An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

Ostrovsky, Olga; Makarewich, Catherine A.; Snapp, Erik L.; Argon, Yair

doi:10.1073/pnas.0902626106

Cited by 62 publications

(70 citation statements)

References 46 publications

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“…However, the mean D value of H53Q ERdj3-sfGFP is still much lower than we would anticipate for a soluble lumenal protein. For example, even the 210 kDa dimer GRP94-mGFP diffuses slightly faster than ERdj3-sfGFP (Ostrovsky et al, 2009). Thus, association with BiP contributes to low ERdj3-sfGFP mobility, but is not the primary regulator of ERdj3-sfGFP diffusion in the ER.…”

Section: Erdj3 Binds the Sec61 Translocon 1431mentioning

confidence: 99%

“…Thus, even modest changes in D values can reflect biologically significant changes (Lai et al, 2010). This approach has been successfully applied to study other ER chaperones, including calreticulin, GRP94, and BiP (Lai et al, 2010;Lai et al, 2012;Lajoie et al, 2012;Ostrovsky et al, 2009;Snapp et al, 2006)In addition, an inert probe, ER-RFP that does not interact with other proteins in the ER lumen, is used to report on changes in ER crowdedness (Dayel et al, 1999;Lai et al, 2010;Snapp et al, 2006).…”

Section: Rationale and Experimental Approachmentioning

confidence: 99%

“…Endoplasmic reticulum (ER) chaperones appear to be mobile and can diffuse to clients (i.e. substrates for ERdj3), even those clients still associated with the relatively immobile translocation channel (Blais et al, 2010;Borgese et al, 2006;Nikonov et al, 2002;Ostrovsky et al, 2009). A number of chaperone co-factors, termed co-chaperones, regulate chaperone activities (Echtenkamp and Freeman, 2012;Laufen et al, 1999;Otero et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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ERdj3 Regulates BiP Occupancy in Living Cells

Guo

Snapp

2013

Journal of Cell Science

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SummaryCo-chaperones regulate chaperone activities and are likely to impact a protein-folding environment as much as the chaperone itself. As co-chaperones are expressed substoichiometrically, the ability of co-chaperones to encounter a chaperone is crucial for chaperone activity. ERdj3, an abundant soluble endoplasmic reticulum (ER) co-chaperone of the Hsp70 BiP, stimulates the ATPase activity of BiP to increase BiP's affinity for client (or substrate) proteins. We investigated ERdj3 availability, how ERdj3 levels impact BiP availability, and the significance of J proteins for regulating BiP binding of clients in living cells. FRAP analysis revealed that overexpressed ERdj3-sfGFP dramatically decreases BiP-GFP mobility in a client-dependent manner. By contrast, ERdj3-GFP mobility remains low regardless of client protein levels. Native gels and co-immunoprecipitations established that ERdj3 associates with a large complex including Sec61a. Translocon binding probably ensures rapid encounters between emerging nascent peptides and stimulates BiP activity in the crucial early stages of secretory protein folding. Importantly, mutant BiP exhibited significantly increased mobility when it could not interact with any ERdjs. Thus, ERdjs appear to play the dual roles of increasing BiP affinity for clients and regulating delivery of clients to BiP. Our data suggest that BiP engagement of clients is enhanced in ER subdomains enriched in ERdj proteins.

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Section: Erdj3 Binds the Sec61 Translocon 1431mentioning

confidence: 99%

Section: Rationale and Experimental Approachmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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ERdj3 Regulates BiP Occupancy in Living Cells

Guo

Snapp

2013

Journal of Cell Science

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“…The function of HSP70 family members appears to revolve around cross-talk between ATPase activity in the nucleotide binding domain and substrate binding in the substrate binding domain. Thus, mutants of HSP70 or of other family members such as the glucose-regulated protein 94 (GRP94) defective in ATP hydrolysis are completely deficient in chaperone activities and unable to support cell survival (11). Although specific substrate proteins of HSP70 are expected and should be uniquely required for survival of transformed cells, the identity of these target proteins remains elusive.…”

