Domain-containing E3 Ubiquitin Protein Ligase Research Articles

Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

WWP2 protects against sepsis-induced cardiac injury through inhibiting cardiomyocyte ferroptosis.

Cardiac injury plays a critical role in contributing to the mortality associated with sepsis, a condition marked by various forms of programmed cell deaths. Previous studies hinted at the WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) involving in heart failure and endothelial injury. However, the precise implications of WWP2 in sepsis-induced cardiac injury, along with the underlying mechanisms, remain enigmatic. Sepsis induced cardiac injury were constructed by intraperitoneal injection of lipopolysaccharide. To discover the function of WWP2 during this process, we designed and performed loss/gain-of-function studies with cardiac-specific vectors and WWP2 knockout mice. Combination experiments were performed to investigate the relationship between WWP2 and downstream signaling in septic myocardium injury. The protein level of WWP2 was downregulated in cardiomyocytes during sepsis. Cardiac-specific overexpression of WWP2 protected heart from sepsis induced mitochondrial oxidative stress, programmed cell death and cardiac injury, while knockdown or knockout of WWP2 exacerbated this process. The protective potency of WWP2 was predominantly linked to its ability to suppress cardiomyocyte ferroptosis rather than apoptosis. Mechanistically, our study revealed a direct interaction between WWP2 and acyl-CoA synthetase long-chain family member 4 (FACL4), through which WWP2 facilitated the ubiquitin-dependent degradation of FACL4. Notably, we observed a notable reduction in ferroptosis and cardiac injury within WWP2 knockout mice after FACL4 knockdown during sepsis. WWP2 assumes a critical role in safeguarding the heart against injury induced by sepsis via regulating FACL4 to inhibit LPS-induced cardiomyocytes ferroptosis.

WWP2 binds to NKRF, enhances the NF-κB signaling, and promotes malignant phenotypes of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is one of the hematological malignancies with a high recurrence rate. WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) is identified as a pivotal regulator of tumor progression. This study aimed to assess the possible role of WWP2 in AML. Analysis of the GEPIA database indicated an elevated WWP2 expression in AML. We established stable WWP2-overexpressed or WWP2-silenced cells using lentivirus loaded with cDNA encoding WWP2 mRNA or shRNA targeting WWP2. Notably, WWP2 overexpression facilitated cell proliferation and cell cycle progression, which was manifested as the increase of colony formation number, S-phase percentage and cell cycle related protein levels. As observed, WWP2 knockdown presented opposite effects, leading to inhibition of tumorigenicity. Strikingly, WWP2 knockdown induced apoptosis, accompanied by upregulation of pro-apoptosis proteins cleaved caspase-9, Bax and cleaved caspase-3 and downregulation of anti-apoptosis protein Bcl-2. Functionally, we further confirmed that WWP2 overexpression enhanced the NF-κB signaling and upregulated the levels of downstream genes, which may contribute to aggravating the development of AML. More importantly, by co-immunoprecipitation assay, we verified that WWP2 bound to NF-κB-repressing factor (NKRF) and promoted NKRF ubiquitylation. Dramatically, NKRF overexpression abolished the role of WWP2 in facilitating the process of AML. Overall, our observations confirm that WWP2 exerts a critical role in the tumorigenicity of AML, and NKRF is regarded as an essential factor in the WWP2-mediated AML progression. WWP2 may be proposed as a promising target of AML.

African swine fever virus MGF-360-10L is a novel and crucial virulence factor that mediates ubiquitination and degradation of JAK1 by recruiting the E3 ubiquitin ligase HERC5.

African swine fever virus (ASFV) causes acute hemorrhagic infectious disease in pigs. The ASFV genome encodes various proteins that enable the virus to escape innate immunity; however, the underlying mechanisms are poorly understood. The present study found that ASFV MGF-360-10L significantly inhibits interferon (IFN)-β-triggered STAT1/2 promoter activation and the production of downstream IFN-stimulated genes (ISGs). ASFV MGF-360-10L deletion (ASFV-Δ10L) replication was impaired compared with the parental ASFV CN/GS/2018 strain, and more ISGs were induced by the ASFV-Δ10L in porcine alveolar macrophages in vitro. We found that MGF-360-10L mainly targets JAK1 and mediates its degradation in a dose-dependent manner. Meanwhile, MGF-360-10L also mediates the K48-linked ubiquitination of JAK1 at lysine residues 245 and 269 by recruiting the E3 ubiquitin ligase HERC5 (HECT and RLD domain-containing E3 ubiquitin protein ligase 5). The virulence of ASFV-Δ10L was significantly lower than that of the parental strain in vivo, which indicates that MGF-360-10L is a novel virulence factor of ASFV. Our findings elaborate the novel mechanism of MGF-360-10L on the STAT1/2 signaling pathway, expanding our understanding of the inhibition of host innate immunity by ASFV-encoded proteins and providing novel insights that could contribute to the development of African swine fever vaccines. IMPORTANCE African swine fever outbreaks remain a concern in some areas. There is no effective drug or commercial vaccine to prevent African swine fever virus (ASFV) infection. In the present study, we found that overexpression of MGF-360-10L strongly inhibited the interferon (IFN)-β-induced STAT1/2 signaling pathway and the production of IFN-stimulated genes (ISGs). Furthermore, we demonstrated that MGF-360-10L mediates the degradation and K48-linked ubiquitination of JAK1 by recruiting the E3 ubiquitin ligase HERC5. The virulence of ASFV with MGF-360-10L deletion was significantly less than parental ASFV CN/GS/2018. Our study identified a new virulence factor and revealed a novel mechanism by which MGF-360-10L inhibits the immune response, thus providing new insights into the vaccination strategies against ASFV.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals.

