The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

USP38 promotes deubiquitination of K11-linked polyubiquitination of HIF1α at Lys769 to enhance hypoxia signaling.

HIF1α is one of the master regulators of the hypoxia signaling pathway and its activation is regulated by multiple post-translational modifications (PTMs). Deubiquitination mediated by deubiquitylating enzymes (DUBs) is an essential PTM that mainly modulates the stability of target proteins. USP38 belongs to the ubiquitin-specific proteases (USPs). However, whether USP38 can affect hypoxia signaling is still unknown. In this study, we used quantitative real-time PCR assays to identify USPs that can influence hypoxia-responsive gene expression. We found that overexpression of USP38 increased hypoxia-responsive gene expression, but knockout of USP38 suppressed hypoxia-responsive gene expression under hypoxia. Mechanistically, USP38 interacts with HIF1α to deubiquitinate K11-linked polyubiquitination of HIF1α at Lys769, resulting in stabilization and subsequent activation of HIF1α. In addition, we show that USP38 attenuates cellular ROS and suppresses cell apoptosis under hypoxia. Thus, we reveal a novel role for USP38 in the regulation of hypoxia signaling.

Cul-4 inhibition rescues spastin levels and reduces defects in hereditary spastic paraplegia models.

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-Ring E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation.

Reciprocal regulation of IL-33 receptor-mediated inflammatory response and pulmonary fibrosis by TRAF6 and USP38.

The warning cytokine interleukin-33 receptor (IL-33R) mediates local inflammatory responses and plays crucial roles in the pathogenesis of immune diseases such as pulmonary fibrosis and rheumatoid arthritis. Whether and how IL-33R is regulated remain enigmatic. Here, we identified ubiquitin-specific protease 38 (USP38) as a negative regulator of IL-33R­mediated signaling. USP38 deficiency promotes interleukin-33 (IL-33)­induced downstream proinflammatory responses in vitro and in vivo. Usp38−/− mice are more susceptible to inflammatory damage and death and developed more serious pulmonary fibrosis after bleomycin treatment. USP38 is constitutively associated with IL-33R and deconjugates its K27-linked polyubiquitination at K511, resulting in its autophagic degradation. We further show that the E3 ubiquitin ligase tumor necrosis factor receptor­associated factor 6 (TRAF6) catalyzes K27-linked polyubiquitination of IL-33R at K511, and that deficiency of TRAF6 inhibits IL-33­mediated signaling. Our findings suggest that K27-linked polyubiquitination and deubiquitination of IL-33R by TRAF6 and USP38 reciprocally regulate IL-33R level and signaling, which represents a critical mechanism in the regulation of IL-33­triggered lung inflammatory response and pulmonary fibrosis.

The ubiquitin ligase HERC4 suppresses MafA transcriptional activity triggered by GSK3ß in myeloma by atypical K63-linked polyubiquitination.

MafA and c-Maf are close members of the Maf transcription factor family and indicators of poor prognosis of multiple myeloma (MM). Our previous study finds that the ubiquitin ligase HERC4 induces c-Maf degradation but stabilizes MafA, and the mechanism is elusive. In the present study, we find that HERC4 interacts with MafA and mediates its K63-linked polyubiquitination at K33. Moreover, HERC4 inhibits MafA phosphorylation and its transcriptional activity triggered by glycogen synthase kinase 3ß (GSK3ß). The K33R MafA variant prevents HERC4 from inhibiting MafA phosphorylation and increases MafA transcriptional activity. Further analyses reveal that MafA can also activate the STAT3 signaling, but it is suppressed by HERC4. Lastly, we demonstrate that lithium chloride, a GSK3ß inhibitor, can upregulate HERC4 and synergizes dexamethasone, a typical anti-MM drug, in inhibiting MM cell proliferation and xenograft growth in nude mice. These findings thus highlight a novel regulation of MafA oncogenic activity in MM and provide the rationale by targeting HERC4/GSK3ß/MafA for the treatment of MM.

