GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the lAP family members and is required for GIDE＇s E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE- induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over-or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.     

TRIP12 functions as an E3 ubiquitin ligase of APP-BP1

The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hip-pel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Identification of a novel E3 ubiquitin ligase that is required for suppression of premature senescence in Arabidopsis

During leaf senescence, resources are recycled by redistribution to younger leaves and reproductive organs. Candidate pathways for the regulation of onset and progression of leaf senescence include ubiquitin‐dependent turnover of key proteins. Here, we identified a novel plant U‐box E3 ubiquitin ligase that prevents premature senescence in Arabidopsis plants, and named it SENESCENCE‐ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1). Using in vitro ubiquitination assays, we show that SAUL1 has E3 ubiquitin ligase activity. We isolated two alleles of saul1 mutants that show premature senescence under low light conditions. The visible yellowing of leaves is accompanied by reduced chlorophyll content, decreased photochemical efficiency of photosystem II and increased expression of senescence genes. In addition, saul1 mutants exhibit enhanced abscisic acid (ABA) biosynthesis. We show that application of ABA to Arabidopsis is sufficient to trigger leaf senescence, and that this response is abolished in the ABA‐insensitive mutants abi1‐1 and abi2‐1, but enhanced in the ABA‐hypersensitive mutant era1‐3. We found that increased ABA levels coincide with enhanced activity of Arabidopsis aldehyde oxidase 3 (AAO3) and accumulation of AAO3 protein in saul1 mutants. Using label transfer experiments, we showed that interactions between SAUL1 and AAO3 occur. This suggests that SAUL1 participates in targeting AAO3 for ubiquitin‐dependent degradation via the 26S proteasome to prevent premature senescence.     

TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase

Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the “USP-like-TRIM” which regulates the activity of associated TRIM proteins.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

AtCHIP functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits and alters plant response to abscisic acid treatment

CHIP proteins are E3 ubiquitin ligases that promote degradation of Hsp70 and Hsp90 substrate proteins through the 26S proteasome in animal systems. A CHIP-like protein in Arabidopsis, AtCHIP, also has E3 ubiquitin ligase activity and has important roles to play under conditions of abiotic stress. In an effort to study the mode of action of AtCHIP in plant cells, proteins that physically interact with it were identified. Like its animal orthologs, AtCHIP interacts with a unique class of ubiquitin-conjugating enzymes (UBC or E2) that belongs to the stress-inducible UBC4/5 class in yeast. AtCHIP also interacts with other proteins, including an A subunit of protein phosphatase 2A (PP2A). This PP2A subunit appears to be a substrate of AtCHIP, because it can be ubiquitylated by AtCHIP in vitro and because the activity of PP2A is increased in AtCHIP-overexpressing plants in the dark or under low-temperature conditions. Unlike the rcn1 mutant, that has reduced PP2A activity due to a mutation in one of the A subunit genes of PP2A, AtCHIP-overexpressing plants are more sensitive to ABA treatment. Since PP2A was previously shown to be involved in low-temperature responses in plants, the low-temperature-sensitive phenotype observed in AtCHIP-overexpressing plants might be partly due to the change in PP2A activity. These data suggest that the E3 ubiquitin ligase AtCHIP may function upstream of PP2A in stress-responsive signal transduction pathways under conditions of low temperature or in the dark.     

Apoptosis by aloe-emodin is mediated through down-regulation of calpain-2 and ubiquitin-protein ligase E3A in human hepatoma Huh-7 cells

Natural flavonoids are associated with anti-proliferation of cancer growth. However, the antioxidant and anti-proliferation effects of AE (aloe-emodin) have not been well studied. We have investigated how AE affects the proliferation of hepatic hepatocellular carcinoma cells and exerts an anti-cancer effect. The cytotoxic effect of AE was demonstrated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Huh-7 cells were inhibited by AE treatment in both dose- and time-dependent manners. The IC(50) level of AE was ～75 μM. AE also has anti-proliferative effects via induction of DNA damage and apoptosis. 2-DE (two-dimensional electrophoresis) revealed that several proteins were related to the anti-cancer effects of AE. CAPN2 (calpain-2) and UBE3A (ubiquitin-protein ligase E3A), which are associated with the apoptosis signalling pathway, were verified by Western blotting. AE exhibited potent anti-proliferative effects on Huh-7 cells via down-regulation of CAPN2 and UBE3A. The findings support the possibility of AE being a chemopreventative agent.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

Mammalian Numb is a target protein of Mdm2, ubiquitin ligase

Drosophila Numb protein functions as an antagonist against Notch signal. The expression of this protein is asymmetrical in divided cells and thought to be involved in the neural cell differentiation and/or cell fate. Human homologue of Numb (hNumb) was cloned as Mdm2-binding protein by yeast two-hybrid screening. Since Mdm2 is an oncoprotein and has ubiquitin ligase activity toward tumor suppressor p53, we assessed to find out whether Mdm2 ubiquitinylates the hNumb protein. The recombinant hNumb expressed in Sf-9 cells using baculovirus protein expression system bound to Mdm2 in vitro. When hNumb was subjected to in vitro ubiquitinylation assay system, which contains E1, E2, or UbcH5c, and Mdm2, hNumb was ubiquitinylated as efficiently as the p53 protein. However, when the Ring-finger domain mutant of Mdm2 was used in place of wild-type Mdm2, hNumb was not ubiquitinylated. Furthermore, when U2OS cells were co-transfected with hNumb and Mdm2, the hNumb protein was ubiquitinylated and degraded. These data strongly suggest that Mdm2 functions as the ubiquitin ligase toward hNumb and that it induces its degradation in intact cells.     

Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin

Trim32 belongs to the tripartite motif (TRIM) protein family, which is characterized by a common domain structure composed of a RING-finger, a B-box, and a coiled-coil motif. In addition to these motifs, Trim32 possesses six C-terminal NHL-domains. A point mutation in one NHL domain (D487N) has been linked to two forms of muscular dystrophy called limb girdle muscular dystrophy type 2H and sarcotubular myopathy. In the present study we demonstrate that Trim32 is an E3 ubiquitin ligase that acts in conjunction with ubiquitin-conjugating enzymes UbcH5a, UbcH5c, and UbcH6. Western blot analysis showed that Trim32 is expressed primarily in skeletal muscle, and revealed its differential expression from one muscle to another. The level of Trim32 expression was elevated significantly in muscle undergoing remodeling due to changes in weight bearing. Furthermore, expression of Trim32 was induced in myogenic differentiation. Thus, variability in Trim32 expression in different skeletal muscles could be due to induction of Trim32 expression upon changes in physiological conditions. We show that Trim32 associates with skeletal muscle thick filaments, interacting directly with the head and neck region of myosin. Our data indicate that myosin is not a substrate of Trim32; however, Trim32 was found to ubiquitinate actin in vitro and to cause a decrease in the level of endogenous actin when transfected into HEK293 cells. In conclusion, our results demonstrate that Trim32 is a ubiquitin ligase that is expressed in skeletal muscle, can be induced upon muscle unloading and reloading, associates with myofibrils and is able to ubiquitinate actin, suggesting its likely participation in myofibrillar protein turnover, especially during muscle adaptation.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

CK2 phosphorylation of SAG at Thr10 regulates SAG stability,but not its E3 ligase activity

Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAG^Thr10. Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG’s stability, rather than its enzymatic activity directly.