1
|
Amoah DP, Hussein SK, Johnson JL, LaPointe P. Ordered ATP hydrolysis in the Hsp90 chaperone is regulated by Aha1 and a conserved post-translational modification. Protein Sci 2025; 34:e5255. [PMID: 39665290 PMCID: PMC11635476 DOI: 10.1002/pro.5255] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2024] [Revised: 11/25/2024] [Accepted: 11/28/2024] [Indexed: 12/13/2024]
Abstract
Hsp90 is a dimeric molecular chaperone that is important for the folding, stabilization, activation, and maturation of hundreds of protein substrates called "clients" in cells. Dozens of co-chaperones and hundreds of post-translational modifications (PTMs) regulate the ATP-dependent client activation cycle. The Aha1 co-chaperone is the most potent stimulator of the ATPase cycle of Hsp90 and phosphorylation of threonine 22 in Hsp90 can regulate the recruitment of Aha1 in cells. We report here that phosphorylation of threonine 22 regulates specific aspects of Aha1 function after recruitment occurs. The phosphomimetic substitution, T22E, neutralizes the action of the Aha1 NxNNWHW motif. Moreover, this substitution can exert this effect from only one protomer of the Hsp90 dimer. This work sheds light on how asymmetric modifications in the Hsp90 dimer can functionalize individual protomers and fine-tune the Hsp90 cycle.
Collapse
Affiliation(s)
- Desmond Prah Amoah
- Department of Cell Biology, Faculty of Medicine & DentistryUniversity of AlbertaEdmontonAlbertaCanada
| | - Solomon K. Hussein
- Department of Cell Biology, Faculty of Medicine & DentistryUniversity of AlbertaEdmontonAlbertaCanada
| | - Jill L. Johnson
- Department of Biological Sciences and the Center for Reproductive BiologyUniversity of IdahoMoscowIdahoUSA
| | - Paul LaPointe
- Department of Cell Biology, Faculty of Medicine & DentistryUniversity of AlbertaEdmontonAlbertaCanada
| |
Collapse
|
2
|
Fedotcheva TA, Fedotcheva NI, Shimanovsky NL. Progesterone as an Anti-Inflammatory Drug and Immunomodulator: New Aspects in Hormonal Regulation of the Inflammation. Biomolecules 2022; 12:biom12091299. [PMID: 36139138 PMCID: PMC9496164 DOI: 10.3390/biom12091299] [Citation(s) in RCA: 49] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 09/10/2022] [Accepted: 09/11/2022] [Indexed: 11/25/2022] Open
Abstract
The specific regulation of inflammatory processes by steroid hormones has been actively studied in recent years, especially by progesterone (P4) and progestins. The mechanisms of the anti-inflammatory and immunomodulatory P4 action are not fully clear. The anti-inflammatory effects of P4 can be defined as nonspecific, associated with the inhibition of NF-κB and COX, as well as the inhibition of prostaglandin synthesis, or as specific, associated with the regulation of T-cell activation, the regulation of the production of pro- and anti-inflammatory cytokines, and the phenomenon of immune tolerance. The specific anti-inflammatory effects of P4 and its derivatives (progestins) can also include the inhibition of proliferative signaling pathways and the antagonistic action against estrogen receptor beta-mediated signaling as a proinflammatory and mitogenic factor. The anti-inflammatory action of P4 is accomplished through the participation of progesterone receptor (PR) chaperones HSP90, as well as immunophilins FKBP51 and FKBP52, which are the validated targets of clinically approved immunosuppressive drugs. The immunomodulatory and anti-inflammatory effects of HSP90 inhibitors, tacrolimus and cyclosporine, are manifested, among other factors, due to their participation in the formation of an active ligand–receptor complex of P4 and their interaction with its constituent immunophilins. Pharmacological agents such as HSP90 inhibitors can restore the lost anti-inflammatory effect of glucocorticoids and P4 in chronic inflammatory and autoimmune diseases. By regulating the activity of FKBP51 and FKBP52, it is possible to increase or decrease hormonal signaling, as well as restore it during the development of hormone resistance. The combined action of immunophilin suppressors with steroid hormones may be a promising strategy in the treatment of chronic inflammatory and autoimmune diseases, including endometriosis, stress-related disorders, rheumatoid arthritis, and miscarriages. Presumably, the hormone receptor- and immunophilin-targeted drugs may act synergistically, allowing for a lower dose of each.
Collapse
Affiliation(s)
- Tatiana A. Fedotcheva
- Science Research Laboratory of Molecular Pharmacology, Medical Biological Faculty, Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Ostrovityanova St. 1, Moscow 117997, Russia
- Correspondence: ; Tel.: +7-9169353196
| | - Nadezhda I. Fedotcheva
- Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya Str. 3, Pushchino 142290, Russia
| | - Nikolai L. Shimanovsky
- Science Research Laboratory of Molecular Pharmacology, Medical Biological Faculty, Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Ostrovityanova St. 1, Moscow 117997, Russia
| |
Collapse
|
3
|
KAWANO K, YANAGAWA Y, NAGANO M, KATAGIRI S. Effects of heat stress on the endometrial epidermal growth factor profile and fertility in dairy cows. J Reprod Dev 2022; 68:144-151. [PMID: 35095040 PMCID: PMC8979802 DOI: 10.1262/jrd.2021-120] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The endometrial epidermal growth factor (EGF) profile is an indicator of uterine function and fertility in cattle. The present study aimed to investigate the effects of heat stress on the
endometrial EGF profile and fertility in lactating Holstein cows. The endometrial EGF profiles of 365 cows in the Hokkaido and Kyushu regions were examined between June and September (heat
stress period, n = 211) and between October and January (control period, n = 154). EGF profiles were investigated using uterine endometrial tissues obtained by biopsy 3 days after estrus
(Day 3). The proportion of cows with an altered EGF profile was higher between June and September than between October and January (41.2 vs. 16.2%, P < 0.05). The effects
of rectal temperature on Days 0 and 3 on the endometrial EGF profile were also assessed in cows (n = 79) between June and September in the Kyushu region. A single embryo was transferred to
cow on Day 7 to evaluate fertility (n = 67). Regardless of the rectal temperature on Day 3, the proportion of cows with an altered EGF profile was higher (64.1 vs. 30.0%, P
< 0.05) and the pregnancy rate after embryo transfer (ET) was lower (26.7 vs. 51.4%, P < 0.05) in cows with a rectal temperature ≥ 39.5°C on Day 0 than in cows with a
rectal temperature < 39.5°C on Day 0. The present results indicate that alterations in the endometrial EGF profile induced by an elevated body temperature on Day 0 contributed to
reductions in fertility in lactating dairy cows during the heat stress period.