MiR-17∼92 is involved in NF-κB activation via targeting the ubiquitin-editing proteins to mediate RIP1 complex polyubiquitinations in ABC-DLBCL.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation, but whether miR-17∼92 contributes to this activation remains unclear. Herein, we sought to evaluate the role of miR-17∼92 in the process of NF-κB activation in ABC-DLBCL. We found that the expression of miR-17∼92 primary transcript was positively correlated with NF-κB activity, miR-17∼92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17∼92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17∼92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17∼92 expression was also associated with poorer outcome in ABC-DLBCL patients. Overall, our results showed that miR-17∼92 selectively activated the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome in ABC-DLBCL. These findings uncovered an innovative function of miR-17∼92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17∼92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

A conserved methyltransferase active site residue of Zika virus NS5 is required for the restriction of STING activation and interferon expression.

Zika virus (ZIKV) is a re-emerging RNA virus and causes major public health events due to its link to severe neurological complications in foetuses and neonates. The cGAS-STING signalling pathway regulates innate immunity and plays an important role in the invasion of DNA and RNA viruses. This study reveals a distinct mechanism by which ZIKV restricts the cGAS-STING signalling to repress IFN-ß expression. ZIKV attenuates IFN-ß expression induced by DNA viruses (herpes simplex virus type 1, HSV-1) or two double-stranded DNAs (dsDNA90 and HSV120) in mouse embryonic fibroblasts (MEFs). Notably, ZIKV NS5, the viral RNA-dependent RNA polymerase, was responsible for the repression of IFN-ß. NS5 interacts with STING in the cytoplasm, suppresses IRF3 phosphorylation and nucleus localization and promotes the cleavage of STING K48-linked polyubiquitination. Furthermore, the NS5 methyltransferase (MTase) domain interacts with STING to restrict STING-induced IFN-ß expression. Interestingly, point mutation analyses of conserved methyltransferase active site residue D146 indicate that it is critical for repressing IFN-ß expression induced by STING stimulation in cGAS-STING signalling.

E3 ubiquitin ligase RNF187 promotes growth of spermatogonia via lysine 48-linked polyubiquitination-mediated degradation of KRT36/KRT84.

Ubiquitination is the most common post-translational modification and is essential for various cellular regulatory processes. RNF187, which is known as RING domain AP1 coactivator-1, is a member of the RING finger family. RNF187 can promote the proliferation and migration of various tumor cells. However, whether it has a similar role in regulating spermatogonia is not clear. This study explored the role and molecular mechanism of RNF187 in a mouse spermatogonia cell line (GC-1). We found that RNF187 knockdown reduced the proliferation and migration of GC-1 cells and promoted their apoptosis. RNF187 overexpression significantly increased the proliferation and migration of GC-1 cells. In addition, we identified Keratin36/Keratin84 (KRT36/KRT84) as interactors with RNF187 by co-immunoprecipitation and mass spectrometry analyses. RNF187 promoted GC-1 cell growth by degrading KRT36/KRT84 via lysine 48-linked polyubiquitination. Subsequently, we found that KRT36 or KRT84 overexpression significantly attenuated proliferation and migration of RNF187-overexpressing GC-1 cells. In summary, our study explored the involvement of RNF187 in regulating the growth of spermatogonia via lysine 48-linked polyubiquitination-mediated degradation of KRT36/KRT84. This may provide a promising new strategy for treating infertility caused by abnormal spermatogonia development.

Tudor-SN exacerbates pathological vascular remodeling by promoting the polyubiquitination of PTEN via NEDD4-1.

