E3 ubiquitin ligase SIAH1 mediates ubiquitination and degradation of TRB3

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.     

Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara

Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara.     

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.     

E3 ubiquitin ligase Grail promotes hepatic steatosis through Sirt1 inhibition

In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.Subject terms: Cell signalling, Metabolic disorders     

LRSAM1 E3 ubiquitin ligase promotes proteasomal clearance of E6-AP protein

Numerous proteins participate and actively contribute to the various cellular mechanisms, where several of them are crucial for regular metabolism, including survival. Thus, to maintain optimal cellular physiology, cells govern protein quality control functions with the assistance of comprehensive actions of molecular chaperones, the ubiquitin-proteasome system, and autophagy. In the ubiquitin-proteasome pathway, few quality control E3 ubiquitin ligases actively participate against misfolded protein aggregation generated via stress conditions. But how these quality control E3s active expression levels returned to basal levels when cells achieved re-establishment of proteostasis is still poorly understood. Our current study demonstrated that LRSAM1 E3 ubiquitin ligase promotes the proteasomal degradation of quality control E3 ubiquitin ligase E6-AP. We have observed the co-localization and recruitment of LRSAM1 with E6-AP protein and noticed that LRSAM1 induces the endogenous turnover of E6-AP. Partial depletion of LRSAM1 elevates the levels of E6-AP and affects overall cell cycle regulatory proteins (p53 and p27) expression, including the rate of cellular proliferation. The current finding also provides an excellent opportunity to better understand the basis of the E6-AP associated pathomechanism of Angelman Syndrome disorder. Additionally, this study touches upon the novel potential molecular strategy to regulate the levels of one quality control E3 ubiquitin ligase with another E3 ubiquitin ligase and restore proteostasis and provide a possible therapeutic approach against abnormal protein aggregation diseases.     

Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.     

MEX is a testis-specific E3 ubiquitin ligase that promotes death receptor-induced apoptosis

In the present study, we report the identification and characterization of MEX (MEKK1-related protein X), a protein with homology to MEKK1 that is expressed uniquely in the testis. MEX is comprises four putative zinc-binding domains including an N-terminal SWIM (SWI2/SNF2 and MuDR) domain of unknown function and two RING (really interesting new gene) fingers separated by a ZZ zinc finger domain. Biochemical analyses revealed that MEX is self-ubiquitinated and targeted for degradation through the proteasome pathway. MEX can act as an E3, Ub (ubiquitin) ligase, through the E2, Ub-conjugating enzymes UbcH5a, UbcH5c or UbcH6. A region of MEX that contains the RING fingers and the ZZ zinc finger was required for interaction with UbcH5a and MEX self-association, whereas the SWIM domain was critical for MEX ubiquitination. The expression of MEX promoted apoptosis that was induced through Fas, DR (death receptor) 3 and DR4 signalling, but not that mediated by the BH3 (Bcl-2 homology 3)-only protein BimEL or the chemotherapeutic drug adriamycin. The enhancement of apoptosis by MEX required a functional SWIM domain, suggesting that MEX ubiquitination is critical for the enhancement of apoptosis. These results indicate that MEX acts as an E3 Ub ligase, an activity that is dependent on the SWIM domain and suggest a role for MEX in the regulation of death receptor-induced apoptosis in the testes.     

CHIP functions an E3 ubiquitin ligase of Runx1

Runx1 is a key factor in the generation and maintenance of hematopoietic stem cells. Improper expression and mutations in Runx1 are frequently implicated in human leukemia. Here, we report that CHIP, the carboxyl terminus of Hsc70-interacting protein, also named Stub1, physically interacts with Runx1 through the TPR and Charged domains in the nucleus. Over-expression of CHIP directly induced Runx1 ubiquitination and degradation through the ubiquitin-proteasome pathway. Interestingly, we found that CHIP-mediated degradation of Runx1 is independent of the molecular chaperone Hsp70/90. Taken together, we propose that CHIP serves as an E3 ubiquitin ligase that regulates Runx1 protein stability via an ubiquitination and degradation mechanism that is independent of Hsp70/90.     

The E3 ubiquitin ligase WVIP2 highlights the versatility of protein ubiquitination

Plant cells regulate many cellular processes controlling the half-life of critical proteins through ubiquitination. Previously, we characterized two interacting RING-type E3 ubiquitin ligases of Triticum durum, TdRF1 and WVIP2. We revealed their role in tolerance to dehydration, and existing knowledge about their partners also indicated their involvement in the regulation of some aspects of plant development. Here we located WVIP2 in the regulation of the ABA signaling, based on sequence similarities. Further we acquired general evidence about the versatility of ubiquitination in plant cells. A protein can be target of different E3 ligases for a perfect tuning of its abundance as well as the same E3 ligase can ubiquitinate different and unrelated proteins, thus representing a cross-connections between different signaling pathways for a global coordination of cellular processes.     

Regulation of interleukin-10 receptor ubiquitination and stability by beta-TrCP-containing ubiquitin E3 ligase

Interleukin-10 (IL-10) initiates potent anti-inflammatory effects via activating its cell surface receptor, composed of IL-10R1 and IL-10R2 subunits. The level of IL-10R1 is a major determinant of the cells' responsiveness to IL-10. Here, via a series of biochemical analyses using 293T cells reconstituted with IL-10R1, we identify the latter as a novel substrate of βTrCP-containing ubiquitin E3 ligase. Within the intracellular tail of IL-10R1, a canonical ((318)DpSGFGpS) and a slightly deviated ((369)DpSGICLQEP) βTrCP recognition motif can additively recruit βTrCP in a phosphorylation-dependent manner. βTrCP recruitment leads to ubiquitination, endocytosis and degradation of IL-10R1, subsequently reducing the cellular responsiveness to IL-10. Our study uncovers a novel negative regulatory mechanism that may potentially affect IL-10 function in target cells under physiological or pathological conditions.     

Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase

Genetic studies identified Itch, which is a homologous to the E6-associated protein carboxyl terminus (Hect) domain-containing E3 ubiquitin-protein ligase that is disrupted in non-agouti lethal mice or Itchy mice. Itch-deficiency results in abnormal immune responses and constant itching in the skin. Here, Itch was shown to associate with Notch, a protein involved in cell fate decision in many mammalian cell types, including cells in the immune system. Itch binds to the N-terminal portion of the Notch intracellular domain via its WW domains and promotes ubiquitination of Notch through its Hect ubiquitin ligase domain. Thus, Itch may participate in the regulation of immune responses by modifying Notch-mediated signaling.     

Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

Background  

Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.