Collapse
Affiliation(s)
- Kohei KAWANO
- Laboratory of Theriogenology, Department of Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
| | - Yojiro YANAGAWA
- Laboratory of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
| | - Masashi NAGANO
- Laboratory of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
| | - Seiji KATAGIRI
- Laboratory of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
| |
Collapse
|
4
|
Nakatani Y, Miyazaki Y, Hara S. Cytosolic Prostaglandin E Synthase Is Involved in c-Fos Expression in Rat Fibroblastic 3Y1 Cells. Biol Pharm Bull 2017; 40:1963-1967. [PMID: 29093345 DOI: 10.1248/bpb.b17-00518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cytosolic prostaglandin (PG) E synthase (cPGES/p23) plays a role in the biosynthesis of PGE2 and in the molecular chaperone machinery. Studies of knockout mice lacking cPGES/p23 have demonstrated that cPGES/p23 is essential in fetal mouse development. A cDNA microarray analysis revealed that a lack of cPGES/p23 decreases the expression of some immediate early genes, such as c-fos and activating transcription factor 3 (ATF3). Here we report the involvement of cPGES/p23 in c-Fos expression. A stable knockdown of cPGES/p23 in cultured fibroblasts not only reduced serum-induced c-Fos expression, but also decreased the phosphorylation of extracellular signal regulated kinase (ERK). These results suggest that cPGES/p23 is involved in the activation of ERK to promote c-Fos expression.
Collapse
Affiliation(s)
- Yoshihito Nakatani
- Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University
| | - Yuki Miyazaki
- Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University
| | - Shuntaro Hara
- Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University
| |
Collapse
|
5
|
Gellersen B, Brosens JJ. Cyclic decidualization of the human endometrium in reproductive health and failure. Endocr Rev 2014; 35:851-905. [PMID: 25141152 DOI: 10.1210/er.2014-1045] [Citation(s) in RCA: 713] [Impact Index Per Article: 64.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Decidualization denotes the transformation of endometrial stromal fibroblasts into specialized secretory decidual cells that provide a nutritive and immunoprivileged matrix essential for embryo implantation and placental development. In contrast to most mammals, decidualization of the human endometrium does not require embryo implantation. Instead, this process is driven by the postovulatory rise in progesterone levels and increasing local cAMP production. In response to falling progesterone levels, spontaneous decidualization causes menstrual shedding and cyclic regeneration of the endometrium. A growing body of evidence indicates that the shift from embryonic to maternal control of the decidual process represents a pivotal evolutionary adaptation to the challenge posed by invasive and chromosomally diverse human embryos. This concept is predicated on the ability of decidualizing stromal cells to respond to individual embryos in a manner that either promotes implantation and further development or facilitates early rejection. Furthermore, menstruation and cyclic regeneration involves stem cell recruitment and renders the endometrium intrinsically capable of adapting its decidual response to maximize reproductive success. Here we review the endocrine, paracrine, and autocrine cues that tightly govern this differentiation process. In response to activation of various signaling pathways and genome-wide chromatin remodeling, evolutionarily conserved transcriptional factors gain access to the decidua-specific regulatory circuitry. Once initiated, the decidual process is poised to transit through distinct phenotypic phases that underpin endometrial receptivity, embryo selection, and, ultimately, resolution of pregnancy. We discuss how disorders that subvert the programming, initiation, or progression of decidualization compromise reproductive health and predispose for pregnancy failure.
Collapse
Affiliation(s)
- Birgit Gellersen
- Endokrinologikum Hamburg (B.G.), 20251 Hamburg, Germany; and Division of Reproductive Health (J.J.B.), Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom
| | | |
Collapse
|
6
|
Graf C, Lee CT, Eva Meier-Andrejszki L, Nguyen MTN, Mayer MP. Differences in conformational dynamics within the Hsp90 chaperone family reveal mechanistic insights. Front Mol Biosci 2014; 1:4. [PMID: 25988145 PMCID: PMC4428384 DOI: 10.3389/fmolb.2014.00004] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2014] [Accepted: 05/21/2014] [Indexed: 12/28/2022] Open
Abstract
The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90β (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.
Collapse
Affiliation(s)
- Christian Graf
- Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany
| | - Chung-Tien Lee
- Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany
| | - L Eva Meier-Andrejszki
- Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany
| | - Minh T N Nguyen
- Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany
| | - Matthias P Mayer
- Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany
| |
Collapse
|
7
|
Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters JA, Harmar AJ. The Concise Guide to PHARMACOLOGY 2013/14: enzymes. Br J Pharmacol 2013; 170:1797-867. [PMID: 24528243 PMCID: PMC3892293 DOI: 10.1111/bph.12451] [Citation(s) in RCA: 415] [Impact Index Per Article: 34.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. Enzymes are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates.
Collapse
Affiliation(s)
- Stephen PH Alexander
- School of Life Sciences, University of Nottingham Medical SchoolNottingham, NG7 2UH, UK
| | - Helen E Benson
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Elena Faccenda
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Adam J Pawson
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | - Joanna L Sharman
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| | | | - John A Peters
- Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of DundeeDundee, DD1 9SY, UK
| | - Anthony J Harmar
- The University/BHF Centre for Cardiovascular Science, University of EdinburghEdinburgh, EH16 4TJ, UK
| |
Collapse
|
8
|
Li J, Richter K, Reinstein J, Buchner J. Integration of the accelerator Aha1 in the Hsp90 co-chaperone cycle. Nat Struct Mol Biol 2013; 20:326-31. [PMID: 23396352 DOI: 10.1038/nsmb.2502] [Citation(s) in RCA: 99] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2012] [Accepted: 12/27/2012] [Indexed: 02/07/2023]
Abstract
Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that associates dynamically with various co-chaperones during its chaperone cycle. Here we analyzed the role of the activating co-chaperone Aha1 in the progression of the yeast Hsp90 chaperone cycle and identified a critical ternary Hsp90 complex containing the co-chaperones Aha1 and Cpr6. Aha1 accelerates the intrinsically slow conformational transitions of Hsp90 to an N-terminally associated state but does not fully close the nucleotide-binding pocket yet. Cpr6 increases the affinity between Aha1 and Hsp90 and further stimulates the Hsp90 ATPase activity. Synergistically, Aha1 and Cpr6 displace the inhibitory co-chaperone Sti1 from Hsp90. To complete the cycle, Aha1 is released by the co-chaperone p23. Thus, at distinct steps during the Hsp90 chaperone cycle, co-chaperones selectively trap statistically distributed Hsp90 conformers and thus turn Hsp90 into a deterministic machine.
Collapse
Affiliation(s)
- Jing Li
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, München, Germany
| | | | | | | |
Collapse
|
9
|
The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J 2012; 31:1506-17. [PMID: 22227520 DOI: 10.1038/emboj.2011.472] [Citation(s) in RCA: 172] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2011] [Accepted: 11/30/2011] [Indexed: 01/11/2023] Open
Abstract
Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.
