The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

Hernandez, Mayda; Sullivan, William P.; Toft, David O.

doi:10.1074/jbc.m206566200

Cited by 171 publications

(180 citation statements)

References 75 publications

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“…The HSP70/HSP90 complex functions cooperatively with other key proteins in order to organize protein folding and renature damaged proteins (Schumacher et al, 1996;Hernandez et al, 2002). HSP90 and HSP70 are both intimately involved in centrosome regulation and mitotic spindle formation and organization (Liang and MacRae, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Both HSP90 and HSP70 are located at and help regulate the centrosome during mitosis (Rattner, 1991;de Carcer et al, 2001). HSP70 and HSP90 work cooperatively in a complex (Schumacher et al, 1996;Hernandez et al, 2002). Thus, direct inhibition of HSP90 may have a much more profound effect on arsenite sensitivity and centrosome regulation than preventing induction of HSP70i.…”

Section: Disruption Of Hsp70/hsp90 Activity Enhances Effectiveness Ofmentioning

confidence: 99%

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Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Taylor

McNeely

Miller

et al. 2008

Toxicology and Applied Pharmacology

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Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of α-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of α-tubulin in mitotic cells showed spindle formation in arsenite-and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. γ-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.

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Section: Discussionmentioning

confidence: 99%

Section: Disruption Of Hsp70/hsp90 Activity Enhances Effectiveness Ofmentioning

confidence: 99%

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Taylor

McNeely

Miller

et al. 2008

Toxicology and Applied Pharmacology

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“…It is possible that only one molecule of hsp70 is bound directly to and interacting productively with the receptor, and that under these conditions of assembly with purified proteins the receptor-bound hsp70 can associate with other molecules of hsp70 to form dimers and trimers, much as purified hsp70 and its homologs have been shown to do in solution (32)(33)(34). It is perhaps important to note that analysis of both the native and assembled hsp90⅐Hop⅐hsp70 machinery has revealed a molar ratio of 1 hsp70 per hsp90 dimer (6,18). If the preassembled machinery can interact with the receptor to produce receptor⅐hsp90 heterocomplexes, then it is reasonable to predict that the initial binding to the receptor and the priming step is by one molecule of hsp70 interacting with one molecule of receptor.…”

Section: Fig 4 Release Of Primed Gr⅐hsp70 Complex and Visualizationmentioning

confidence: 99%

“…Although Hop is not essential for GR⅐hsp90 heterocomplex assembly, when Hop is present to form a machinery, assembly is much faster (16). Hop does not act solely as a tie rod to bring the essential chaperones together; it also influences the conformational state and function of each protein (18). The hsp90⅐Hop⅐hsp70 complexes also contain small amounts of the hsp70 co-chaperone hsp40 (14), and together they form the hsp90/hsp70-based chaperone machinery.…”

mentioning

confidence: 99%

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

Murphy

Morishima

Chen

et al. 2003

Journal of Biological Chemistry

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A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR)⅐hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR⅐hsp70 complex that primes the receptor for subsequent ATPdependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR⅐hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size~1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR⅐hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/ 554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/ 554C, and it is the subsequent assembly step with hsp90 that is defective.Glucocorticoid receptors (GR) 1 are recovered from hormonefree cells as large multiprotein complexes containing a dimer of hsp90 (for review see Refs. 1 and 2). Hsp90 binds to the ligand binding domain (LBD) of the GR, and the steroid binding activity of the GR is absolutely hsp90-dependent (3, 4). When the GR is stripped of its associated hsp90, it immediately loses its ability to bind steroid, and steroid binding activity is regenerated when GR⅐hsp90 heterocomplexes are reformed by the hsp90/hsp70-based chaperone machinery (5, 6). The steroids bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand, such that the LBD must change its conformation to allow entry of ligand (7). The hsp90/hsp70-dependent chaperone machinery carries out the ATP-dependent opening of the binding cleft in the GR LBD such that it can be accessed by steroid. In addition to opening the steroid binding cleft, formation of a complex with hsp90 is accompanied by conformational changes that increase the sensitivity of the GR LBD to attack by thiol-derivatizing agents and trypsin (8 -10).The multiprotein chaperone machinery that forms receptor⅐hsp90 heterocomplexes was originally identified in reticulocyte lysate (11,12). This machinery has been reconstituted (13), and a mixture of five purified proteins (hsp90, hsp70, 2 Hop, hsp40, and p23) is now used to achieve efficient receptor⅐hsp90 heterocomplex assembly (14, 15). The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR⅐hsp90 heterocomplex assembly (16). Hop b...

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“…Hop optimises the functional cooperation between Hsp70 and Hsp90 which requires highly co-ordinated interactions for the folding and conformational regulation of a variety of proteins [5]. The molecular chaperone complex of Hop, Hsp70 and Hsp90 is involved in the folding and maturation of key regulatory proteins involved in cell viability [6], such as steroid hormone receptors, transcription factors and kinases (some of which are involved in cancer progression).…”

Section: Introductionmentioning

confidence: 99%

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

et al. 2011

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Keywords:Hop/STIP1/STI1 (Hsp70/Hsp90-organising protein) Pancreatic cancer Immunohistochemistry In vitro invasion MMP-2 a b s t r a c tWe previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered.In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours.Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

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The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

Cited by 171 publications

References 75 publications

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

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