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.     

The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

Oxidative stress injury is involved in many cardiovascular diseases, like hypertension and myocardial infarction. Ubiquitination is a ubiquitous protein post‐translational modification that controls a wide range of biological functions and plays a crucial role in maintaining the homeostasis of cells in physiology and disease. Many studies have shown that oxidative stress damage is inextricably linked to ubiquitination. We demonstrate that Smurf2, an E3 ubiquitinated ligase, is involved in HUVEC apoptosis induced by oxidative stress to alleviate H₂O₂‐induced reactive oxygen species (ROS) production and the apoptosis of human umbilical vein endothelial cells (HUVECs). At the same time, we found that Smurf2 can bind the poly(ADP‐ribose) polymerase‐1(PARP1), and the interaction is enhanced under the stimulation of oxidative stress. We further study and prove that Smurf2 can promote PARP1 ubiquitination and degradation. Collectively, we demonstrate Smurf2 degradation of overactivated PARP1 by ubiquitin‐proteasome pathway to protect HUVEC and alleviate oxidative stress injury.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

Tripartite motif (TRIM) proteins comprise a large family of RING‐type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled‐coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher‐order oligomerization of the basal coiled‐coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.     

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.     

The ubiquitin E3 ligase TRIM10 promotes STING aggregation and activation in the Golgi apparatus

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E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.     

Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The development of a polarised morphology with multiple dendrites and a single axon is an essential step in the differentiation of neurons. The establishment of neuronal polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process during the establishment of neuronal polarity where it specifies axonal identity. Here, we show that the ubiquitin ligases Smad ubiquitination regulatory factor-1 (Smurf1) and Smurf2 are essential for neurite growth and neuronal polarity, respectively, and regulate the GTPases Rho and Rap1B in hippocampal neurons. Smurf2 is required for the restriction of Rap1B to a single neurite. Smurf2 ubiquitinates inactive Rap1B and initiates its degradation through the ubiquitin/proteasome pathway (UPS). Degradation of Rap1B restricts it to a single neurite and thereby ensures that neurons extend a single axon.     

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.     

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.     

Arrestin domain‐containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the β2‐adrenergic receptor

Prolonged stimulation of the β2‐adrenergic receptor (β2AR) leads to receptor ubiquitination and downregulation. Using a genome‐wide RNA interference screen, we identified arrestin domain‐containing 3 (ARRDC3) as a gene required for β2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co‐localizes with activated β2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and β2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)‐mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for β2AR ubiquitination.     

TRIM32 ubiquitin E3 ligase,one enzyme for several pathologies: From muscular dystrophy to tumours

TRIM32 is a member of the TRIpartite Motif family characterised by the presence of an N-terminal three-domain-module that includes a RING domain, which confers E3 ubiquitin ligase activity, one or two B-box domains and a Coiled-Coil region that mediates oligomerisation. Several TRIM32 substrates were identified including muscular proteins and proteins involved in cell cycle regulation and cell motility. As ubiquitination is a versatile post-translational modification that can affect target turnover, sub-cellular localisation or activity, it is likely that diverse substrates may be differentially affected by TRIM32-mediated ubiquitination, reflecting its multi-faceted roles in muscle physiology, cancer and immunity. With particular relevance for muscle physiology, mutations in TRIM32 are associated with autosomal recessive Limb-Girdle Muscular Dystrophy 2H, a muscle-wasting disease with variable clinical spectrum ranging from almost asymptomatic to wheelchair-bound patients. In this review, we will focus on the ability of TRIM32 to mark specific substrates for proteasomal degradation discussing how the TRIM32-proteasome axis may (i) be important for muscle homeostasis and for the pathogenesis of muscular dystrophy; and (ii) define either an oncogenic or tumour suppressive role for TRIM32 in the context of different types of cancer.     

E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation

Psoriasis is mainly characterized by abnormal hyperplasia of keratinocytes and immune cells infiltrating into the dermis and epidermis. Neural precursor cell expressed developmentally downregulated 4‐like (NEDD4L) is a highly conserved HECT type E3 ligase that plays an important role in regulating physiological and pathological processes. Here, we identify NEDD4L as a negative regulator of psoriasis. Nedd4l significantly inhibits imiquimod (IMQ)‐induced skin hyperplasia, and this effect is attributed to the inhibitory effect of NEDD4L on IL‐6/GP130 signaling in keratinocytes. Mechanistically, NEDD4L directly interacts with GP130 and mediates its Lys‐27‐linked ubiquitination and proteasomal degradation. Moreover, the expression of NEDD4L is downregulated in the epidermis from IMQ‐treated mice and psoriasis patients and negatively correlates with the protein levels of GP130 and p‐STAT3 in clinical samples. Collectively, we uncover an inhibitory role of NEDD4L in the pathogenesis of psoriasis and suggest a new therapeutic strategy for the treatment of psoriasis.