Collapse
|
10
|
Pretheeban T, Gordon MB, Singh R, Rajamahendran R. Comparison of expression levels of candidate genes in endometrium of dairy heifers and lactating dairy cows. CANADIAN JOURNAL OF ANIMAL SCIENCE 2011. [DOI: 10.4141/cjas2010-012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Pretheeban, T., Gordon, M. B., Singh, R. and Rajamahendran, R. 2011. Comparison of expression levels of candidate genes in endometrium of dairy heifers and lactating dairy cows. Can. J. Anim. Sci. 91: 255–264. Pregnancy rates (PR) in high-producing lactating dairy cows have declined drastically over the past several decades, but those of heifers have remained constant. Reduced PR could be due to multiple causes, and the underlying pathophysiological mechanisms are still unclear. A compromised maternal uterine environment could be one of factors that could affect the PR. This study was performed to compare the nature of the uterine environment in dairy heifers and lactating dairy cows (2nd/3rd parity) by analyzing the expression levels of selected endometrial genes. Estrus was synchronized in heifers (n=5) and lactating dairy cows (n=5) and endometrial biopsies were performed during the mid luteal phase (day 11) of the estrous cycle. Real-time polymerase chain reaction (Q-RT PCR) and immunohistochemistry were performed to analyse the mRNA and protein levels of genes respectively. Relative abundance of BCL2, HSPA1A, IL1A, TNF, IGF1, FGF2 and SERPINA14 transcripts and the protein expression of IL1A, TNF and FGF2 were significantly higher in heifers in comparison with lactating dairy cows. Our findings suggest an altered endometrial environment in lactating dairy cows compared with heifers. However, whether these differences play a role in pregnancy outcomes should be further investigated.
Collapse
Affiliation(s)
- T. Pretheeban
- Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T1Z4
| | - M. B. Gordon
- Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T1Z4
| | - R. Singh
- Translational Research Labs, Tom Baker Cancer Centre, Calgary, Alberta, Canada T2N4N2
| | - R. Rajamahendran
- Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T1Z4
| |
Collapse
|
11
|
Preechaphol R, Klinbunga S, Yamano K, Menasveta P. Molecular cloning and expression of progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp Penaeus monodon. Gen Comp Endocrinol 2010; 168:440-9. [PMID: 20566361 DOI: 10.1016/j.ygcen.2010.06.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2009] [Revised: 06/01/2010] [Accepted: 06/01/2010] [Indexed: 01/03/2023]
Abstract
Knowledge on molecular mechanisms of steroid hormonal induction on oocyte development may lead to the possible ways to effectively induce ovarian maturation in shrimp. In this study, progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp (Penaeus monodon) initially identified by EST analysis was further characterized. The full-length cDNA of Pgmrc1 was 2015bp in length containing an ORF of 573bp corresponding to a polypeptide of 190 amino acids. Northern blot analysis revealed a single form of Pgmrc1 in ovaries of P. monodon. Quantitative real-time PCR indicated that the expression level of Pgmrc1 mRNA in ovaries of both intact and eyestalk-ablated broodstock was greater than that of juveniles (P<0.05). Pgmrc1 was up-regulated in mature (stage IV) ovaries of intact broodstock (P<0.05). Unilateral eyestalk ablation resulted in an earlier up-regulation of Pgmrc1 since the vitellogenic (II) ovarian stage. Moreover, the expression level of Pgmrc1 in vitellogenic, early cortical rod and mature (II-IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock (P<0.05). Pgmrc1 mRNA was clearly localized in the cytoplasm of follicular cells, previtellogenic and early vitellogenic oocytes. Immunohistochemistry revealed the positive signals of the Pgmrc1 protein in the follicular layers and cell membrane of follicular cells and various stages of oocytes. Taken the information together, Pgmrc1 gene products seem to play the important role on ovarian development and may be used as the bioindicator for monitoring progression of oocyte maturation of P. monodon.
Collapse
Affiliation(s)
- Rachanimuk Preechaphol
- Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
| | | | | | | |
Collapse
|
12
|
Retzlaff M, Hagn F, Mitschke L, Hessling M, Gugel F, Kessler H, Richter K, Buchner J. Asymmetric activation of the hsp90 dimer by its cochaperone aha1. Mol Cell 2010; 37:344-54. [PMID: 20159554 DOI: 10.1016/j.molcel.2010.01.006] [Citation(s) in RCA: 201] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2009] [Revised: 08/14/2009] [Accepted: 12/07/2009] [Indexed: 01/01/2023]
Abstract
The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.
Collapse
Affiliation(s)
- Marco Retzlaff
- Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, D-85747 Garching, Germany
| | | | | | | | | | | | | | | |
Collapse
|
13
|
Chadli A, Felts SJ, Wang Q, Sullivan WP, Botuyan MV, Fauq A, Ramirez-Alvarado M, Mer G. Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23. J Biol Chem 2009; 285:4224-4231. [PMID: 19996313 DOI: 10.1074/jbc.m109.081018] [Citation(s) in RCA: 72] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.
Collapse
Affiliation(s)
- Ahmed Chadli
- From the Center for Molecular Chaperone Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, Georgia 30912.
| | - Sara J Felts
- the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and
| | - Qin Wang
- From the Center for Molecular Chaperone Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, Georgia 30912
| | - William P Sullivan
- the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and
| | - Maria Victoria Botuyan
- the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and
| | - Abdul Fauq
- the Organic Chemistry Laboratory, Mayo Clinic College of Medicine, Jacksonville, Florida 32224
| | - Marina Ramirez-Alvarado
- the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and
| | - Georges Mer
- the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and
| |
Collapse
|
14
|
p23/Sba1p protects against Hsp90 inhibitors independently of its intrinsic chaperone activity. Mol Cell Biol 2008; 28:3446-56. [PMID: 18362168 DOI: 10.1128/mcb.02246-07] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The molecular chaperone Hsp90 assists a subset of cellular proteins and is essential in eukaryotes. A cohort of cochaperones contributes to and regulates the multicomponent Hsp90 machine. Unlike the biochemical activities of the cochaperone p23, its in vivo functions and the structure-function relationship remain poorly understood, even in the genetically tractable model organism Saccharomyces cerevisiae. The SBA1 gene that encodes the p23 ortholog in this species is not an essential gene. We found that in the absence of p23/Sba1p, yeast and mammalian cells are hypersensitive to Hsp90 inhibitors. This protective function of Sba1p depends on its abilities to bind Hsp90 and to block the Hsp90 ATPase and inhibitor binding. In contrast, the protective function of Sba1p does not require the Hsp90-independent molecular chaperone activity of Sba1p. The structure-function analysis suggests that Sba1p undergoes considerable structural rearrangements upon binding Hsp90 and that the large size of the p23/Sba1p-Hsp90 interaction surface facilitates maintenance of high affinity despite sequence divergence during evolution. The large interface may also contribute to preserving a protective function in an environment in which Hsp90 inhibitory compounds can be produced by various microorganisms.
Collapse
|
15
|
Flom G, Behal R, Rosen L, Cole D, Johnson J. Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions. Biochem J 2007; 404:159-67. [PMID: 17300223 PMCID: PMC1868838 DOI: 10.1042/bj20070084] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.