BACKGROUND: Dysregulation of vascular homeostasis can induce cardiovascular diseases and increase global mortality rates. Although lineage tracing studies have confirmed the pivotal role of modulated vascular smooth muscle cells (VSMCs) in the progression of pathological vascular remodeling, the underlying mechanisms are still unclear. METHODS: The expression of Tudor-SN was determined in VSMCs of artery stenosis, PDGF-BB-treated VSMCs and atherosclerotic plaque. Loss- and gain-of-function approaches were used to explore the role of Tudor-SN in the modulation of VSMCs phenotype both in vivo and in vitro. RESULTS: In this study, we demonstrate that Tudor-SN expression is significantly elevated in injury-induced arteries, atherosclerotic plaques, and PDGF-BB-stimulated VSMCs. Tudor-SN deficiency attenuates, but overexpression aggravates the synthetic phenotypic switching of VSMCs and pathological vascular remodeling. Loss of Tudor-SN also reduces atherosclerotic plaque formation and increases plaque stability. Mechanistically, PTEN, the major regulator of the MAPK and PI3K-AKT signaling pathways, plays a vital role in Tudor-SN-mediated regulation on proliferation and migration of VSMCs. Tudor-SN facilitates the polyubiquitination and degradation of PTEN via NEDD4-1, thus exacerbating vascular remodeling under pathological conditions. BpV (HOpic), a specific inhibitor of PTEN, not only counteracts the protective effect of Tudor-SN deficiency on proliferation and migration of VSMCs, but also abrogates the negative effect of carotid artery injury-induced vascular remodeling in mice. CONCLUSIONS: Our findings reveal that Tudor-SN deficiency significantly ameliorated pathological vascular remodeling by reducing NEDD4-1-dependent PTEN polyubiquitination, suggesting that Tudor-SN may be a novel target for preventing vascular diseases.

TRIM34 modulates influenza virus-activated programmed cell death by targeting Z-DNA-binding protein 1 for K63-linked polyubiquitination.

Z-DNA-binding protein 1 (ZBP1) is an innate sensor of influenza A virus (IAV) that participates in IAV-induced programmed cell death. Nevertheless, little is known about the upstream signaling pathways regulating ZBP1. We found that a member of the tripartite motif (TRIM) family, TRIM34, interacted with ZBP1 to promote its K63-linked polyubiquitination. Using a series of genetic approaches, we provide in vitro and in vivo evidence indicating that IAV triggered cell death and inflammatory responses via dependent on TRIM34/ZBP1 interaction. TRIM34 and ZBP1 expression and interaction protected mice from death during IAV infection owing to reduced inflammatory responses and epithelial damage. Additionally, analysis of clinical samples revealed that TRIM34 associates with ZBP1 and mediates ZBP1 polyubiquitination in vivo. Higher levels of proinflammatory cytokines correlated with higher levels of ZBP1 in IAV-infected patients. Taken together, we conclude that TRIM34 serves as a critical regulator of IAV-induced programmed cell death by mediating the K63-linked polyubiquitination of ZBP1.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Tripartite motif-containing 68-stabilized modulator of apoptosis-1 retards the proliferation and metastasis of lung cancer.

Non-small cell lung cancer (NSCLC) is a major global health threat with high incidence and mortality. Modulator of apoptosis-1 (MOAP1), also named MAP-1, belongs to the PNMA gene family and plays a key role in regulating apoptosis and tumor growth. However, its influences on NSCLC are largely unclear, and thus were explored in our present study, particularly the underlying mechanisms. Here, we initially find that MOAP1 expression is significantly decreased in NSCLC patients compared with the normal ones, and negatively correlated with the TNM and pathologic stages among patients. Additionally, MOAP1 low expression predicts a poorer prognosis than that of the NSCLC patients expressing higher MOAP1. Our in vitro studies confirm much lower MOAP1 expression in NSCLC cell lines. Of note, promoting MOAP1 expression strongly reduces the proliferation and induces apoptosis in NSCLC cells, accompanied with cell cycle arrest distributed in G0/G1 phase. Moreover, we find that MOAP1 has a negative correlation with Th2 cells' infiltration, but a positive correlation with the infiltration levels of eosinophils. Epithelial mesenchymal transition (EMT) process is also greatly restrained in NSCLC cells with MOAP1 over-expression, as proved by the reduced migration and invasion of cells. We further identify a positive correlation between MOAP1 and tripartite motif-containing 68 (TRIM68) in patients with NSCLC. Further analysis shows that TRIM68 directly interacts with MOAP1 and stabilizes MOAP1. Importantly, TRIM68 can activate MOAP1 by inducing the K63-linked polyubiquitination of MOAP1. Finally, animal studies verify that promoting MOAP1 efficiently suppresses tumor growth and lung metastasis in the nude mice. Collectively, our results reveal a novel mechanism through which MOAP1 stabilized by TRIM68 inhibits NSCLC development and targeting MOAP1 for its up-regulation may be a promising therapeutic strategy for NSCLC treatment.

