HRI did not bind Hsp90 and transformed HRI was resistant to stress-induced aggregation. The Hsp90 binding drug geldanamycin disrupted the interaction of Hsp90 with HRI, inhibited the maturation of HRI into a form that was competent to activate, and inhibited the transformation of HRI into a stable
heme-regulatable kinase. Thus, Hsp90 plays an obligatory role in HRI acquiring and maintaining a transformation-competent conformation. The mechanism through which cisplatin inhibits protein synthesis in RRL was characterized. Polyribosome profiles and northern blot analysis of cisplatin-inhibited RRL indicated that cisplatin arrests the elongation stage of protein synthesis by causing extensive derivatization of RNA in translating ribosomes. We suggest that elongation arrest contributes to the cytotoxic action of cisplatin.
Impacts
(N/A)
Publications
- UMA, S., HARTSON, S. D., CHEN, J.-J., AND MATTS, R. L. (1997) Hsp90 is Obligatory for the Heme-regulated eIF-2a Kinase to Acquire and Maintain an Activatable Conformation J. Biol. Chem. 272, 11648-11656
- XU, Z., PAL, J. K., THULASIRAMAN, V., HAJIN, H. P., CHEN, J.-J., AND MATTS, R. L. (1997) The Role of the 90-kDa Heat Shock Protein and its Associated Cohorts in Stabilizing the Heme-Regulated eIF-2a Kinase in Reticulocyte Lysates during Heat Stress.Eur. J. Biochem 246, 461-470.
- HEMINGER, K. A., ROGERS, J., HARTSON,S. D., AND MATTS, R. L. (1997) Cisplatin Inhibits Protein Synthesis in Rabbit Reticulocyte Lysate by Causing an Arrest in Elongation. Arch. Biochem. Biophys. 344,
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Progress 10/01/95 to 09/30/96
Outputs
cDNAs encoding the e-subunit of eIF-2B were isolated from rabbit, human and rat cDNA libraries. Rat genomic sequences coding for eIF-2Be were isolated. The nucleotide and amino acid sequences were highly conserved. Northern and western blot analyses of rabbit and rat tissue extracts indicated that eIF-2Be was ubiquitously expressed. Part of the anti-proliferative effect of interferon (IFN) is due to its modulation of translation. A transient autocrine production of IFN, activation of the double-stranded RNA dependent eIF-2a kinase, diminished eIF-2B activity and reduced translation were observed to occur during specific periods of growth in differentiating mouse 3T3 cells. Hemin-induced differentiation of K562 cells converted polio virus infection from persistent to lytic. Differentiation is accompanied by the induction of the synthesis of heat shock proteins (HSP). Induction of HSP synthesis was not the cause of the lytic phase, as other inducers of the heat shock
response did not convert polio virus infection of K562 cells from persistent to lytic. Hsp90 function was found to be absolutely required for the folding of the lymphoid cell p56lck kinase into an active conformation in vivo and in vitro. Hsp90 function was also required to maintain p56lck in an active form. The hsp90 inhibitor geldanamycin was demonstrated to disrupt the normal interaction of hsp90 with denatured luciferase and inhibit its renaturation. The mechanism of this effect was analyzed.
Impacts
(N/A)
Publications
- Hartson, S.D., Barrett, D.J., Burn, P., and Matts, R.L. 1996. Hsp-90-mediated Folding of the Lymphoid Cell Kinase p56lck. Biochemistry 35: 13451-15459.
- Asuru, A.I.,Mellor, H., Thomas, N.S.B., Yu, L., Chen, J.J., Crosby, J.S., Harson, S.D. Kimball, S.R., Jefferson, L.S., and Matts, R.L. 1996. Cloning and Characterization of cDNAs Encolding the e-Subunit of Eukaryotic Initiation Factor-2B fr
- Flowers, K.M., Mellor, H., Matts, R.L., Kimball, S.R., and Jefferson, L.S. 1996.Cloning and Characterization of Complementary and Genomic DNAs Encolding the e-subunit of the Rat Translation Initiation Factor-2B. Biochim. Biophys. Acta 1307
- Petryshyn, R., Chen, J.J., Daniel, L., and Matts, R.L. 1996. Effect of Inteferonon Protein Translation During Growth Stages of 3T3 Cells. Arch. Biochem. Biophys. 328: 209-297.
