E3 Ligase Activity Research Articles

Molecular basis for PHF7-mediated ubiquitination of histone H3.

The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.

Overexpression of a C3HC4‐type E3‐ubiquitin ligase contributes to salinity tolerance by modulating Na+ homeostasis in rice

AbstractSoil salinity has a negative effect on crop yield. Therefore, plants have evolved many strategies to overcome decreases in yield under saline conditions. Among these, E3‐ubiquitin ligase regulates salt tolerance. We characterized Oryza sativa Really Interesting New Gene (RING) Finger C3HC4‐type E3 ligase (OsRFPHC‐4), which plays a positive role in improving salt tolerance. The expression of OsRFPHC‐4 was downregulated by high NaCl concentrations and induced by abscisic acid (ABA) treatment. GFP‐fused OsRFPHC‐4 was localized to the plasma membrane of rice protoplasts. OsRFPHC‐4 encodes a cellular protein with a C3HC4‐RING domain with E3 ligase activity. However, its variant OsRFPHC‐4C161A does not possess this activity. OsRFPHC‐4‐overexpressing plants showed enhanced salt tolerance due to low accumulation of Na+ in both roots and leaves, low Na+ transport in the xylem sap, high accumulation of proline and soluble sugars, high activity of reactive oxygen species (ROS) scavenging enzymes, and differential regulation of Na+/K+ transporter expression compared to wild‐type (WT) and osrfphc‐4 plants. In addition, OsRFPHC‐4‐overexpressing plants showed higher ABA sensitivity under exogenous ABA treatment than WT and osrfphc‐4 plants. Overall, these results suggest that OsRFPHC‐4 contributes to the improvement of salt tolerance and Na+/K+ homeostasis via the regulation of changes in Na+/K+ transporters.

Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Nimbolide targets RNF114 to induce the trapping of PARP1 and synthetic lethality in BRCA-mutated cancer.

Recent studies have pointed to PARP1 trapping as a key determinant of the anticancer effects of PARP1 inhibitors (PARPi). We identified RNF114, as a PARylation-dependent, E3 ubiquitin ligase involved in DNA damage response. Upon sensing genotoxicity, RNF114 was recruited, in a PAR-dependent manner, to DNA lesions, where it targeted PARP1 for degradation. The blockade of this pathway interfered with the removal of PARP1 from DNA lesions, leading to profound PARP1 trapping. We showed that a natural product, nimbolide, inhibited the E3 ligase activity of RNF114 and thus caused PARP1 trapping. However, unlike conventional PARPi, nimbolide treatment induced the trapping of both PARP1 and PARylation-dependent DNA repair factors. Nimbolide showed synthetic lethality with BRCA mutations, and it overcame intrinsic and acquired resistance to PARPi, both in vitro and in vivo. These results point to the exciting possibility of targeting the RNF114-PARP1 pathway for the treatment of homologous recombination-deficient cancers.

Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.

E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

BackgroundAlterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC).MethodsWe assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the KrasLSL−G12D/+; Tp53fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry.ResultsOur findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs.ConclusionThe present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

The deubiquitinase ZRANB1 is an E3 ubiquitin ligase for SLC7A11 and regulates ferroptotic resistance.

The dependency of cancer cells on iron increases their susceptibility to ferroptosis, thus providing new opportunities for patients with treatment-resistant tumors. However, we show that lipid peroxidation, a hallmark of ferroptosis, was found in various areas of patient samples, indicating the potential resistance of ferroptosis. Using whole deubiquitinases (DUBs) sgRNA screening, we found that loss of ZRANB1 confers cancer cell resistance to ferroptosis. Intriguingly, functional studies revealed that ZRANB1 ubiquitinates and represses SLC7A11 expression as an E3 ubiquitin ligase and that ZRANB1 inhibits glutathione (GSH) synthesis through SLC7A11 degradation, leading to elevated lipid peroxidation and ferroptosis. Deletion of the region (residues 463-584) abolishes the E3 activity of ZRANB1. Moreover, we show that ZRANB1 has lower expression in tumors, which is positively correlated with lipid peroxidation. Collectively, our results demonstrate the role of ZRANB1 in ferroptosis resistance and unveil mechanisms involving modulation of E3 ligase activity through an unconventional catalytic domain.