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confidence: 99%

HSP70 Protein Promotes Survival of C6 and U87 Glioma Cells by Inhibition of ATF5 Degradation

Guan

et al. 2011

Journal of Biological Chemistry

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Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.Glioblastoma is the most common form of primary brain tumor that responds poorly to conventional therapy. The average survival time from diagnosis is less than 1 year (1, 2). These tumors, arising either from astrocytes or their progenitor cells, escape most normal cell growth and cell death control mechanisms (3).The stress-inducible HSP70 2 (also known as HSP72 or iHSP70) is a molecular chaperone that plays an essential role in protein folding, stability, and turnover. HSP70 is highly expressed in malignant tumors of various origins, and its expression correlates with increased cell proliferation, poor differentiation, lymph node metastases, and poor therapeutic outcome in human cancer (4 -7). In support of its role in promoting tumorigenesis via its prosurvival function, overexpression of HSP70 effectively inhibits cell death induced by a wide range of stimuli, including several cancer-related stresses like hypoxia, inflammatory cytokines, monocytes, irradiation, oxidative stress, and anticancer drugs. Indicating a cancer-specific cell survival function of HSP70, depletion of HSP70 induces massive apoptotic death in tumorigenic cells but not in nontumorigenic epithelial cells or embryonic fibroblasts (8, 9). These lines of evidence suggest that HSP70 is required for protecting certain vulnerable proteins whose function is essential and specific for the survival of cancer cells. The HSP70 molecule, as all other members of the HSP70 family, has a modular structure that consists of an N-terminal nucleotide binding domain and a C-terminal substrate binding domain, connected by a short linker (10). The...

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“…61 The most important biological activity of GRP94 is its role as a master molecular chaperone, directing the folding and/or assembly of secreted or membrane proteins. This essential role is dependent on GRP94 ATPase activity in vivo 62 and results in the folding of a select group of client proteins -the insulin-like growth factors (IGFs) I-III, immunoglobulins, some integrins, and TLRs 55 and lipoprotein receptor-related protein (LRP) 6.…”

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confidence: 99%

Hsp90 as a therapeutic target in endocrinology: current evidence

Ratajczak¹,

Ward²,

Walsh³

et al. 2015

RRED

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Abstract:The ability of heat shock protein 90 (Hsp90) to modulate many growth and signaling pathways simultaneously makes it an attractive target in the field of cancer therapeutics and provided the initial impetus for significant efforts over the past decade to identify Hsp90 inhibitors, several of which are now showing promise in the clinic for cancer treatment. The four known human Hsp90 members are compartmentalized: Hsp90α and β in the cytoplasm, GRP94 in the endoplasmic reticulum, and TRAP1 in the mitochondrial matrix. While these isoforms share a similar N-terminal domain adenosine triphosphate-binding pocket, structural variations allow unique interactions for inhibitors targeting this binding site, providing an avenue for the development of paralog-selective drugs with different biological effects applicable therapeutically to a wide range of diseases. At the same time, the conformational flexibility of the Hsp90 molecular chaperone has unveiled multiple small-molecule target sites within all subdomains of the protein, greatly expanding opportunities for viable drug development. This review summarizes the function, expression, and clinical significance of the Hsp90 isoforms and elaborates on the inhibitors and modulators that impact Hsp90 chaperone activity. Finally, the review focuses on the therapeutic utility of a range of Hsp90-modulating agents in the treatment of specific diseases associated with the endocrine system.

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An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

Cited by 62 publications

References 46 publications

ERdj3 Regulates BiP Occupancy in Living Cells

ERdj3 Regulates BiP Occupancy in Living Cells

HSP70 Protein Promotes Survival of C6 and U87 Glioma Cells by Inhibition of ATF5 Degradation

Hsp90 as a therapeutic target in endocrinology: current evidence

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