The Cytoplasmic Hsp70 Chaperone Machinery Subjects Misfolded and Endoplasmic Reticulum Import-incompetent Proteins to Degradation via the Ubiquitin–Proteasome System

Park, Sae-Hun; Bolender, Natalia; Eisele, Frederik; Kostova, Zlatka; Takeuchi, Junko; Coffino, Philip; Wolf, Dieter H.

doi:10.1091/mbc.e06-04-0338

Cited by 149 publications

(166 citation statements)

References 93 publications

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“…S2 B, C, and E). This was contrary to earlier published work concerning Sse1 and CPY ‡ -GFP (14). Using the in vitro assay, we directly tested the role of Sse1 in Ubr1-mediated ubiquitination of tGnd1.…”

mentioning

confidence: 67%

“…CPY ‡ was fused to GFP, yielding the optically detectable misfolded cytoplasmic protein CPY ‡ -GFP (13,14) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

“…It has been previously reported that degradation of cytoplasmic proteins requires chaperones (5,14), and the Ubr1/San1-dependent substrates appropriately showed a strong dependence on Hsp70 and the Sse1 (Hsp110) cochaperone. However, the role of chaperones in the action of each was distinguishable.…”

mentioning

confidence: 98%

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Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Heck

Cheung

Hampton

2009

Proc. Natl. Acad. Sci. U.S.A.

249

383

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Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the "N-end rule." In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis.chaperone | proteostasis | misfolding P rotein quality control (QC) functions to ensure that damaged and misfolded proteins are maintained at acceptable levels to limit their stress-causing, or proteotoxic, effects. One strategy of protein QC is the selective degradation of misfolded proteins. For degradative QC pathways to be effective, they must be specific for aberrant proteins; sufficiently general to recognize selectively common structural hallmarks shared by numerous unrelated proteins; and physiologically important, better allowing the cell to survive proteotoxic stress. Because protein QC underlies many pressing maladies, such as parkinsonism, cystic fibrosis, and aging, discovery of the rules of substrate selectivity and destruction is a key step in understanding these conditions and designing appropriate therapeutical interventions to combat them.In eukaryotes, the ubiquitin proteasome system is employed in the selective degradation of many proteins (1). A substrate protein is marked for degradation by assembly of a polyubiquitin chain, initiated by covalent addition of the small (7.6 kDa) protein ubiquitin to a lysine in an isopeptide bond, followed by iterative addition of the next ubiquitin to the previously added one to create a polyubiquitin chain that is uniquely recognized by the 26S proteasome. Protein ubiquitination is catalyzed by a three-enzyme cascade. The single E1 ubiquitin-activating enzyme hydrolyzes ATP to acquire ubiquitin in labile thioester linkage, which is then transferred in thioester linkage to one of a small group of E2s or ubiquitin-conjugating enzymes (UBCs). E2-bound ubiquitin is finally transferred to an isopeptide linkage on the target protein or the growing polyubiquitin chain by the action of the E3 ubiquitin ligase. It is the E3 ubiquitin ligase that determines the specificity of a given ubiquitination process; identifying and understanding the E3s involved in a degradative pathway are thus key parts of understanding the mechanisms of substrate selection and modification.E3s ...

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mentioning

confidence: 67%

“…CPY ‡ was fused to GFP, yielding the optically detectable misfolded cytoplasmic protein CPY ‡ -GFP (13,14) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Heck

Cheung

Hampton

2009

Proc. Natl. Acad. Sci. U.S.A.

249

383

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“…During ER translocation, yeast cytosolic chaperones target ER importincompetent proteins to the proteasome (31). In mammalian cells, roles for Hsc70 have been demonstrated in CFTR triage (32) and in cotranslocational destruction of apoB that has stalled in the import translocon (33).…”

Section: Discussionmentioning

confidence: 99%

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Spooner

Hart

Cook

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.Hsc70 ͉ Hsp90 ͉ ricin E ndoplasmic-reticulum (ER) associated protein degradation (ERAD) comprises coordinated disposal systems that recognize and remove misfolded and unassembled proteins in the ER, dislocating them across the ER membrane to the cytosol for proteasomal destruction. Degradation is normally facilitated by polyubiquitylation, usually on internal lysyl residues of the target protein. Both membrane-bound and soluble ER proteins can be disposed of by ERAD (1, 2).The plant cytotoxin ricin traffics to the ER lumen of mammalian cells where it is reduced to its RTA and RTB subunits before RTA dislocation (3). RTA does not penetrate the ER membrane directly; instead it exploits pre-existing proteinconducting channels as a nonnative species, mimicking ER proteins dispatched via ERAD (4, 5). Thus, it enters the cytosol in a form susceptible to proteolysis or aggregation. A proportion must evade these fates to gain a catalytic conformation that depurinates target ribosomes, stopping protein synthesis. The paucity of lysine residues in RTA may facilitate uncoupling from ERAD by reducing the potential for polyubiquitylation, thereby hampering proteasomal degradation (6). This, in turn, may provide opportunities for spontaneous or chaperone-assisted folding not normally sanctioned for ERAD substrates.We show an interaction of RTA with the cytosolic heat shock (cognate) protein Hsc70. From the chaperone-bound state, nonnative cytosolic RTA can achieve a catalytic conformation or can be inactivated. Its ultimate fate depends on the activities of ...

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“…HSP70, the chaperone protein upregulated following treatment with GA, plays a key role in this process. [21][22][23][24] We therefore investigated if the observed increase in cytopathic effect was due to upregulation of the measles H protein expression on the cell surface. Immunoblotting of cell surface-biotinylated proteins immunoprecipitated with an anti-FLAG antibody showed no evidence of increased H expression in combination-treated cells.…”

Section: Combining Measles Virus With Heat Shock Protein Inhibitors Cmentioning

confidence: 99%

Heat shock protein inhibitors increase the efficacy of measles virotherapy

Liu

Erlichman

et al. 2008

Gene Ther

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Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combinationtreated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GAtreated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

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The Cytoplasmic Hsp70 Chaperone Machinery Subjects Misfolded and Endoplasmic Reticulum Import-incompetent Proteins to Degradation via the Ubiquitin–Proteasome System

Cited by 149 publications

References 93 publications

Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Heat shock protein inhibitors increase the efficacy of measles virotherapy

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