E3 Ubiquitin Ligase Activity Research Articles

Molecular characterization and functional analysis of TRIM37 from black tiger shrimp (Penaeus monodon).

The family of TRIM proteins with E3 ubiquitin ligase activity served important roles in the regulation of innate immune processes, in particular antiviral and proinflammatory cytokine responses. In this study, a novel TRIM37 homolog was identified from Penaeus monodon (named PmTRIM37). The PmTRIM37 protein contained three conserved domains (one RING finger domain, a B-box, and one Coiled-coil region) at its N-terminal and one Meprin and MATH domain at its C-terminal. The MATH domain was the characteristic of TRIM37 family. PmTRIM37 has relatively high expression in immune-related tissues such as hepatopancreas, gills, lymphoid organs and hemocytes. The expression levels of PmTRIM37 in hepatopancreas and lymphoid organs were significantly up-regulated after white spot syndrome virus (WSSV) infection. Knock down of PmTRIM37 promoted WSSV replication and VP28 expression, suggesting that PmTRIM37 played a negative role in WSSV infection. Further studies revealed that PmTRIM37 positively regulated the NF-κB pathway and Antimicrobial peptides (AMP) expression during WSSV infection. These findings indicated that PmTRIM37 might restrict WSSV replication by positively regulating NF-κB pathway during WSSV infection in P. monodon.

STAT1 suppresses the transcriptional activity of TRIM21 in gastric cancer.

Tripartite motif-containing protein 21 (TRIM21) has E3 ubiquitin ligase activity and is involved in the regulation of various biological processes in vivo. TRIM21 has been found to have strong associations with various cancers. However, its role in gastric cancer is unclear. The TCGA database was screened to obtain TRIM21 using WGCNA and PPI analyses. The TCGA database was used to evaluate the correlation of TRIM21 expression with patients' clinical characteristics, prognosis, functional enrichment and immune cell infiltration. The role of TRIM21 in cell proliferation, apoptosis and invasion was verified by in vivo and in vitro assays. The UCSC and JASPAR databases were used to evaluate the regulatory role of STAT1 on TRIM21 transcription. Finally, dual-luciferase reporter assay was used to confirm the regulation of TRIM21 transcriptional activity by STAT1. As a key gene, high expression of TRIM21 inhibited the gastric cancer growth and was significantly enriched in apoptosis, cell proliferation, and JAK/STAT signaling pathways. TRIM21 expression was positively correlated with a variety of TICs, including T cells, NK cells, and DCs. In vivo assays, TRIM21 inhibited functions in gastric cancer cell lines, including inhibition of proliferation and migration, and promotion of apoptosis. Database analysis and dual-luciferase reporter assay showed that STAT1 inhibited the transcriptional activity of TRIM21. In vivo assays confirmed that TRIM21 inhibited tumor growth, and STAT1 expression was negatively correlated with STAT1. TRIM21 is a tumor-suppressive gene in gastric cancer, and its transcriptional activity is inhibited by STAT1.

Tripartite Motif-Containing Protein 32 (TRIM32): What Does It Do for Skeletal Muscle?

Tripartite motif-containing protein 32 (TRIM32) is a member of the tripartite motif family and is highly conserved from flies to humans. Via its E3 ubiquitin ligase activity, TRIM32 mediates and regulates many physiological and pathophysiological processes, such as growth, differentiation, muscle regeneration, immunity, and carcinogenesis. TRIM32 plays multifunctional roles in the maintenance of skeletal muscle. Genetic variations in the TRIM32 gene are associated with skeletal muscular dystrophies in humans, including limb-girdle muscular dystrophy type 2H (LGMD2H). LGMD2H-causing genetic variations of TRIM32 occur most frequently in the C-terminal NHL (ncl-1, HT2A, and lin-41) repeats of TRIM32. LGMD2H is characterized by skeletal muscle dystrophy, myopathy, and atrophy. Surprisingly, most patients with LGMD2H show minimal or no dysfunction in other tissues or organs, despite the broad expression of TRIM32 in various tissues. This suggests more prominent roles for TRIM32 in skeletal muscle than in other tissues or organs. This review is focused on understanding the physiological roles of TRIM32 in skeletal muscle, the pathophysiological mechanisms mediated by TRIM32 genetic variants in LGMD2H patients, and the correlations between TRIM32 and Duchenne muscular dystrophy (DMD).

Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Ethanol Extract of Citrus grandis 'Tomentosa' Exerts Anticancer Effects by Targeting Skp2/p27 Pathway in Non-Small Cell Lung Cancer.

