BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.     

E3 ubiquitin ligase Cullin4B mediated polyubiquitination of p53 for its degradation

Controlled protein ubiquitination through E3 ubiquitin ligases and degradation via 26S proteasome machinery is required for orderly progression through cell cycle, chromatin remodeling, DNA repair, and development. Each cullin-dependent ubiquitin ligase (E3) complex can recruit various substrates for their degradation. Cullin 4A (CUL4A) and Cullin 4B (CUL4B) are members of cullin family proteins that mediate ubiquitin dependent proteolysis. Though, these two cul4 genes are functionally redundant, Cullin 4B is not a substitute for all the Cullin 4A functions. Published report has shown that CUL4A interacts with p53 and induces its decay. Although, CUL4A has been known to control several cellular processes, little is known about CUL4B functions. Therefore, in this study, we analyzed the role of CUL4B on p53 polyubiquitination. Our stable cell line and transient transfection studies show that CUL4B indeed interacts with p53 and induces its polyubiquitination. Importantly, both CUL4A and CUL4B overexpressing cells show almost equal levels of p53 polyubiquitination. Moreover, we observed an increased level of polyubiquitination on p53 in CUL4B overexpressing stable cell line upon treatment with siRNA specific for CUL4A indicating that CUL4B plays a vital role in p53 stability. In addition, we have observed the differential expression of CUL4B in various eukaryotic cell lines and mouse tissues suggesting the important role of CUL4B in various tissues. Together, these observations establish an important negative regulatory role of CUL4B on p53 stability.     

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.     

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.     

CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s.     

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.     

UHRF2, another E3 ubiquitin ligase for p53

UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.     

The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation

The folding of both wild-type and mutant forms of the cystic-fibrosis transmembrane-conductance regulator (CFTR), a plasma-membrane chloride-ion channel, is inefficient. Most nascent CFTR is retained in the endoplasmic reticulum and degraded by the ubiquitin proteasome pathway. Aberrant folding and defective trafficking of CFTRDeltaF508 is the principal cause of cystic fibrosis, but how the endoplasmic-reticulum quality-control system targets CFTR for degradation remains unknown. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. Here we show that CHIP functions with Hsc70 to sense the folded state of CFTR and targets aberrant forms for proteasomal degradation by promoting their ubiquitination. The U-box appeared essential for this process because overexpresion of CHIPDeltaU-box inhibited the action of endogenous CHIP and blocked CFTR ubiquitination and degradation. CHIP is a co-chaperone that converts Hsc70 from a protein-folding machine into a degradation factor that functions in endoplasmic-reticulum quality control.     

The poxviral RING protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories

The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence.     

Interactions between the quality control ubiquitin ligase CHIP and ubiquitin conjugating enzymes

Background  

Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.     

Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor invitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.     

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.     

The p73 tumor suppressor is targeted by Pirh2 RING finger E3 ubiquitin ligase for the proteasome-dependent degradation

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.