Collapse
Affiliation(s)
- Gary Flom
- Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
| | - Robert H. Behal
- Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
| | - Luke Rosen
- Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
| | - Douglas G. Cole
- Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
| | - Jill L. Johnson
- Department of Microbiology, Molecular Biology and Biochemistry, and the Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, U.S.A
- To whom correspondence should be addressed (email )
| |
Collapse
|
16
|
Picard D, Suslova E, Briand PA. 2-color photobleaching experiments reveal distinct intracellular dynamics of two components of the Hsp90 complex. Exp Cell Res 2006; 312:3949-58. [PMID: 17010336 DOI: 10.1016/j.yexcr.2006.08.026] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2006] [Revised: 08/21/2006] [Accepted: 08/26/2006] [Indexed: 11/24/2022]
Abstract
The abundant molecular chaperone Hsp90 functions in association with co-chaperones including p23 to promote the folding and maturation of a subset of cytosolic proteins. "Fluorescence recovery after photobleaching" (FRAP) experiments showed that the dynamics of p23 in live cells is dictated by Hsp90. Since Hsp90 is present in large excess over p23, the mobility of Hsp90 could conceivably be quite different. To facilitate the analysis and to allow a direct comparison with p23, we developed a 2-color FRAP technique. Two test proteins are expressed as fusion proteins with the two spectrally separable fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP). The 2-color FRAP technique is powerful for the concomitant recording of two proteins located in the same area of a cell, two components of the same protein complex, or mutant and wild-type versions of the same protein under identical experimental conditions. 2-color FRAP of Hsp90 and p23 is virtually indistinguishable, consistent with the notion that they are both engaged in a multitude of large protein complexes. However, when Hsp90-p23 complexes are disrupted by the Hsp90 inhibitor geldanamycin, p23 moves by free diffusion while Hsp90 maintains its low mobility because it remains bound in remodeled multicomponent complexes.
Collapse
Affiliation(s)
- Didier Picard
- Département de Biologie Cellulaire, Université de Genève, Sciences III, 30 quai Ernest-Ansermet, CH-1211 Genève 4, Switzerland
| | | | | |
Collapse
|
17
|
Mukherjee K, Syed V, Ho SM. Estrogen-induced loss of progesterone receptor expression in normal and malignant ovarian surface epithelial cells. Oncogene 2005; 24:4388-400. [PMID: 15806153 DOI: 10.1038/sj.onc.1208623] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
While estrogens are suspected risk factors for epithelial ovarian cancer (OCa), progesterone (P4) has been shown to exert protective effects. The biological actions of P4 in target cells are mediated by progesterone receptors (PRs) that exist principally as A- and B-isoforms. We observed overexpression of PR-A and PR-B protein in two lines of OCa cells when compared to two lines of nontumorigenic, normal human ovarian surface epithelial (HOSE) cells. Treatment of HOSE or OCa cells with estrone or 17beta-estradiol at 10(-8) M for a period of 72 h induced significant loss of PR-A and PR-B mRNA and protein expression, with the regulation primarily controlled at the transcriptional level. In contrast, breast cancer cells (line MCF-7) exposed to estrogens upregulated PR-A and PR-B expression. Of significance, both the inhibitory and stimulatory actions of estrogens were blocked by the specific ER-antagonist ICI 182,780 (ICI, 10(-5) M), confirming estrogen specificity. Co-treatment of estrogen-exposed HOSE, OCa, and MCF-7 cell lines with inhibitors of type 1- and type 2-17beta hydroxysteroid dehydrogenase did not affect the previously observed changes in PR expression, suggesting that the action of each estrogen is direct and not mediated via conversion to its metabolic counterpart. Green fluorescence protein (GFP)-PR-A and GFP-PR-B were localized in the cytoplasmic compartment of untreated HOSE cells and translocated to the nucleus after P4 treatment, while both chimera PRs resided in the nuclei of OCa cells in a ligand-independent manner. In OCa cell cultures, P4 (10(-6) M), but not RU486 (10(-5) M), induced apoptosis that was blocked by co-treatment with the antiprogestin but enhanced by co-treatment with ICI. In sharp contrast, P4 induced proliferation, while ICI and RU486 caused cell death in MCF-7 cells. In conclusion, this study is first to demonstrate estrogens as negative regulators of PR expression in HOSE/OCa cells and to provide a mechanistic basis upon which to explain the antagonism of estrogens on the anti-OCa action of progestins. It also raises the possibility of using progestin and ICI as a combinational therapy for OCa treatment.
Collapse
Affiliation(s)
- Kasturi Mukherjee
- Department of Surgery, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | | | | |
Collapse
|
18
|
Barzilay E, Ben-Califa N, Shahar M, Kashman Y, Neumann D. Generation of a novel anti-geldanamycin antibody. Biochem Biophys Res Commun 2005; 330:561-4. [PMID: 15796919 DOI: 10.1016/j.bbrc.2005.03.014] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2005] [Indexed: 11/30/2022]
Abstract
Geldanamycin (GA) and herbimycin A are benzoquinone ansamycins (BAs) that inhibit the molecular chaperone HSP90. The central role of HSP90 in maintaining the conformation, stability, and function of key oncogenic proteins involved in signal transduction pathways renders BAs attractive candidates for clinical development. Two GA derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin are currently evaluated in clinical trials. The present study demonstrates generation of a polyclonal antibody elicited against GA that was conjugated to keyhole limpet hemocyanin via its 17 position. The anti-GA antibody recognizes GA as well as other BAs, suggesting its possible application for monitoring plasma levels of GA derivatives. The specificity of the antibody towards BAs is demonstrated by its inability to recognize radicicol, an HSP90 inhibitor not related to BAs. This antibody thus presents a novel research tool as well as a possible alternative approach for monitoring drug levels in patients.
Collapse
Affiliation(s)
- Eran Barzilay
- Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel
| | | | | | | | | |
Collapse
|
19
|
Zhang C, Guy CL. Co-immunoprecipitation of Hsp101 with cytosolic Hsc70. PLANT PHYSIOLOGY AND BIOCHEMISTRY : PPB 2005; 43:13-8. [PMID: 15763661 DOI: 10.1016/j.plaphy.2004.10.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/28/2003] [Accepted: 10/20/2004] [Indexed: 05/24/2023]
Abstract
In animals and yeast, cytosolic Hsp70s function in concert with other molecular chaperones. Hsp70 is a major chaperone in the Hsp90 multi-chaperone complexes that participate in maturation of steroid receptors and several other proteins. Hsp70s also appear to form a complex with Hsp90 and Hsp110/sHsp. A 100 kDa protein was co-immunoprecipitated with cytosolic Hsc70 from maize seedlings (Zea mays). The presence of this complex was further confirmed using gel-filtration chromatography. Mass spectrometric analysis showed that the 100 kDa protein is homologous with Arabidopsis Hsp101. Treatment with apyrase enhanced the co-immunoprecipitation of Hsp101 with Hsc70, while ATP had the opposite effect. In the presence of carboxymethylated alpha-lactalbumin (CMLA), which is permanently unfolded, the complex dissociated. Based on these observations, it is concluded that Hsc70 and Hsp101 are present in a complex in the plant cytosol.
Collapse
Affiliation(s)
- Chun Zhang
- Plant Molecular and Cellular Biology Program, Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611-0675, USA
| | | |
Collapse
|
20
|
Richter K, Walter S, Buchner J. The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle. J Mol Biol 2004; 342:1403-13. [PMID: 15364569 DOI: 10.1016/j.jmb.2004.07.064] [Citation(s) in RCA: 123] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2004] [Revised: 07/16/2004] [Accepted: 07/19/2004] [Indexed: 10/26/2022]
Abstract
The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.