Systematic characterization of Brassica napus UBC13 genes involved in DNA-damage response and K63-linked polyubiquitination.

BACKGROUND: Ubc13 is the only known ubiquitin conjugating enzyme (Ubc/E2) dedicated to promoting Lys (K)63-linked polyubiquitination, and this process requires a Ubc/E2 variant (UEV). Unlike conventional K48-linked polyubiquitination that targets proteins for degradation, K63-linked polyubiquitination, which is involved in several cellular processes, does not target proteins for degradation but alter their activities. RESULTS: In this study we report the identification and functional characterization of 12 Brassica napus UBC13 genes. All the cloned UBC13 gene products were able to physically interact with AtUev1D, an Arabidopsis UEV, to form stable complexes that are capable of catalyzing K63-linked polyubiquitination in vitro. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects in spontaneous mutagenesis and DNA-damage responses, suggesting that BnUBC13s can replace yeast UBC13 in mediating K63-linked polyubiquitination and error-free DNA-damage tolerance. CONCLUSION: Collectively, this study provides convincing data to support notions that B. napus Ubc13s promote K63-linked polyubiquitination and are probably required for abiotic stress response. Since plant Ubc13-UEV are also implicated in other developmental and stress responses, this systematic study sets a milestone in exploring roles of K63-linked polyubiquitination in this agriculturally important crop.

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Ubiquitination in the regulation of autophagy.

Autophagy, an efficient and effective approach to clear rapidly damaged organelles, macromolecules, and other harmful cellular components, enables the recycling of nutrient materials and supply of nutrients to maintain cellular homeostasis. Ubiquitination plays an important regulatory role in autophagy. This paper summarizes the most recent progress in ubiquitin modification in various stages of autophagy, including initiation, elongation, and termination. Moreover, this paper shows that ubiquitination is an important way through which selective autophagy achieves substrate specificity. Furthermore, we note the distinction between monoubiquitination and polyubiquitination in the regulation of autophagy. Compared with monoubiquitination, polyubiquitination is a more common strategy to regulate the activity of the autophagy molecular machinery. In addition, the role of ubiquitination in the closure and fusion of autophagosomes warrants further study. This article not only clarifies the regulatory mechanism of autophagy but also contributes to a deeper understanding of the importance of ubiquitination modification.

Ubiquitination of TLR3 by TRIM3 signals its ESCRT-mediated trafficking to the endolysosomes for innate antiviral response.

Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation. Trim3-/- cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses.

ß-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of ß-TrCP2 in response to genotoxic stress.

F-box proteins ß-TrCP1 and ß-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of ß-TrCP1 and ß-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that ß-TrCP1 and ß-TrCP2 communicate during genotoxic stress. We found that expression levels of ß-TrCP1 are significantly increased while levels of ß-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of ß-TrCP1 and ß-TrCP2 via the phosphorylation of ß-TrCP1 at Ser158. Phosphorylated ß-TrCP1 potently promotes the proteasomal degradation of ß-TrCP2 and MDM2, resulting in the activation of p53. Additionally, ß-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by ß-TrCP2. Thus, ß-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of ß-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed ß-TrCP1 as a key player in maintaining the genome integrity through the attenuation of ß-TrCP2 levels in response to genotoxic stress.