CBX4 promotes antitumor immunity by suppressing Pdcd1 expression in T cells.

E3 SUMO-protein ligase CBX4 (CBX4), a key component of polycomb-repressive complexes 1 (PRC1), has been reported to regulate a variety of genes implicated in tumor growth, metastasis, and angiogenesis. However, its role in T-cell-mediated antitumor immunity remains elusive. To shed light on this issue, we generated mice with T-cell-specific deletion of Cbx4. Tumor growth was increased in the knockout mice. Additionally, their tumor-infiltrating lymphocytes exhibited impaired tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) production, with an elevated programmed cell death protein 1 (PD-1) level. In fact, dysregulated Pdcd1 expression was observed in all major subsets of peripheral T cells from the knockout mice, which was accompanied by a functional defect in response to T-cell receptor (TCR) stimulation. In support of a direct link between CBX4 and PD-1, Cbx4 overexpression resulted in the downregulation of Pdcd1 expression. Epigenetic analyses indicated that Cbx4 deficiency leads to diminished accumulation of inhibitory histone modifications at conserved region (CR)-C and CR-B sites of the Pdcd1 promoter, namely mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3). Moreover, inhibition of either the E3 ligase activity of polycomb-repressive complexes 1 (PRC1) or the methyltransferase activity of polycomb-repressive complexes 2 (PRC2) restores Pdcd1 expression in Cbx4-transfected cells. Cumulatively, this study reveals a novel function of CBX4 in the regulation of T-cell function and expands our understanding of the epigenetic control of Pdcd1 expression.

A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution

The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.

A conserved microtubule-binding region in Xanthomonas XopL is indispensable for induced plant cell death reactions.

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity.

Feedback regulation of ubiquitination and phase separation of HECT E3 ligases

Lipid homeostasis is essential for normal cellular functions and dysregulation of lipid metabolism is highly correlated with human diseases including neurodegenerative diseases. In the ubiquitin-dependent autophagic degradation pathway, Troyer syndrome-related protein Spartin activates and recruits HECT-type E3 Itch to lipid droplets (LDs) to regulate their turnover. In this study, we find that Spartin promotes the formation of Itch condensates independent of LDs. Spartin activates Itch through its multiple PPAY-motif platform generated by self-oligomerization, which targets the WW12 domains of Itch and releases the autoinhibition of the ligase. Spartin-induced activation and subsequent autoubiquitination of Itch lead to liquid-liquid phase separation (LLPS) of the poly-, but not oligo-, ubiquitinated Itch together with Spartin and E2 both in vitro and in living cells. LLPS-mediated condensation of the reaction components further accelerates the generation of polyubiquitin chains, thus forming a positive feedback loop. Such Itch-Spartin condensates actively promote the autophagy-dependent turnover of LDs. Moreover, we show that the catalytic HECT domain of Itch is sufficient to interact and phase separate with poly-, but not oligo-ubiquitin chains. HECT domains from other HECT E3 ligases also exhibit LLPS-mediated the promotion of ligase activity. Therefore, LLPS and ubiquitination are mutually interdependent and LLPS promotes the ligase activity of the HECT family E3 ligases.

Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation

Inflammation is essential for healthy immune function, wound healing, and resolution of infection. RIG-I is a key RNA sensor that initiates an immune response, with activation and termination of RIG-I signaling reliant on its modification with ubiquitin. The RING E3 ubiquitin ligase, RNF125, has a critical role in the attenuation of RIG-I signaling, yet it is not known how RNF125 promotes ubiquitin transfer or how its activity is regulated. Here we show that the E3 ligase activity of RNF125 relies on the first zinc finger (ZF1) as well as the RING domain. Surprisingly, ZF1 helps recruit the E2, while residues N-terminal to the RING domain appear to activate the E2∼Ub conjugate. These discoveries help explain how RNF125 brings about the termination of RIG-I dependent inflammatory responses, and help account for the contribution of RNF125 to disease. This study also reveals a new role for ZF domains in E3 ligases.