Collapse
Affiliation(s)
- Klaus Richter
- Department für Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
| | | | | |
Collapse
|
21
|
Masuda Y, Shima G, Aiuchi T, Horie M, Hori K, Nakajo S, Kajimoto S, Shibayama-Imazu T, Nakaya K. Involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in apoptosis induced by beta-hydroxyisovalerylshikonin. J Biol Chem 2004; 279:42503-15. [PMID: 15292218 DOI: 10.1074/jbc.m404256200] [Citation(s) in RCA: 115] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.
Collapse
Affiliation(s)
- Yutaka Masuda
- Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan.
| | | | | | | | | | | | | | | | | |
Collapse
|
22
|
Sreedhar AS, Csermely P. Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review. Pharmacol Ther 2004; 101:227-57. [PMID: 15031001 DOI: 10.1016/j.pharmthera.2003.11.004] [Citation(s) in RCA: 313] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Heat shock proteins (Hsp) form the most ancient defense system in all living organisms on earth. These proteins act as molecular chaperones by helping in the refolding of misfolded proteins and assisting in their elimination if they become irreversibly damaged. Hsp interact with a number of cellular systems and form efficient cytoprotective mechanisms. However, in some cases, wherein it is better if the cell dies, there is no reason for any further defense. Programmed cell death is a widely conserved general phenomenon helping in many processes involving the reconstruction of multicellular organisms, as well as in the elimination of old or damaged cells. Here, we review some novel elements of the apoptotic process, such as its interrelationship with cellular senescence and necrosis, as well as bacterial apoptosis. We also give a survey of the most important elements of the apoptotic machinery and show the various modes of how Hsp interact with the apoptotic events in detail. We review caspase-independent apoptotic pathways and anoikis as well. Finally, we show the emerging variety of pharmacological interventions inhibiting or, just conversely, inducing Hsp and review the emergence of Hsp as novel therapeutic targets in anticancer protocols.
Collapse
Affiliation(s)
- Amere Subbarao Sreedhar
- Department of Medical Chemistry, Semmelweis University, P.O. Box 260, H-1444 Budapest, Hungary
| | | |
Collapse
|
23
|
Botos J, Xian W, Smith DF, Smith CL. Progesterone receptor deficient in chromatin binding has an altered cellular state. J Biol Chem 2004; 279:15231-9. [PMID: 14744870 DOI: 10.1074/jbc.m309718200] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligand-independent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.
Collapse
MESH Headings
- Animals
- Benzoquinones
- Blotting, Western
- Cell Line, Tumor
- Cell Nucleus/metabolism
- Chromatin/chemistry
- Chromatin/metabolism
- Cytosol/metabolism
- DNA/chemistry
- DNA/metabolism
- Dimerization
- Dose-Response Relationship, Drug
- Enzyme Inhibitors/pharmacology
- Fluorescent Antibody Technique, Indirect
- Genes, Viral
- HSP90 Heat-Shock Proteins/metabolism
- Hormones/metabolism
- In Situ Hybridization, Fluorescence
- Intramolecular Oxidoreductases
- Lactams, Macrocyclic
- Ligands
- Luciferases/metabolism
- Mammary Tumor Virus, Mouse/genetics
- Mice
- Microscopy, Fluorescence
- Models, Biological
- Molecular Chaperones/metabolism
- Phosphoproteins/metabolism
- Phosphorylation
- Precipitin Tests
- Promoter Regions, Genetic
- Prostaglandin-E Synthases
- Protein Binding
- Protein Conformation
- Quinones/pharmacology
- Receptors, Progesterone/chemistry
- Receptors, Progesterone/metabolism
- Tacrolimus Binding Proteins/chemistry
- Tacrolimus Binding Proteins/metabolism
- Transcription, Genetic
- Transfection
- beta-Galactosidase/metabolism
Collapse
Affiliation(s)
- Jeannine Botos
- Laboratory of Receptor Biology and Gene Expression, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
| | | | | | | |
Collapse
|
24
|
Abstract
Molecular chaperones are a functionally defined set of proteins which assist the structure formation of proteins in vivo. Without certain protective mechanisms, such as binding nascent polypeptide chains by molecular chaperones, cellular protein concentrations would lead to misfolding and aggregation. In the mammalian system, the molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins, like steroid hormone receptors, transcription factors, and kinases, some of which are involved in cancer progression. Hsp70 and Hsp90 form a multichaperone complex, in which both are connected by a third protein called Hop. The connection of and the interplay between the two chaperone machineries is of crucial importance for cell viability. This review provides a detailed view of the Hsp70 and Hsp90 machineries, their cofactors and their mode of regulation. It summarizes the current knowledge in the field, including the ATP-dependent regulation of the Hsp70/Hsp90 multichaperone cycle and elucidates the complex interplay and their synergistic interaction.
Collapse
Affiliation(s)
- H Wegele
- Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany
| | | | | |
Collapse
|
25
|
Sumanasekera WK, Tien ES, Turpey R, Vanden Heuvel JP, Perdew GH. Evidence that peroxisome proliferator-activated receptor alpha is complexed with the 90-kDa heat shock protein and the hepatitis virus B X-associated protein 2. J Biol Chem 2003; 278:4467-73. [PMID: 12482853 DOI: 10.1074/jbc.m211261200] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPARalpha mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPARalpha plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPARalpha activity, a monoclonal antibody (mAb) was made against PPARalpha and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPARalpha was tested in immunoprecipitations using in vitro translated PPAR subtypes. The mAb 3B6/PPAR recognized PPARalpha, failed to bind to PPARbeta or PPARgamma, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPARalpha in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPARalpha in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPARalpha/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPARalpha in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPARalpha resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPARalpha is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPARalpha is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.
Collapse
Affiliation(s)
- Wasana K Sumanasekera
- Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, Pennsylvania 16802, USA
| | | | | | | | | |
Collapse
|
26
|
Zhang Z, Quick MK, Kanelakis KC, Gijzen M, Krishna P. Characterization of a plant homolog of hop, a cochaperone of hsp90. PLANT PHYSIOLOGY 2003; 131:525-35. [PMID: 12586877 PMCID: PMC166829 DOI: 10.1104/pp.011940] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/28/2002] [Revised: 09/09/2002] [Accepted: 11/08/2002] [Indexed: 05/19/2023]
Abstract
The 90-kD molecular chaperone hsp90 is the key component of a multiprotein chaperone complex that facilitates folding, stabilization, and functional modulation of a number of signaling proteins. The components of the animal chaperone complex include hsp90, hsp70, hsp40, Hop, and p23. The animal Hop functions to link hsp90 and hsp70, and it can also inhibit the ATPase activity of hsp90. We have demonstrated the presence of an hsp90 chaperone complex in plant cells, but not all components of the complex have been identified. Here, we report the isolation and characterization of soybean (Glycine max) GmHop-1, a soybean homolog of mammalian Hop. An analysis of soybean expressed sequence tags, combined with preexisting data in literature, suggested the presence of at least three related genes encoding Hop-like proteins in soybean. Transcripts corresponding to Hop-like proteins in soybean were detected under normal growth conditions, and their levels increased further in response to stress. A recombinant GmHop-1 bound hsp90 and its binding to hsp90 could be blocked by the tetratricopeptide repeat (TPR) domain of rat (Rattus norvegicus) protein phosphatase 5. Deletion of amino acids 325 to 395, adjacent to the TPR2A domain in GmHop-1, resulted in loss of hsp90 binding. In a minimal assembly system, GmHop-1 was able to stimulate mammalian steroid receptor folding. These data show that plant and animal Hop homologs are conserved in their general characteristics, and suggest that a Hop-like protein in plants is an important cochaperone of plant hsp90.