This study aims to investigate the anticancer properties of Citrus grandis 'Tomentosa' (CGT) in non-small cell lung cancer (NSCLC). The ethanol extract of CGT (CGTE) is prepared by using anhydrous ethanol and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), revealing that the main chemical components in CGTE are flavonoids and coumarins, such as naringin, rhoifolin, apigenin, bergaptol, and osthole. CGTE at concentrations without inducing cell death significantly inhibits cell proliferation via inducing cell cycle G1 phase arrest by MTT, colony formation, and flow cytometry assays, implying that CGT has anticancer potential. CGTE markedly inhibits the activity of Skp2-SCF E3 ubiquitin ligase, decreases the protein level of Skp2, and promotes the accumulation of p27 by co-immunoprecipitation (co-IP) and in vivo ubiquitination assay; whereas Skp2 overexpression rescues the effects of CGTE in NSCLC cells. In subcutaneous LLC allograft and A549 xenograft mouse models, CGTE, without causing obvious side effects in mice, significantly inhibits lung tumor growth by targeting the Skp2/p27 signaling pathway. These findings demonstrate that CGTE efficiently inhibits NSCLC proliferation both in vitro and in vivo by targeting the Skp2/p27 signaling pathway, suggesting that CGTE may serve as a therapeutic candidate for NSCLC treatment.

E3 ubiquitin ligase-dependent regulatory mechanism of TRIM family in carcinogenesis

<p class="MsoNormal" style="text-align: justify;"><span lang="EN-US" style="mso-bidi-font-size: 10.5pt; font-family: Nunito; color: #212529; background: white;">Tripartite motif-containing (TRIM) proteins consist of over 80 proteins, the majority of which exhibit E3 ubiquitin ligase activity. E3 ligases have a critical role in various cellular processes by specifically recognizing and ubiquitinating substrate proteins to promote their proteasomal degradation or alter their activities. Numerous studies have indicated that TRIMs are involved in carcinogenesis through various mechanisms. However, the regulatory mechanisms delimitating TRIMs’ function as E3 ligases has not yet been specifically addressed in a previous review article. In this review, we focus on recent advancements in understanding how certain TRIMs function solely as E3 ligases during cancer cell proliferation, apoptosis, and metastasis. We comprehensively summarize the target proteins of TRIMs involved in disordered signaling pathways such as Wnt/</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt; font-family: 'Cambria',serif; mso-bidi-font-family: Cambria; color: #212529; background: white;">β</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt; font-family: Nunito; color: #212529; background: white;">-catenin, PI3K/AKT, NF-</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt; font-family: 'Cambria',serif; mso-bidi-font-family: Cambria; color: #212529; background: white;">κ</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt; font-family: Nunito; color: #212529; background: white;">B, p53, ERK, and STAT3, as well as those regulating the cell cycle and glycolysis. Following ubiquitination modification by TRIM E3 ligases, these target proteins either undergo proteasome-mediating degradation, maintain steady levels, or get activated/inactivated. This review provides a foundation for the development of E3 ligase-based cancer treatments.</span></p>

E3 ubiquitin ligase-dependent regulatory mechanism of TRIM family in carcinogenesis

<p>Tripartite motif-containing (TRIM) proteins consist of over 80 proteins, the majority of which exhibit E3 ubiquitin ligase activity. E3 ligases have a critical role in various cellular processes by specifically recognizing and ubiquitinating substrate proteins to promote their proteasomal degradation or alter their activities. Numerous studies have indicated that TRIMs are involved in carcinogenesis through various mechanisms. However, the regulatory mechanisms delimitating TRIMs’ function as E3 ligases has not yet been specifically addressed in a previous review article. In this review, we focus on recent advancements in understanding how certain TRIMs function solely as E3 ligases during cancer cell proliferation, apoptosis, and metastasis. We comprehensively summarize the target proteins of TRIMs involved in disordered signaling pathways such as Wnt/β-catenin, PI3K/AKT, NF-κB, p53, ERK, and STAT3, as well as those regulating the cell cycle and glycolysis. Following ubiquitination modification by TRIM E3 ligases, these target proteins either undergo proteasome-mediating degradation, maintain steady levels, or get activated/inactivated. This review provides a foundation for the development of E3 ligase-based cancer treatments.</p>

GhSINA1, a SEVEN in ABSENTIA ubiquitin ligase, negatively regulates fiber development in Upland cotton

Protein ubiquitination is essential for plant growth and responses to the environment. The SEVEN IN ABSENTIA (SINA) ubiquitin ligases have been extensively studied in plants, but information on their roles in fiber development is limited. Here, we identified GhSINA1 in Upland cotton (Gossypium hirsutum), which has a conserved RING finger domain and SINA domain. Quantitative real-time PCR (qRT-PCR) analysis showed that GhSINA1 was preferentially expressed during fiber initiation and elongation, especially during initiation in the fuzzless-lintless cotton mutant. Subcellular localization experiments indicated that GhSINA1 localized to the nucleus. In vitro ubiquitination analysis revealed that GhSINA1 has E3 ubiquitin ligase activity. Ectopic overexpression of GhSINA1 in Arabidopsis thaliana reduced the number and length of root hairs and trichomes. Yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) assays demonstrated that the GhSINA1 proteins could interact with each other to form homodimers and heterodimers. Overall, these results suggest that GhSINA1 may act as a negative regulator in cotton fiber development through homodimerization and heterodimerization.