The TRIM37 variants in Mulibrey nanism patients paralyze follicular helper T cell differentiation

The Mulibrey (Muscle–liver–brain–eye) nanism caused by loss-of-function variants in TRIM37 gene is an autosomal recessive disorder characterized by severe growth failure and constrictive pericarditis. These patients also suffer from severe respiratory infections, co-incident with an increased mortality rate. Here, we revealed that TRIM37 variants were associated with recurrent infection. Trim37 FINmajor (a representative variant of Mulibrey nanism patients) and Trim37 knockout mice were susceptible to influenza virus infection. These mice showed defects in follicular helper T (TFH) cell development and antibody production. The effects of Trim37 on TFH cell differentiation relied on its E3 ligase activity catalyzing the K27/29-linked polyubiquitination of Bcl6 and its MATH domain-mediated interactions with Bcl6, thereby protecting Bcl6 from proteasome-mediated degradation. Collectively, these findings highlight the importance of the Trim37-Bcl6 axis in controlling the development of TFH cells and the production of high-affinity antibodies, and further unveil the immunologic mechanism underlying recurrent respiratory infection in Mulibrey nanism.

Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation

The BRCA1-interacting protein Obg-like ATPase 1 (OLA1) functions in centriole duplication. In this study, we show the role of the mitotic kinase Aurora A in the reduction of centrosomal OLA1. Aurora A binds to and polyubiquitinates OLA1, targeting it for proteasomal degradation. NIMA-related kinase 2 (NEK2) phosphorylates the T124 residue of OLA1, increases binding of OLA1 to Aurora A and OLA1 polyubiquitination by Aurora A, and reduces centrosomal OLA1 in G2 phase. The kinase activity of Aurora A suppresses OLA1 polyubiquitination. The decrease in centrosomal OLA1 caused by Aurora A-mediated polyubiquitination promotes the recruitment of pericentriolar material proteins in G2 phase. The E3 ligase activity of Aurora A is critical for centrosome amplification induced by its overexpression. The results suggest a dual function of Aurora A as an E3 ubiquitin ligase and a kinase in the regulation of centrosomal OLA1, which is essential for proper centrosome maturation in G2 phase.

LncRNA NRON promotes tumorigenesis by enhancing MDM2 activity toward tumor suppressor substrates.

The E3 ligase MDM2 promotes tumor growth and progression by inducing ubiquitin-mediated degradation of P53 and other tumor-suppressing proteins. Here, we identified an MDM2-interacting lncRNA NRON, which promotes tumor formation by suppressing both P53-dependent and independent pathways. NRON binds to MDM2 and MDMX (MDM4) via two different stem-loops, respectively, and induces their heterogenous dimerization, thereby enhancing the E3 ligase activity of MDM2 toward its tumor-suppressing substrates, including P53, RB1, and NFAT1. NRON knockdown dramatically inhibits tumor cell growth in vitro and in vivo. More importantly, NRON overexpression promotes oncogenic transformation by inducing anchorage-independent growth in vitro and facilitating tumor formation in immunocompromised mice. Clinically, NRON expression is significantly associated with poor clinical outcome in breast cancer patients. Together, our data uncover a pivotal role of lncRNA that induces malignant transformation of epithelial cells by inhibiting multiple tumor suppressor proteins.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

PRPF8 controls alternative splicing of PIRH2 to modulate the p53 pathway and survival of human ESCs.

Human embryonic stem cells (hESCs) have great potential for developmental biology and regenerative medicine. However, extensive apoptosis often occurs when hESCs respond to various stresses or injuries. Understanding the molecular control and identifying new factors associated with hESC survival are fundamental to ensure the high quality of hESCs. In this study, we report that PRPF8, an RNA spliceosome component, is essential for hESC survival. PRPF8 knockdown (KD) induces p53 protein accumulation and activates the p53 pathway, leading to apoptosis in hESCs. Strikingly, silencing of p53 rescues PRPF8 KD-induced apoptosis, indicating that PRPF8 KD triggers hESC apoptosis through activating the p53 pathway. In search for the mechanism by which p53 pathway is activated by PRPF8 KD, we find that PRPF8 KD alters alternative splicing of many genes, including PIRH2 which encodes an E3 ubiquitin ligase of p53. PIRH2 has several isoforms such as PIRH2A, PIRH2B, and PIRH2C. Intriguingly, PRPF8 KD specifically increases the transcript level of the PIRH2B isoform, which lacks a RING domain and E3 ligase activity. Functionally, PIRH2B KD partially rescues the reduction in cell numbers and upregulation of P21 caused by PRPF8 KD in hESCs. The finding suggests that PRPF8 controls alternative splicing of PIRH2 to maintain the balance of p53 pathway activity and survival of hESCs. The PRPF8/PIRH2/p53 axis identified here provides new insights into how p53 pathway and hESC survival are precisely regulated at multiple layers, highlighting an important role of posttranscriptional machinery in supporting hESC survival.