Collapse
Affiliation(s)
- Zhongming Zhang
- Department of Biology, The University of Western Ontario, London, Ontario, Canada N6A 5B7
| | | | | | | | | |
Collapse
|
27
|
Hernández MP, Sullivan WP, Toft DO. The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex. J Biol Chem 2002; 277:38294-304. [PMID: 12161444 DOI: 10.1074/jbc.m206566200] [Citation(s) in RCA: 143] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K(d) = 90 nm), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K(d) = 1.3 microm) on its own, but this affinity is increased (K(d) = 250 nm) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.
Collapse
Affiliation(s)
- M Patricia Hernández
- Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA
| | | | | |
Collapse
|
28
|
Hernández MP, Chadli A, Toft DO. HSP40 binding is the first step in the HSP90 chaperoning pathway for the progesterone receptor. J Biol Chem 2002; 277:11873-81. [PMID: 11809754 DOI: 10.1074/jbc.m111445200] [Citation(s) in RCA: 102] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The progesterone receptor (PR) can be isolated in its native conformation able to bind hormone, yet its ligand-binding domain rapidly loses its activity at elevated temperature. However, an in vitro chaperoning system consisting of five proteins (HSP40, HSP70, HOP, HSP90, and p23) with ATP is capable of restoring this function. The first step of this chaperoning mechanism is usually thought to be the binding of HSP70 to PR. Our findings here show that the binding of HSP40 to PR is, instead, the first step. HSP40 binding occurred rapidly and was not dependent on ATP or other proteins. The stoichiometry of HSP40 to native PR in these complexes was approximately 1:1. HSP40 bound specifically and with a high affinity to native PR (K(d) = 77 nm). The binding of HSP40 to PR was sustained and did not interact in the highly dynamic fashion that has been observed previously for HSP90 in this system. The HSP40 small middle dotPR complex could be isolated as a functional unit that could, after the addition of the other chaperones, progress to a PR complex capable of hormone binding. These results indicate that HSP40 initiates the entry of PR into the HSP90 pathway.
Collapse
Affiliation(s)
- M Patricia Hernández
- Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA
| | | | | |
Collapse
|
29
|
Li TK, Baksh S, Cristillo AD, Bierer BE. Calcium- and FK506-independent interaction between the immunophilin FKBP51 and calcineurin. J Cell Biochem 2002; 84:460-71. [PMID: 11813252 DOI: 10.1002/jcb.10026] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
FKBP51 is a member of the immunophilin family having intrinsic peptidyl-prolyl cis-trans-isomerase (PPIase) activity. Its enzymatic activity is inhibited by binding either immunosuppressive agent FK506 or rapamycin. Similar to FKBP12, but at higher concentrations of FK506, FKBP51 has been shown to inhibit the serine/threonine phosphatase activity of calcineurin in the presence of calcium and calmodulin. Here we show that a glutathione S-transferase (GST) fusion protein of FKBP51 on glutathione-Sepharose beads precipitated both purified calcineurin from bovine brain and calcineurin from murine T cell lysates. Surprisingly, the binding of GST-FKBP51 to calcineurin was FK506-independent and independent of a requirement for calcium or exogenous calmodulin. Unlike FKBP12, FKBP51 transiently expressed in COS-7 cells was precipitated by calcineurin bound to calmodulin-Sepharose beads in the absence of either FK506 or rapamycin. Unlike FKBP12, however, overexpression of FKBP51 in Jurkat T cells did not significantly affect the transcriptional activation of nuclear factor of activated T cells (NFAT) upon physiological stimulation, nor did it affect the ability of FK506 to inhibit NFAT-driven transcription. We generated a series of FKBP51 mutations to map the interaction of FKBP51 with calcineurin. Deletion of the aminoterminal, FKBP12-like domain of FKBP51 did not affect the ability of FKBP51 to bind to purified calcineurin, while deletion of the FKBP51 carboxyterminal domain abrogated the ability of FKBP51 to bind to calcineurin. Taken together, these results demonstrate a novel interaction between calcineurin and the immunophilin FKBP51 that is independent of calcium, calmodulin, and drug. The binding site on calcineurin for FKBP51 is separable from the immunophilin PPIase-active and drug-binding site.
Collapse
Affiliation(s)
- Ta-Kai Li
- Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA
| | | | | | | |
Collapse
|
30
|
Richter K, Muschler P, Hainzl O, Buchner J. Coordinated ATP hydrolysis by the Hsp90 dimer. J Biol Chem 2001; 276:33689-96. [PMID: 11441008 DOI: 10.1074/jbc.m103832200] [Citation(s) in RCA: 163] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.
Collapse
Affiliation(s)
- K Richter
- Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, Garching 85747, Germany
| | | | | | | |
Collapse
|
31
|
Abstract
Hsp90 is an ATP dependent molecular chaperone involved in the folding and activation of an unknown number of substrate proteins. These substrate proteins include protein kinases and transcription factors. Consistent with this task, Hsp90 is an essential protein in all eucaryotes. The interaction of Hsp90 with its substrate proteins involves the transient formation of multiprotein complexes with a set of highly conserved partner proteins. The specific function of each component in the processing of substrates is still unknown. Large ATP-dependent conformational changes of Hsp90 occur during the hydrolysis reaction and these changes are thought to drive the chaperone cycle. Natural inhibitors of the ATPase activity, like geldanamycin and radicicol, block the processing of Hsp90 substrate proteins. As many of these substrates are critical elements in signal transduction, Hsp90 seems to introduce an additional level of regulation.
Collapse
Affiliation(s)
- K Richter
- Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany
| | | |
Collapse
|
32
|
Johnson BD, Chadli A, Felts SJ, Bouhouche I, Catelli MG, Toft DO. Hsp90 chaperone activity requires the full-length protein and interaction among its multiple domains. J Biol Chem 2000; 275:32499-507. [PMID: 10913439 DOI: 10.1074/jbc.m005195200] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Hsp90 is an abundant and ubiquitous protein involved in a diverse array of cellular processes. Mechanistically we understand little of the apparently complex interactions of this molecular chaperone. Recently, progress has been made in assigning some of the known functions of hsp90, such as nucleotide binding and peptide binding, to particular domains within the protein. We used fragments of hsp90 and chimeric proteins containing functional domains from hsp90 or its mitochondrial homolog, TRAP1, to study the requirements for this protein in the folding of firefly luciferase as well as in the prevention of citrate synthase aggregation. In agreement with others who have found peptide binding and limited chaperone ability in fragments of hsp90, we see that multiple fragments from hsp90 can prevent the aggregation of thermally denatured citrate synthase, a measure of passive chaperoning activity. However, in contrast to these results, the luciferase folding assay was found to be much more demanding. Here, folding is mediated by hsp70 and hsp40, requires ATP, and thus is a measure of active chaperoning. Hsp90 and the co-chaperone, Hop, enhance this process. This hsp90 activity was only observed using full-length hsp90 indicating that the cooperation of multiple functional domains is essential for active, chaperone-mediated folding.