- Benton, P.A., Barret, D.J., Matts, R.L., Lloyd, R.E. 1996. Hemin-induced Differentiation of K562 Cells Converts Poliovirus Infections from Persistent to Lytic. J. Virology 70: 5525-5532.
- Thulasiraman, V. and Matts, R.L. 1996. Effect of Geldanamycin on the Kinetics of Chaperone-mediated Renaturation of Firefly Luciferase in Rabbit Reticulocyte Lysate 35: 13443-13450.
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Progress 10/01/94 to 09/30/95
Outputs
A cDNA was isolated from a rabbit reticulocyte cDNA library determined to encodethe e-subunit of eukaryotic initiation factor eIF-2B. The cDNA was used as a probe to isolate partial cDNAs coding for human and rat eIF-2Be, and genomic sequences coding for rat eIF-2Be. The nucleotide and amino acid sequences were highly conserved between the rabbit, rat and human cDNAs, showing 90% sequence identity within the open reading frame. Northern and western blot analyses of rabbit and rat tissue extracts indicated that eIF-2Be was ubiquitously expressed. A transient autocrine production of IFN during specific periods of growth of mouse 3T3 cells was observed. Treatment of pre-confluent mouse 3T3 cells with IFN activated of the double-stranded RNA dependent eIF-2a kinase, and reduced translation in these cells. This translational inhibition was associated with diminished eIF-2B guanine nucleotide exchange activity. Complexes between hsp90 and the cellular src-family kinase
p56lck were detected in cytosolic lysates of LSTRA cells. In fibroblasts transformed by a lck gene, the hsp90-inhibitor geldanamycin reduces expression of p56lck kinase activity by an indirect mechanism specifically reverts transformation. In reticulocyte lysate, geldanamycin disrupts the stable interaction of hsp90 with nascent p56lck and leads to production of kinase molecules which lack tyrosine kinase activity and are hypersensitive to limited proteolysis.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
An assay for chaperone-mediated protein folding in rabbit reticulocyte lysate was developed. Dilution of thermally denatured firefly luciferase into reticulocyte lysate results in the ATP-dependent recovery of >60% of the enzyme activity. Renaturation was blocked by heat shock or the addition of denatured proteins. Reconstitution of the activity with purified components indicates that the chaperoning activity involves the cooperative action of hsp70 and hsp90. To overcome limitations inherent to whole-cell systems or reconstitution procedures, we have synthesized nascent cellular src-family kinases in rabbit reticulocyte lysate to examine the role for hsp90 in modulating the structure and function of these kinases. Following their synthesis, fast- and slow-sedimenting forms of monocyte-specific p59(superscript fgr), B cell-specific p59(superscript fgr), and p56(superscript lck) can be separated on glycerol gradients. Anti-hsp90 monoclonal antibodies co-immunabsorb the
fast-sedimenting, but not the slow-sedimenting forms of these kinases. Additionally, anti-p56(superscript lck) antibodies specifically co-immunoabsorb hsp90 from protein synthesis reactions programmed with lck RNA. The fast sedimenting, hsp90-bound form of p56(superscript lck) is deficient in autophosphorylation activity and phosphorylates an exogenous substrate, acid-treated enolase, less efficiently that does the monomeric form.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
The heme-regulated eIF-2(alpha) kinase (HRI) is activated in heme-deficient reticulocyte lysates as well as in hemin-supplemented lysates treated by heat-shock or N-ethylmaleimide (NEM). Heat shock proteins, hsp90, hsp70 and p59, interact with HRI; the association of HRI with hsp90 and p59, but not hsp70, is dependent on hemin. The association of hsp70 with HRI in hemin-supplemented reticulocyte lysates is blocked upon activation of HRI in response to heat shock or addition of denatured proteins, while the association of HRI with hsp90 is strengthened during heat-shock or NEM-treatment. These findings indicate that dissociation of hsp90 form HRI, which occurs during heme-deficiency, is not a prerequisite for HRI activation. In the presence of hemin the addition of hsp90 to HRI stimulates of its eIF-2(alpha) kinase activity in vitro. An ATP-dependent assay for chaperone-mediated protein folding was developed in reticulocyte lysate. Heat shock or addition of denatured