The deubiquitinase USP10 restores PTEN activity and inhibits non-small cell lung cancer cell proliferation.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a key player in tumorigenesis of non-small cell lung cancer (NSCLC) and was recently found to be inactivated by tripartite motif containing 25 (TRIM25)-mediated K63-linked polyubiquitination. However, the deubiquitinase (Dub) coordinate TRIM25 in PTEN ubiquitination is still elusive. In the present study, we found that this K63-linked polyubiquitination could be ablated by the ubiquitin-specific protease 10 (USP10) in a screen against a panel of Dubs. We found using coimmununoprecipitation/immunoblotting that USP10 interacted with PTEN and reduced the K63-linked polyubiquitination of PTEN mediated by TRIM25 in NSCLC cells. Moreover, USP10, but not its inactive C424A deubiquitinating mutant or other Dubs, abolished PTEN from K63-linked polyubiquitination mediated by TRIM25. In contrast to TRIM25, USP10 restored PTEN phosphatase activity and reduced the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate, thereby inhibiting AKT/mammalian target of rapamycin progrowth signaling transduction in NSCLC cells. Moreover, USP10 was downregulated in NSCLC cell lines and primary tissues, whereas TRIM25 was upregulated. Consistent with its molecular activity, re-expression of USP10 suppressed NSCLC cell proliferation and migration, whereas knockout of USP10 promoted NSCLC cell proliferation and migration. In conclusion, the present study demonstrates that USP10 coordinates TRIM25 to modulate PTEN activity. Specifically, USP10 activates PTEN by preventing its K63-linked polyubiquitination mediated by TRIM25 and suppresses the AKT/mammalian target of rapamycin signaling pathway, thereby inhibiting NSCLC proliferation, indicating that it may be a potential drug target for cancer treatment.

OsUEV1B, an Ubc enzyme variant protein, is required for phosphate homeostasis in rice.

Phosphorus is a crucial macronutrient for plant growth and development. The mechanisms for maintaining inorganic phosphate (Pi) homeostasis in rice are not well understood. The ubiquitin-conjugating enzyme variant protein OsUEV1B was previously found to interact with OsUbc13 and mediate lysine63-linked polyubiquitination. In the present study, we found OsUEV1B was specifically inhibited by Pi deficiency, and was localized in the nucleus and cytoplasm. Both osuev1b mutant and OsUEV1B-RNA interference (RNAi) lines displayed serious symptoms of toxicity due to Pi overaccumulation. Some Pi starvation inducible and phosphate transporter genes were upregulated in osuev1b mutant and OsUEV1B-RNAi plants in association with enhanced Pi acquisition, and representative Pi starvation responses, including stimulation of acid phosphatase activity and root hair growth, were also activated in the presence of sufficient Pi. A yeast two-hybrid screen revealed an interaction between OsUEV1B and OsVDAC1, which was confirmed by bimolecular fluorescence complementation and firefly split-luciferase complementation assays. OsVDAC1 encoded a voltage-dependent anion channel protein localized in the mitochondria, and OsUbc13 was shown to interact with OsVDAC1 via yeast two-hybrid and bimolecular fluorescence complementation assays. Under sufficient Pi conditions, similar to osuev1b, a mutation in OsVDAC1 resulted in significantly greater Pi concentrations in the roots and second leaves, improved acid phosphatase activity, and enhanced expression of the Pi starvation inducible and phosphate transporter genes compared with wild-type DongJin, whereas overexpression of OsVDAC1 had the opposite effects. OsUEV1B or OsVDAC1 knockout reduced the mitochondrial membrane potential and adenosine triphosphate levels. Moreover, overexpression of OsVDAC1 in osuev1b partially restored its high Pi concentration to a level between those of osuev1b and DongJin. Our results indicate that OsUEV1B is required for rice phosphate homeostasis.

Functions and mechanisms of the Ubc13-UEV complex and lysine 63-linked polyubiquitination in plants.

Ubiquitination is one of the best-known post-translational modifications in eukaryotes, in which different linkage types of polyubiquitination result in different outputs of the target proteins. Distinct from the well-characterized K48-linked polyubiquitination that usually serves as a signal for degradation of the target protein, K63-linked polyubiquitination often requires a unique E2 heterodimer Ubc13-UEV and alters the target protein activity instead of marking it for degradation. This review focuses on recent advances on the roles of Ubc13-UEV-mediated K63-linked polyubiquitination in plant growth, development, and response to environmental stresses.