Collapse
Affiliation(s)
- B D Johnson
- Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905, USA
| | | | | | | | | | | |
Collapse
|
33
|
Rosser MF, Nicchitta CV. Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding. J Biol Chem 2000; 275:22798-805. [PMID: 10816561 DOI: 10.1074/jbc.m001477200] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.
Collapse
Affiliation(s)
- M F Rosser
- Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, North Carolina 27710, USA
| | | |
Collapse
|
34
|
Meyer BK, Petrulis JR, Perdew GH. Aryl hydrocarbon (Ah) receptor levels are selectively modulated by hsp90-associated immunophilin homolog XAP2. Cell Stress Chaperones 2000; 5:243-54. [PMID: 11005382 PMCID: PMC312890 DOI: 10.1379/1466-1268(2000)005<0243:aharla>2.0.co;2] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
The aryl hydrocarbon receptor (AhR) is a ligand-inducible transcription factor that mediates biological responses to halogenated aromatic hydrocarbons. The unliganded AhR is a cytoplasmic, tetrameric complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). The role of XAP2 as a member of the AhR core complex is poorly understood. XAP2 shares significant homology with the immunophilins FKBP12 and FKBP52, including a highly conserved, C-terminal, tetratricopeptide repeat (TPR) domain. XAP2 forms a complex with hsp90 and the AhR but can also bind to both independently. This binding is mediated by the conserved TPR domain. Single-point mutations in this region are sufficient to disrupt the association of XAP2 with both the AhR and hsp90 in cells. Cotransfection of the AhR and XAP2 in COS-1 cells results in increased AhR levels compared with cells transfected with the AhR alone. In contrast, coexpression of the AhR with the TPR containing proteins FKBP52, protein phosphatase 5 (PP5), or XAP2 TPR-mutants deficient in binding to the AhR and hsp90 does not affect AhR levels and coexpression of the AhR with the TPR domain of PP5 results in AhR down-regulation. These results demonstrate that XAP2 is apparently unique among hsp90-binding proteins in its ability to enhance AhR levels. A yellow fluorescent protein (YFP)-XAP2-FLAG was constructed and biochemically characterized, and no loss of function was detected. YFP-XAP2-FLAG was transiently transfected into NIH 3T3 and was found to localize in both the nucleus and the cytoplasm when visualized by fluorescence microscopy. Treatment of Hepa-1 cells with the hsp90-binding benzoquinone ansamycin, geldanamycin, and the macrocyclic antifungal compound radicicol resulted in AhR but not XAP2 or FKBP52 turnover. Taken together, these results suggest that XAP2/hsp90 and FKBP52/hsp90 complexes are similar yet exhibit unique functional specificity.
Collapse
Affiliation(s)
- Brian K Meyer
- Graduate Program in Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - John R Petrulis
- Center for Molecular Toxicology and the Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Gary H Perdew
- Center for Molecular Toxicology and the Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802
- Correspondence to: Gary H. Perdew, Tel: 814 865-0400; Fax: 814 863-6140; .
| |
Collapse
|
35
|
Felts SJ, Owen BA, Nguyen P, Trepel J, Donner DB, Toft DO. The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties. J Biol Chem 2000; 275:3305-12. [PMID: 10652318 DOI: 10.1074/jbc.275.5.3305] [Citation(s) in RCA: 277] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.
Collapse
Affiliation(s)
- S J Felts
- Department of Biochemistry, Mayo Graduate School, Rochester, Minnesota 55905, USA.
| | | | | | | | | | | |
Collapse
|
36
|
Scheibel T, Weikl T, Rimerman R, Smith D, Lindquist S, Buchner J. Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae. Mol Microbiol 1999; 34:701-13. [PMID: 10564510 DOI: 10.1046/j.1365-2958.1999.01632.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.
Collapse
Affiliation(s)
- T Scheibel
- Institut für Organische Chemie und Biochemie, Technische Universität München, 85747 Garching, Germany
| | | | | | | | | | | |
Collapse
|
37
|
Schulte TW, Akinaga S, Murakata T, Agatsuma T, Sugimoto S, Nakano H, Lee YS, Simen BB, Argon Y, Felts S, Toft DO, Neckers LM, Sharma SV. Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones. Mol Endocrinol 1999; 13:1435-48. [PMID: 10478836 DOI: 10.1210/mend.13.9.0339] [Citation(s) in RCA: 102] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.
Collapse
Affiliation(s)
- T W Schulte
- Medicine Branch, National Cancer Institute, National Institutes of Health, Rockville, Maryland 20850, USA
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
38
|
Wordinger RJ, Clark AF. Effects of glucocorticoids on the trabecular meshwork: towards a better understanding of glaucoma. Prog Retin Eye Res 1999; 18:629-67. [PMID: 10438153 DOI: 10.1016/s1350-9462(98)00035-4] [Citation(s) in RCA: 175] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Glucocorticoid effects on the human trabecular meshwork can be used as a model system in which to study glaucomatous damage to the trabecular meshwork. One of the most important risk factors for glaucoma is an elevated intraocular pressure. The administration of glucocorticoids also can cause elevated intraocular pressure in some individuals. In addition, there is suggestive evidence linking glucocorticoids with the development of glaucoma. Glucocorticoids cause multiple effects on the human trabecular meshwork including changes in extracellular matrix metabolism, organisation of the cytoskeleton, and changes in gene expression and cell function. New discoveries on the molecular mechanisms of glucocorticoid receptor action provide new opportunities to study the possible role of this receptor in the development of glaucoma. For example, alternate spliced forms of the glucocorticoid receptor, glucocorticoid receptor response element half-sites, numerous modulatory factors, and direct effects of nuclear transcription factors have been recently described. Other recent information has shown that the new glaucoma gene (GLC1A/myocilin) is induced in the human trabecular meshwork by glucocorticoids. Although the exact function of myocilin is currently unknown, it offers the opportunity to dissect the molecular pathways regulating aqueous humor outflow. Future challenges include determining (1) which glucocorticoid effects in the human trabecular meshwork are responsible for elevated intraocular pressure; and (2) the significance of these findings to the development of glaucoma.
Collapse
Affiliation(s)
- R J Wordinger
- Department of Anatomy and Cell Biology, University of North Texas, Health Science Center, Fort Worth 76107, USA.
| | | |
Collapse
|
39
|
Hon T, Hach A, Tamalis D, Zhu Y, Zhang L. The yeast heme-responsive transcriptional activator Hap1 is a preexisting dimer in the absence of heme. J Biol Chem 1999; 274:22770-4. [PMID: 10428861 DOI: 10.1074/jbc.274.32.22770] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.