proteins inhibited refolding of denatured luciferase. Purified hsp90 and hsp70 along with an ATP-regenerating system refolded luciferase to greater than 20% of its original activity. Stress-induced accumulation of denatured protein apparently sequesters hsp70, inhibiting chaperone-mediated renaturation of luciferase, leading to the activation of HRI. The interaction of hsp90 with HRI apparently facilitates HRI activation under these conditions.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants and heat stress. Activated HRI inhibits translation by catalyzing the phosphorylation of the (alpha)-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the arrest of translation in response to heat stress is uncharacterized. HRI interacts with the 90 and 70 kDa heat shock proteins (hsp), and a 56-kDa protein (p56) in hemin-supplemented lysate. We now demonstrate that denatured proteins induce the activation of HRI and inhibit protein synthesis in hemin-supplemented rabbit reticulocyte lysate. Denatured bovine serum albumin (BSA), but not native BSA, binds hsp 70 and blocks the interaction of hsp 70 with HRI, suggesting that the interaction of hsp 70 with HRI is required to maintain HRI in an inactive conformation. The association of
hsp 90 and p56 with HRI was maintained during heat stress and in the presence of denatured protein. We proposed that denatured protein, through its capacity to sequester hsp 70, may be the "signal" that leads to inhibition of translation in response to heat shock. The 82-kDa subunit of the eukaryotic initiation factor (eIF-2B), which is responsible for the recycling of eIF-2 has been cloned and sequenced.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
Translational inhibition in rabbit reticulocyte lysate occurs in response to heme-deficiency, addition of oxidants (toxic metal ions), and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which phosphorylates the (alpha)-subunit of the eukaryotic initiation factor eIF-2. HRI was demonstrated to be present in hemin-supplemented reticulocyte lysate in a complex with heat shock protein (hsp) 90, hsp 70 and a 56-58 kDa protein (p56). p56 reacted with the EC1 monoclonal antibody, which recognizes a 56-59 kDa protein that is also associated with several steroid hormone receptor.hsp 90 complexes. The association of HRI with hsp 90 and p56 was hemin-dependent, while hsp 70 was associated with HRI in both heme-deficient and hemin-supplemented lysates. The immunological properties of hsp 70 indicated that it is the hsc 73 member of the hsp 70 protein family. The extent to which translation was inhibited in
hemin-supplemented lysates in response to heat or oxidative stress correlated inversely with the relative levels of hsp 70 and p56 present in lysates. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates correlated roughly with the levels of hsp 90 present.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Toxic heavy metal ions inhibit protein synthesis in rabbit reticulocyte lysates. The inhibition of protein synthesis occurs due to the activation of the heme-regulated protein kinase (HRI), which specifically phosphorylates the alpha-subunit of eukaryotic initiation factor eIF-2. Heavy metal ions were found to inhibit the capacity of reticulocyte lysates to reduce disulfide bonds. This inhibition was not due to the depletion of lysate glu-6-P, or NADPH. The inhibition has characteristics of the metal ions binding directly to vicinal sulfhydryl groups, possibly those present in the active site of thioredoxin and/or thioredoxin reductase. Addition of metallothioneins (MT) and their apoproteins (apoMT) have been demonstrated to protect or restore protein synthesis in rabbit reticulocyte lysates in a manner which reflects their metal binding properties derived in vito. ApoMTs in particular are capable of restoring proteins synthesis in heavy metal ion inhibited lysates
upon reactivation of the lysates capacity to reduce disulfide bonds, which results in the inactivation of HRI. Binding of metal ions to the alpha-domain of MT correlates with their ability to detoxify heavy metal ions. Recent results suggest that HRI interacts with a complex of proteins consisting of hsp 90, hsp 70, and p59.
Impacts
(N/A)
Publications
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Progress 10/01/88 to 09/30/89
Outputs
The effect of toxic heavy metal ions (Hg,Pb and Cd protein synthesis in hemin-supplemented rabbit reticuloctee lysates was further characterized. Hg was found to inhibit reticulocyte thioredoxin/thioredoxin reductase activity, as measured by the inability of Hg-treated lysates to reduced insulin. Apomentallothionein I and II were found to restore thioredoxin/thioredoxin reductase activity in Hg-treated lysates in conjunction with restoration of protein synthesis. The observations that the addition of glucose-6-phosphate to Hg-treated lysates was found to have no protective or restorative effect on protein synthesis, and the level of NADPH present was not found to vary significantly from the control, suggest that Hg may have a direct inhibitory effect on the reticulocyte lysate thioredoxin/thioredoxin reductase system. An active thioredoxin/thioredoxin reductase system is known to be required for the maintenance of active protein synthesis in the reticulocyte lysate.