Genome-wide analysis and identification of TaRING-H2 gene family and TaSDIR1 positively regulates salt stress tolerance in wheat

Salt stress is an abiotic stress factor that limits high yields, and thus identifying salt tolerance genes is very important for improving the tolerance of salt in wheat. In this study we identified 274 TaRING-H2 family members and analyzed their gene positions, gene structures, conserved structural domains, promoter cis-acting elements and covariance relationships. And we investigated TaRING-H2-120 (TaSDIR1) in salt stress. Transgenic lines exhibited higher salt tolerance in the germination and seedling stages. Compared with the wild type, overexpression of TaSDIR1 upregulated the expression of genes encoding enzymes related to the control of reactive oxygen species (ROS), thereby reducing the accumulation of ROS, as well as increased the expression of ion transport-related genes to limit the inward flow of Na+ in vivo and maintain a higher K+/Na+ ratio. The expression levels of these genes were opposite in lines where TaSDIR1 was silenced by BSMV-VIGS, and the silenced wheat exhibited higher salt sensitivity. Arabidopsis mutants and heterologous TaSDIR1 overexpressing lines had similar salt stress tolerance phenotypes. We also demonstrated that TaSDIR1 interacted with TaSDIR1P2 in vivo and in vitro. A sequence of 80–100 amino acids in TaSDIR1P2 encoded a coiled coil domain that was important for the activity of E3 ubiquitin ligase, and it was also the core region for the interaction between TaSDIR1 and TaSDIR1P2. Overall, our results suggest that TaSDIR1 positively regulates salt stress tolerance in wheat.

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.

TRIM17-mediated ubiquitination and degradation of RBM38 promotes cisplatin resistance in non-small cell lung cancer.

Cisplatin (CDDP)-based chemotherapy is commonly used to treat advanced non-small cell lung cancer (NSCLC). However, the efficacy is limited by the development of drug resistance. Tripartite motif (TRIM) proteins typically have E3 ubiquitin ligase activities and modulate protein stability. In the present study, we screened for chemosensitivity-regulating TRIM proteins using CDDP-resistant NSCLC cell lines. We show that TRIM17 is upregulated in CDDP-resistant NSCLC cells and tumors compared to CDDP-sensitive counterparts. NSCLC patients with high TRIM17 expression in tumors have shorter progression-free survival than those with low TRIM17 expression after CDDP chemotherapy. Knockdown of TRIM17 increases the sensitivity of NSCLC cells to CDDP both in vitro and in vivo. In contrast, overexpression of TRIM17 promotes CDDP resistance in NSCLC cells. TRIM17-mediated CDDP resistance is associated with attenuation of reactive oxygen species (ROS) production and DNA damage. Mechanistically, TRIM17 interacts with RBM38 and promotes K48-linked ubiquitination and degradation of RBM38. TRIM17-induced CDDP resistance is remarkably reversed by RBM38. Additionally, RBM38 enhances CDDP-induced production of ROS. In conclusion, TRIM17 upregulation drives CDDP resistance in NSCLC largely by promoting RBM38 ubiquitination and degradation. Targeting TRIM17 may represent a promising strategy for improving CDDP-based chemotherapy in NSCLC.

PcTrim prevents early infection with white spot syndrome virus by inhibiting AP1-induced endocytosis

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes.BjibKXcktbHxsdfgurhU3PVideo

The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD- deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to it its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein (Rb)- phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.

HDAC6 Degrades nsp8 of Porcine Deltacoronavirus through Deacetylation and Ubiquitination to Inhibit Viral Replication.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.

Ubiquitin proteomics identifies RNA polymerase I as a target of the Smc5/6 complex

Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.