Collapse
Affiliation(s)
- T Hon
- Department of Biochemistry, New York University Medical Center, New York, New York 10016, USA
| | | | | | | | | |
Collapse
|
40
|
Liu XL, Xiao B, Yu ZC, Guo JC, Zhao QC, Xu L, Shi YQ, Fan DM. Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells. World J Gastroenterol 1999; 5:199-208. [PMID: 11819430 PMCID: PMC4688469 DOI: 10.3748/wjg.v5.i3.199] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.
METHODS: A 1.03 kb cDNA sequence of Hsp90β was obtained from the primary plasmid phHSP90 by EcoRI and BamHI nuclease diges tion and was cloned to the EcoRI and BamHI site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90β expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protecti on assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC ) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.
RESULTS: In EcoRI and BamHI restriction analysis, the size and the direction of the cloned sequence of Hsp90β remained what had been designed and the gene constructs were named pcDNA-Hsp90. AH-SGC790, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G1 phase cells were increased; S phase and G2 phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G1 phase cells were decreased, G2 phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G1, S and G2 phase cells remai ned unchanged as compared with their parental cell lines. The sensitivity of AH SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P < 0.05).
CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.
Collapse
|
41
|
Hartson SD, Thulasiraman V, Huang W, Whitesell L, Matts RL. Molybdate inhibits hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substrates. Biochemistry 1999; 38:3837-49. [PMID: 10090774 DOI: 10.1021/bi983027s] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
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. We conclude 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 for hsp90 function.
Collapse
Affiliation(s)
- S D Hartson
- Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078-3035, USA.
| | | | | | | | | |
Collapse
|
42
|
Lim CS, Baumann CT, Htun H, Xian W, Irie M, Smith CL, Hager GL. Differential localization and activity of the A- and B-forms of the human progesterone receptor using green fluorescent protein chimeras. Mol Endocrinol 1999; 13:366-75. [PMID: 10076994 DOI: 10.1210/mend.13.3.0247] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.
Collapse
Affiliation(s)
- C S Lim
- Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, Maryland 20892-5055, USA
| | | | | | | | | | | | | |
Collapse
|
43
|
Reddy RK, Kurek I, Silverstein AM, Chinkers M, Breiman A, Krishna P. High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate. PLANT PHYSIOLOGY 1998; 118:1395-401. [PMID: 9847114 PMCID: PMC34756 DOI: 10.1104/pp.118.4.1395] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/17/1998] [Accepted: 09/08/1998] [Indexed: 05/17/2023]
Abstract
In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.
Collapse
Affiliation(s)
- R K Reddy
- Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7
| | | | | | | | | | | |
Collapse
|
44
|
Sőti C, Csermely P. Characterization of the nucleotide binding properties of the 90 kDa heat shock protein (Hsp90). J Biosci 1998. [DOI: 10.1007/bf02936127] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
|
45
|
Csermely P, Schnaider T, Soti C, Prohászka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998; 79:129-68. [PMID: 9749880 DOI: 10.1016/s0163-7258(98)00013-8] [Citation(s) in RCA: 755] [Impact Index Per Article: 28.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The 90-kDa molecular chaperone family (which comprises, among other proteins, the 90-kDa heat-shock protein, hsp90 and the 94-kDa glucose-regulated protein, grp94, major molecular chaperones of the cytosol and of the endoplasmic reticulum, respectively) has become an increasingly active subject of research in the past couple of years. These ubiquitous, well-conserved proteins account for 1-2% of all cellular proteins in most cells. However, their precise function is still far from being elucidated. Their involvement in the aetiology of several autoimmune diseases, in various infections, in recognition of malignant cells, and in antigen-presentation already demonstrates the essential role they likely will play in clinical practice of the next decade. The present review summarizes our current knowledge about the cellular functions, expression, and clinical implications of the 90-kDa molecular chaperone family and some approaches for future research.
Collapse
Affiliation(s)
- P Csermely
- Department of Medical Chemistry, Semmelweis University, Budapest, Hungary
| | | | | | | | | |
Collapse
|
46
|
Affiliation(s)
- I Yahara
- Tokyo Metropolitan Institute of Medical Science, Japan.
| | | | | |
Collapse
|
47
|
Affiliation(s)
- U Gehring
- Institut für Biologische Chemie, Universität Heidelberg, Germany
| |
Collapse
|
48
|
Johnson BD, Schumacher RJ, Ross ED, Toft DO. Hop modulates Hsp70/Hsp90 interactions in protein folding. J Biol Chem 1998; 273:3679-86. [PMID: 9452498 DOI: 10.1074/jbc.273.6.3679] [Citation(s) in RCA: 272] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.
Collapse
Affiliation(s)
- B D Johnson
- Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905, USA
| | | | | | | |
Collapse
|
49
|
Klinge CM, Brolly CL, Bambara RA, Hilf R. hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in vitro. J Steroid Biochem Mol Biol 1997; 63:283-301. [PMID: 9459195 DOI: 10.1016/s0960-0760(97)00091-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [3H]estradiol (E2) or [3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.
Collapse
Affiliation(s)
- C M Klinge
- Department of Biochemistry, The University of Louisville School of Medicine, KY 40292, USA.
| | | | | | | |
Collapse
|
50
|
Gong W, Chávez S, Beato M. Point mutation in the ligand-binding domain of the progesterone receptor generates a transdominant negative phenotype. Mol Endocrinol 1997; 11:1476-85. [PMID: 9280063 DOI: 10.1210/mend.11.10.9991] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
A short conserved alpha-helix in the carboxyl-terminal activation function of the ligand-binding domain of steroid hormone receptors, called AF2, is important for ligand-dependent transactivation of inducible genes. We have generated two AF2 mutants of the B isoform of human progesterone receptor (PRB): a point mutant, PRBE911A, and a short deletion, PRB delta907-913R. The two mutants are expressed at levels comparable to the wild type receptor in transfected cells. The PRBE911A mutant showed similar hormone- and DNA- binding affinities as the wild type receptor, whereas the PRB delta907-913R mutant was defective in hormone and DNA binding. Both mutants were inactive when transiently transfected in CV-1 cells, which do not express endogenous PR. However, the point mutant, but not the deletion mutant, inhibited transactivation by cotransfected wild type PRB in a hormone-dependent fashion. The activity of endogenous PR in T47D cells or of endogenous glucocorticoid receptor in HeLa cells was also inhibited by the PRBE911A, but not by the deletion mutant. The point mutant was less active when introduced into an N-terminal truncated form of PR, where it gave rise to proteins that formed homodimers with poor affinity for DNA, but were able to form heterodimers with PRB. The negative dominant phenotype of the PRBE911A mutant likely originates from competition with wild type receptors for binding to DNA and will be useful for mechanistic studies of receptor function.
Collapse
Affiliation(s)
- W Gong
- Institut für Molekularbiologie und Tumorforschung, Philipps Universität, Marburg, Germany
| | | | | |
Collapse
|