Cbx4 governs HIF-1α to involve in Th9 cell differentiation promoting asthma by its SUMO E3 ligase activity

The potential role of polycomb chromobox 4 (Cbx4), as a small ubiquitin-like ligase (SUMO) E3 ligase, in the development and exacerbation of asthma remains unclear. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor in the cellular response to hypoxia and contributes to the pathogenesis and progression of a range of diseases, including asthma. Here, we aimed to investigate the interaction of Cbx4 with Hypoxia inducible factor-1α (HIF-1α) and the potent mechanism of action in asthma progression. In present study, in vitro and ex vivo results demonstrated that Cbx4 interacts with HIF-1α protein through its SUMO E3 ligase activity and enhances the sumoylation, which increases HIF-1 transactivation through Cbx4 and promotes the differentiation of Th9 cells, then in turn promotes the process of asthma. Treatment of inhibitors targeting SUMO E3 ligase activity of Cbx4 or HIF-1α can effectively reduce HIF-1α activation and differentiation of Th9 cells, which further attenuates the asthma in mouse model. Current results collectively demonstrated Cbx4 can govern HIF-1α to involve in Th9 cell differentiation promoting asthma by its SUMO E3 ligase activity, providing a new direction for clinical treatment of asthma.

Peli3 ablation ameliorates acetaminophen-induced liver injury through inhibition of GSK3β phosphorylation and mitochondrial translocation

The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3−/− knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3β were decreased in primary hepatocytes obtained from Peli3−/− KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3β compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3β. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3−/− KO hepatocytes restored the mitochondrial translocation of GSK3β, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3β polyubiquitination.

Ubiquitin E3 ligase activity of Ralstonia solanacearum effector RipAW is not essential for induction of plant defense in Nicotiana benthamiana.

As one of the most destructive bacterial phytopathogens, Ralstonia solanacearum causes substantial annual yield losses of many important crops. Deciphering the functional mechanisms of type III effectors, the crucial factors mediating R. solanacearum-plant interactions, will provide a valuable basis for protecting crop plants from R. solanacearum. Recently, the NEL (novel E3 ligase) effector RipAW was found to induce cell death on Nicotiana benthamiana in a E3 ligase activity-dependent manner. Here, we further deciphered the role of the E3 ligase activity in RipAW-triggered plant immunity. We found that RipAWC177A, the E3 ligase mutant of RipAW, could not induce cell death but retained the ability of triggering plant immunity in N. benthamiana, indicating that the E3 ligase activity is not essential for RipAW-triggered immunity. By generating truncated mutants of RipAW, we further showed that the N-terminus, NEL domain and C-terminus are all required but not sufficient for RipAW-induced cell death. Furthermore, all truncated mutants of RipAW triggered ETI immune responses in N. benthamiana, confirming that the E3 ligase activity is not essential for RipAW-triggered plant immunity. Finally, we demonstrated that RipAW- and RipAWC177A-triggered immunity in N. benthamiana requires SGT1 (suppressor of G2 allele of skp1), but not EDS1 (enhanced disease susceptibility), NRG1 (N requirement gene 1), NRC (NLR required for cell death) proteins or SA (salicylic acid) pathway. Our findings provide a typical case in which the effector-induced cell death can be uncoupled with immune responses, shedding new light on effector-triggered plant immunity. Our data also provide clues for further in-depth study of mechanism underlying RipAW-induced plant immunity.