AMBRA1 control immune-specific gene expression in T lymphocytes

Abstract Autoimmune lymphoproliferative syndrome (ALPS) is a disease characterized by the dysregulation of cell death in lymphocytes, resulting in symptoms such as hepatomegaly, splenomegaly, and anemia. While most patients with ALPS have a mutation in the FAS gene or within the FAS pathway, which helps mediate cell death, some patients who present with ALPS-like symptoms do not have known causative gene mutations. To identify other genes that confer resistance to cell death, and thus may play a role in diseases such as ALPS, we performed a whole genome CRISPR screen of Fas-mediated cell death and identified AMBRA1, a protein previously studied in autophagy and E3 ubiquitin ligase activity. We verified the screen results and indeed found that AMBRA1 KO cells resist Fas-mediated cell death. To identify the mechanism of how AMBRA1 contributes to cell death resistance, we performed cycloheximide chase assays, qPCR, and proteomic analysis and confirmed that AMBRA1 regulates FAS expression. Furthermore, we found that AMBRA1 itself can be upregulated by TCR stimulation through the CD28-PI3K-mTORC1 pathway. Mass spectrometry analysis and subsequent experiments also confirmed that AMBRA1 regulates other immune genes. Together, these data establish a novel function for AMBRA1 as a critical regulator in T cells and unveil new insights into how AMBRA1 may contribute to ALPS and dysregulated cell death in other immune diseases. This research was supported by the Intramural Research Program of the NIH.

Loss of MuRF1 in Duroc pigs promotes skeletal muscle hypertrophy

Muscle mass development depends on increased protein synthesis and reduced muscle protein degradation. Muscle ring-finger protein-1 (MuRF1) plays a key role in controlling muscle atrophy. Its E3 ubiquitin ligase activity recognizes and degrades skeletal muscle proteins through the ubiquitin-proteasome system. The loss of Murf1, which encodes MuRF1, in mice leads to the accumulation of skeletal muscle proteins and alleviation of muscle atrophy. However, the function of Murf1 in agricultural animals remains unclear. Herein, we bred F1 generation Murf1+/- and F2 generation Murf1-/- Duroc pigs from F0 Murf1-/- pigs to investigate the effect of Murf1 knockout on skeletal muscle development. We found that the Murf1+/- pigs retained normal levels of muscle growth and reproduction, and their percentage of lean meat increased by 6% compared to that of the wild type (WT) pigs. Furthermore, the meat color, pH, water-holding capacity, and tenderness of the Murf1+/- pigs were similar to those of the WT pigs. The drip loss rate and intramuscular fat decreased slightly in the Murf1+/- pigs. However, the cross-sectional area of the myofibers in the longissimus dorsi increased in the adult Murf1+/- pigs. The skeletal muscle proteins MYBPC3 and actin, which are targeted by MuRF1, accumulated in the Murf1+/- and Murf1-/- pigs. Our findings show that inhibiting muscle protein degradation in MuRF1-deficient Duroc pigs increases the size of their myofibers and their percentage of lean meat without influencing their growth or pork quality. Our study demonstrates that Murf1 is a target gene for promoting skeletal muscle hypertrophy in pig breeding.

Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.

The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia

BackgroundE3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-β signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain.ResultsWe found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-β signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding.ConclusionsTaken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

Abstract 5028: The regulation of inflammatory signaling in cancer via A20 phosphorylation

Abstract A20 is an anti-inflammatory protein with a dual ubiquitin-editing activity. Through its N-terminal ovarian tumor (OTU) domain, A20 negatively regulates NF-ĸB by removing the K63-linked ubiquitin chains on RIP1 that act as platforms for NF-ĸB activation downstream of tumor necrosis factor alpha (TNFα). On the other hand, A20 also catalyzes the addition of K48-linked ubiquitin chains on RIP1 via its C-terminal zinc finger domain 4 (ZnF4) to promote its proteasomal degradation. Through both mechanisms, A20 is a means of negative feedback on TNFα-induced NF-ĸB activity. Importantly, inactivation of A20 is frequently found in B-cell lymphomas, where loss-of-function A20 truncations promote cell proliferation and upregulation of NF-ĸB signaling. Among the post-translational modifications (PTMs) reported in A20, there are several phosphorylation events in its C-terminus, including within the ZnF4 domain. Besides its known E3 ubiquitin ligase activity, previous reports show that the C-terminal region of A20 is important for ubiquitin binding, having preference for K63-linked poly-ubiquitin. However, a little is known about the effects of phosphorylation at this region on A20 activity as well as its consequences in downstream signaling. Our current work suggests that phosphorylation in the C-terminus of A20 inhibits its E3 ubiquitin ligase activity and disrupts its interaction with ubiquitin, which may also affect its deubiquitinase activity by preventing its recruitment to ubiquitinated proteins in the TNFα receptor (TNFR) complex. We also propose that the C-terminal region is important for the formation of the A20 ubiquitin-editing complex, necessary to downregulate NF-ĸB-activated inflammatory signals. Based on these observations, we hypothesize that not only gene-inactivating mutations, but also post-translational mechanisms, such as phosphorylation, inhibit A20 to promote cancer development. Our research is now focused on understanding the upstream signaling that regulates A20 phosphorylation. Citation Format: Tania Lopez Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua L. Andersen. The regulation of inflammatory signaling in cancer via A20 phosphorylation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5028.