The mechanism by which the reticulocyte lysate heme-regulated eLF-2a kinase (HRI) is activated in response to toxic heavy metal ions and oxidative stress in currently being investigated. Data obtained using monoclonal antibody probes suggest that HRI is associated with the 90 Kda heat shock protein (hsp90) in an inactive complex in hemin-supplemented lysates, and dissociates from hsp90 upon activation.
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Progress 10/01/87 to 09/30/88
Outputs
The effect of toxic heavy metal ions on protein synthesis in hemin-supplemented reticulocyte lysates was further characterized. Rabbit liver metallothionein I or II protected against and reversed the effects of Cd and Cu on protein synthesis in the reticulocyte lysate, but only marginally protected against the effects of Hg or Pb. Purified apometallothionein I or II protected and reversed the effects of Hg and Pb, as well as Cd and Cu. Metallothionein I preparations, found to be contaminated with proteolytic breakdown products of itself, were found to be highly toxic to protein synthesis in the reticulocyte lysate. Thioredoxin was also found to protect and reverse the effects of heavy metal ions on protein synthesis. The metal chelators, EDTA and DTPA were also found to be capable of stimulating protein synthesis in heavy metal ion inhibited lysates. We have investigated whether RF is expressed in other cell types, tissues and organs. Using immunoblotting
techniques, 10,000 x g supernatants of extracts from rabbit tissues were analyzed for the presence of the 5 subunits of GEF. On a per mg protein basis, GEF was present at the highest levels in the reticulocyte lysate and red blood cell precursors followed by the brain, heart and kidney. GEF was present in lower levels in the liver and whole bone marrow and the spleen.
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Progress 10/01/86 to 09/30/87
Outputs
The mechanisms by which toxic heavy metals (Cd,Hg,Pb) inhibit eukaryotic protein synthesis was studied utilizing the rabbit reticulocyte lysate as a model system. Inhibition of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2, and the loss of guanine nucleotide exchange factor (GEF) activity. Agents which prevented or reversed the inhibition of protein synthesis by heavy metal ions, inhibited eIF-2alpha phosphorylation and restored GEF activity. Anti-serum to reticulocyte heme-regulated eIF-2alpha kinase (HRI) inhibited the phosphorylation of eIF-2alpha catalyzed by Hg-inhibited lysate. Addition of glc-6-P, NADPH, or GSH had no effect on protein ynthesis in heavy metal-ion inhibited lysates; chelating agents, EDTA, DTPA or BAL had variable effects. DTT, thioredoxin, metallothionein II and thionein II prevented and reversed the inhibition of protein synthesis observed in the presence of Cd or
Hg; thionein I had marginal effects, while metallothionein I was inhibitory. Studies on the distribution of eIF-2 and GEF in the lysate indicate that the association of these factors with the 60S ribosomal subunit was not an aftifact of the low salt concentrations employed in the sucrose density gradients. Upon analysis of 5 plaque purified clones isolated from a rat liver lambda GT11 cDNA library, inserts from 0.9-1,3 Kb were found to be present coding for fusion protein products with increased molecular weights between 17-39 KDa.
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Progress 02/01/86 to 09/30/86
Outputs
The effects of heavy metal ions (Hg, Pb, Cd and As(2)) on protein synthesis in the rabbit reticulocyte lysate were examined as a model system for oxidative stress, as these are environmentally important toxins. Heavy metal ions were found to stimulate the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2, leading to the inhibition of initiation and the disaggregation of polyribosomes. These effects were reversed by 2mM MgGTP and 1 mM DTT. The potency of a heavy metal ion in inhibiting protein synthesis was found to correlate directly with its known biological toxicity. Currently studies are underway to determine the mechanism by which heavy metal ions stimulate eIF-2alpha phosphorylation. Immunoblot analysis of the distribution of the eIF-2 recycling factor (RF) in sucrose density gradients of hemin-supplemented reticulocyte lysates demonstrates that RF binds to 60S ribosomal subunits and 80S ribosome couples in the absence of eIF-2alpha
phosphorylation complexes was also detected. Five plaque purified clones in gammaGT11 putitively coding for RF subunits, are currently being analyzed for the length of their cDNA insert and the production of fusion protein.
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