A foldable CFTRΔF508 biogenic intermediate accumulates upon inhibition of the Hsc70–CHIP E3 ubiquitin ligase

Younger, J. Michael; Ren, Hong Yu; Chen, Liling; Fan, Chun Yang; Fields, Andrea; Patterson, Cam; Cyr, Douglas M.

doi:10.1083/jcb.200410065

Cited by 163 publications

(206 citation statements)

References 45 publications

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“…CHIP interacts with HSC70 and HSP90 through its amino-terminal TPR domain, and the ubiquitin ligating reaction is performed by a U-box motif in its carboxy terminus. In association with these chaperones, CHIP plays an essential role in the quality control of misfolded proteins (Tateishi et al, 2004;Younger et al, 2004Younger et al, , 2006Qian et al, 2006).Here, we report that many of the MKKS mutants that are responsible for the MKKS/BBS diseases are degraded in the cell much more rapidly than the wild-type protein and that a CHIP-dependent ubiqutin-proteasome pathway plays a crucial role in this rapid degradation. Although the wildtype MKKS rapidly shuttles between the centrosome and cytosol, rapidly degraded MKKS mutants often fail to localize to the centrosome.…”

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confidence: 72%

“…The CHIP-dependent degradation of misfolded protein has been well-studied in cystic fibrosis transmembrane conductance regulator (CFTR) ⌬508 mutant proteins (Tateishi et al, 2004;Younger et al, 2004Younger et al, , 2006Qian et al, 2006), which make insoluble aggregates in the ER (Meacham et al, 2001). However, the role of CHIP in cystic fibrosis seems to differ from its role in BBS.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

Hirayama

Yamazaki²,

Kitamura³

et al. 2008

MBoC

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McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases. INTRODUCTIONMcKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease predominantly characterized by developmental anomalies, including vaginal atresia with hydrometrocolpos, polydactyly, and congenital heart defects (McKusick et al., 1964). The MKKS gene encodes a 570-amino acid polypeptide with weak but significant similarity to group II chaperonins . Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder characterized by obesity, retinal dystrophy, polydactyly, mental retardation, renal malformation, and hypogenitalism (Beales et al., 1999), and thus MKKS is also called BBS6. The MKKS mRNA is widely expressed in various tissues, including those affected by MKKS and BBS diseases . Although more than 10 mutations in the MKKS gene have been identified in patients (Beales et al., 2001;Slavotinek et al., 2002), little is known about how they cause MKKS and BBS.Twelve of the genes that are responsible for BBS (BBS1-12) have been identified so far, and the products of several of these genes have been suggested to be involved in intraflagellar transport in cilia and intracellular trafficking in the cytosol Badano et al., 2005;Beales, 2005;Blacque and Leroux, 2006). Cargo-carrying motors play crucial roles in intraflagellar and intracellular transport systems by bidirectionally moving on microtubules, and BBS4 interacts with the dynactin complex, which mediates interactions between cargoes and the dynein motor (Kim et al., 2004). Retrograde transport of melanosomes in zebrafish is slowed by knockdown of BBS2 or BBS4-8 (Yen et al., 2006), whereas mutations in Caenorhabditis elegans BBS7 and BBS8 cause delays in intraflagellar transport driven by kinesin motors (Ou et al., 2005). The Chlamydomonas homologue of BBS5 is essential for flagellum formation (Li et al., ...

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confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

Hirayama

Yamazaki²,

Kitamura³

et al. 2008

MBoC

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“…Findings supporting this conclusion include: (a) CspH43Q, a mutant of Csp that binds Hsc70/Hsp70 poorly, retained its ability to co-immunoprecipitate CHIP, and (b) purified CHIP and CspWT or CspH43Q proteins interacted in an in vitro binding assay. This novel interaction of Csp with CHIP suggests that Csp links CFTR to the ubiquitin-proteasome system in a manner similar to the pro-degradative function of Hdj-2 (37).…”

Section: Discussionmentioning

confidence: 99%

Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Schmidt

Watts

Aridor

et al. 2009

Journal of Biological Chemistry

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Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/ Hsp70 and CHIP-mediated CFTR degradation.Cysteine string protein (Csp, DnaJC5) 2 is a member of the DnaJ/Hsp40 protein chaperone family (1, 2). Csp contains a short N-terminal sequence, followed by the J-domain, a linker region, and the central cysteine-rich region, which gives these proteins their name. The cysteine residues in this region are post-translationally modified by the palmitate groups that serve for the membrane attachment of Csp (3). This structure is followed by a C-terminal domain, which is the most variable region among the three Csp paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic junctions of Drosophila neurons (4). Csps are expressed at high levels in neurons and in cell types involved in regulated exocytosis (5-7), where they have been shown to govern exocytic secretory functions. For example, depolarizationinduced synaptic vesicle exocytosis is impaired in neurons from Csp-deficient Drosophila (8), and Csp overexpression produced decreases in stimulated insulin release from ␤-cells and stimulated catecholamine release from chromaffin cells (9 -11). Deletion of the Csp gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder characterized by developing neurodegenerative changes (12, 13).As for other Hsp40 homologs, the J-domain is responsible for the ability of Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity (14). Similar to other chaperone proteins, Csp can be found in many different protein complexes in the...

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“…Ubiquitin ligases have been implicated in CFTR degradation by the ERAD system (9,(19)(20)(21). J. M. Younger et al detected ubiquitination in NBD1 R fragments by in vitro coincubation with chaperones and ubiquitination enzymes (22). However, the ubiquitination of specific lysine residues in the R domain is uncertain.…”

Section: Resultsmentioning

confidence: 99%

Interference with Ubiquitination in CFTR Modifies Stability of Core Glycosylated and Cell Surface Pools

Lee

Henderson

Schiffhauer

et al. 2014

Molecular and Cellular Biology

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It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band C CFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

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A foldable CFTRΔF508 biogenic intermediate accumulates upon inhibition of the Hsc70–CHIP E3 ubiquitin ligase

Cited by 163 publications

References 45 publications

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Interference with Ubiquitination in CFTR Modifies Stability of Core Glycosylated and Cell Surface Pools

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