Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.     

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.     

Biochemical and structural studies of a HECT-like ubiquitin ligase from Escherichia coli O157:H7

Many microbial pathogens deliver effector proteins via the type III secretion system into infected host cells. Elucidating the function of these effectors is essential for our understanding of pathogenesis. Here, we describe biochemical and structural characterization of an effector protein (NleL) from Escherichia coli O157:H7, a widespread pathogen causing severe foodborne diseases. We show that NleL functionally and structurally mimics eukaryotic HECT E3 ligases and catalyzes formation of unanchored polyubiquitin chains using Lys(6) and Lys(48) linkage. The catalytic cysteine residue forms a thioester intermediate with ubiquitin. The structure of NleL contains two domains, a β-helix domain formed by pentapeptide repeats and a bilobed catalytic domain reminiscent of the N- and C-lobe architecture of HECT E3s. Six structures of NleL observed in two crystal forms revealed a large range of different positions of the C-lobe relative to the N-lobe, indicating that the helix linking the two lobes is extremely flexible. Comparing the structure of NleL with that of the Salmonella homolog SopA showed that the orientation of the C-lobes differ by as much as 108°, suggesting that large movements of the C-lobe may be required to facilitate the transfer of ubiquitin from E2 to the substrate. These results provide critical knowledge toward understanding the molecular mechanism by which pathogens utilize the host ubiquitination system during infection.     

Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels

Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.     

Membrane-associated RING-CH 10 (MARCH10 protein) is a microtubule-associated E3 ubiquitin ligase of the spermatid flagella

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.     

Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.     

Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation but also releases other Cbl targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGF receptor signaling.     

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.     

RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins

RBX1 (RING box protein 1), also known as ROC1 (Regulator of Cullin 1), is an essential component of SCF (Skp1/Cullins/F-box) E3 ubiquitin ligases, which target diverse proteins for proteasome-mediated degradation. Our recent study showed that RBX1 silencing triggered a DNA damage response (DDR) leading to G(2)-M arrest, senescence, and apoptosis, with the mechanism remaining elusive. Here, we show that, in human cancer cells, RBX1 silencing causes the accumulation of DNA replication licensing proteins CDT1 and ORC1, leading to DNA double-strand breaks, DDR, G(2) arrest, and, eventually, aneuploidy. Whereas CHK1 activation by RBX1 silencing is responsible for the G(2) arrest, enhanced DNA damage renders cancer cells more sensitive to radiation. In Caenorhabditis elegans, RBX-1 silencing causes CDT-1 accumulation, triggering DDR in intestinal cells, which is largely abrogated by simultaneous CDT-1 silencing. RBX-1 silencing also induces lethality during development of embryos and in adulthood. Thus, RBX1 E3 ligase is essential for the maintenance of mammalian genome integrity and the proper development and viability in C. elegans.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation

Substrate-specific protein degradation mediated by the ubiquitin proteasome system (UPS) is crucial for the proper function of the cell. Proteins are specifically recognized and ubiquitinated by the ubiquitin ligases (E3s) and are then degraded by the proteasome. BTB proteins act as the substrate recognition subunit that recruits their cognate substrates to the Cullin 3-based multisubunit E3s. Recently, it was reported that missense mutations in KLHL7, a BTB-Kelch protein, are related to autosomal dominant retinitis pigmentosa (adRP). However, the involvement of KLHL7 in the UPS and the outcome of the adRP causative mutations were unknown. In this study, we show that KLHL7 forms a dimer, assembles with Cul3 through its BTB and BACK domains, and exerts E3 activity. Lys-48-linked but not Lys-63-linked polyubiquitin chain co-localized with KLHL7, which increased upon proteasome inhibition suggesting that KLHL7 mediates protein degradation via UPS. An adRP-causative missense mutation in the BACK domain of KLHL7 attenuated only the Cul3 interaction but not dimerization. Nevertheless, the incorporation of the mutant as a heterodimer in the Cul3-KLHL7 complex diminished the E3 ligase activity. Together, our results suggest that KLHL7 constitutes a Cul3-based E3 and that the disease-causing mutation inhibits ligase activity in a dominant negative manner, which may lead to the inappropriate accumulation of the substrates targeted for proteasomal degradation.     

HECT-type ubiquitin ligase ITCH targets lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) and prevents LAPTM5-mediated cell death

LAPTM5 (lysosomal-associated protein multispanning transmembrane 5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. Although the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is regulated negatively by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death.     

Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells

We have characterized the interaction of syntrophin with F-actin. Subcellular fractionation of cardiac and skeletal muscle tissues showed that alpha-, beta1- and beta2-syntrophins were present in the soluble and the membrane fraction. Syntrophins are known to bind to the dystrophin-glycoprotein complex (DGC), but since the DGC is not present in the soluble fraction, it was concluded that some syntrophin did not associate with the DGC. Native syntrophins purified from the soluble fraction and recombinant syntrophins were both able to bind to F-actin, and binding occurred through several sites on syntrophin, including the second pleckstrin homology domain and the unique carboxyl-terminal domain. Syntrophin was also able to inhibit actin-activated myosin ATPase activity and actomyosin super-precipitation. alpha-Syntrophin co-localized with cortical F-actin fibers when expressed in Chinese hamster ovary cells, and deletion of the actin-binding region abolished co-localization. Most of exogenous or endogenous syntrophin also co-localized with stress fibers in endothelial and smooth muscle (A7r5) cells. However, syntrophins were mostly localized in the cytosol of serum-starved C2C12 or primary cultured skeletal muscle myotubes, and translocated to the membrane upon treatment with lysophosphatidic acid or the actin-stabilizing agent jasplakinolide. The actin-depolymerizing agent latrunculin-B abolished this syntrophin translocation. These findings suggest that syntrophin is an actin-binding protein the subcellular localization of which is regulated through cytoskeletal reorganization.     

RNF170 protein, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity. Depletion of endogenous RNF170 by RNA interference inhibited stimulus-induced IP(3) receptor ubiquitination, and degradation and overexpression of a catalytically inactive RNF170 mutant suppressed stimulus-induced IP(3) receptor processing. A substantial proportion of RNF170 is constitutively associated with the erlin1/2 (SPFH1/2) complex, which has been shown previously to bind to IP(3) receptors immediately after their activation. Depletion of RNF170 did not affect the binding of the erlin1/2 complex to stimulated IP(3) receptors, whereas erlin1/2 complex depletion inhibited RNF170 binding. These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP(3) receptors where it mediates IP(3) receptor ubiquitination. Thus, RNF170 plays an essential role in IP(3) receptor processing via the ubiquitin-proteasome pathway.     

Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection

The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.     

Identification of a novel Bcl-2-interacting mediator of cell death (Bim) E3 ligase, tripartite motif-containing protein 2 (TRIM2), and its role in rapid ischemic tolerance-induced neuroprotection

We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischemia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol-13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2. Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance.     

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) delays cellular senescence by promoting p27(Kip1) degradation in human diploid fibroblasts

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27(Kip1) and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27(Kip1) expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27(Kip1) in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27(Kip1) by promoting p27(Kip1) degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.