The ubiquitin ligase HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking. This study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk. We analyzed HUWE1 single nucleotide polymorphisms in 190 non-obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA-mediated RARα knockdown. Using C18-4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participated in retinoic acid-mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit-8 assays, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non-obstructive azoospermia and obstructive azoospermia patients using quantitative real-time polymerase chain reaction and immunofluorescence. Three HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non-obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non-obstructive azoospermia patients. An HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non-obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non-obstructive azoospermia pathogenesis.

Down-regulation of WWP2 aggravates Type 2 diabetes mellitus-induced vascular endothelial injury through modulating ubiquitination and degradation of DDX3X

BackgroundEndothelial injury caused by Type 2 diabetes mellitus (T2DM) is considered as a mainstay in the pathophysiology of diabetic vascular complications (DVCs). However, the molecular mechanism of T2DM-induced endothelial injury remains largely unknown. Here, we found that endothelial WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) act as a novel regulator for T2DM-induced vascular endothelial injury through modulating ubiquitination and degradation of DEAD-box helicase 3 X-linked (DDX3X).MethodsSingle-cell transcriptome analysis was used to evaluate WWP2 expression in vascular endothelial cells of T2DM patients and healthy controls. Endothelial-specific Wwp2 knockout mice were used to investigate the effect of WWP2 on T2DM-induced vascular endothelial injury. In vitro loss- and gain-of-function studies were performed to assess the function of WWP2 on cell proliferation and apoptosis of human umbilical vein endothelial cells. The substrate protein of WWP2 was verified using mass spectrometry, coimmunoprecipitation assays and immunofluorescence assays. The mechanism of WWP2 regulation on substrate protein was investigated by pulse-chase assay and ubiquitination assay.ResultsThe expression of WWP2 was significantly down-regulated in vascular endothelial cells during T2DM. Endothelial-specific Wwp2 knockout in mice significantly aggravated T2DM-induced vascular endothelial injury and vascular remodeling after endothelial injury. Our in vitro experiments showed that WWP2 protected against endothelial injury by promoting cell proliferation and inhibiting apoptosis in ECs. Mechanically, we found that WWP2 is down-regulated in high glucose and palmitic acid (HG/PA)-induced ECs due to c-Jun N-terminal kinase (JNK) activation, and uncovered that WWP2 suppresses HG/PA-induced endothelial injury by catalyzing K63-linked polyubiquitination of DDX3X and targeting it for proteasomal degradation.ConclusionOur studies revealed the key role of endothelial WWP2 and the fundamental importance of the JNK-WWP2-DDX3X regulatory axis in T2DM-induced vascular endothelial injury, suggesting that WWP2 may serve as a new therapeutic target for DVCs.

Systemic depletion of WWP1 improves insulin sensitivity and lowers triglyceride content in the liver of obese mice.

Obesity is a metabolic disorder associated with many diseases. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type E3 ubiquitin ligase involved in several diseases. Recently, we found that the level of WWP1 is increased in white adipose tissue in a mouse model of obesity and that obese Wwp1 knockout (KO) mice exhibit improved whole-body glucose metabolism. Here, to determine which insulin-sensitive tissues contribute to this phenotype, we investigated the levels of several insulin signaling markers in white adipose tissue, liver, and skeletal muscle of Wwp1 KO mice, which were fed a normal or high-fat diet and transiently treated with insulin. In obese Wwp1 KO mice, phosphorylated Akt levels were increased in the liver but not in white adipose tissue or skeletal muscle. Moreover, the weight and triglyceride content of the liver of obese Wwp1 KO mice were decreased. These results suggest that systemic deletion of WWP1 improves glucose metabolism via enhanced hepatic insulin signaling and suppressed hepatic fat accumulation. In summary, WWP1 participates in obesity-related metabolic dysfunction and pathologies related to hepatic steatosis via suppressed insulin signaling.

MORC4 plays a tumor-promoting role in colorectal cancer via regulating PCGF1/CDKN1A axis in vitro and in vivo.

MORC family CW-type zinc finger 4 (MORC4) possessing nuclear matrix binding domains has been observed to be involved in multiple cancer development. By digging three gene expression omnibus (GEO) gene microarrays (GSE110223, GSE110224 and GSE24514), we found that MORC4 was overexpressed in colorectal cancer (CRC) samples (log2 Fold change >1, p < 0.05). We aimed to investigate the role of MORC4 in CRC malignant behaviors, with an emphasis on polycomb group ring finger 1 (PCGF1)/cyclin-dependent kinase inhibitor 1A (CDKN1A) axis. Firstly, we confirmed MORC4 as an upregulated gene in 60 pairs of frozen CRC and adjacent normal samples. MORC4 overexpression increased proliferation and metastasis, and decreased apoptosis in SW480 and HT29 cells, which was diminished by the knockdown of PCGF1, a transcriptional repressor of CDKN1A (a potent cyclin-dependent kinase inhibitor). MORC4 was further identified as a novel molecule that interacted with PCGF1 via coimmunoprecipitation. MORC4 itself did not substantially suppress CDKN1A transcriptional activity, but it augmented PCGF1's effect on CDKN1A. Additionally, MORC4 acted as the substrate of HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2 (HECW2) and was degraded through ubiquitin-proteasome system. Collectively, our work suggested that MORC4 accelerated CRC progression via governing PCGF1/CDKN1A signaling.

Identification of prognostic biomarkers of invasive ductal carcinoma by an integrated bioinformatics approach

Background: Invasive ductal carcinoma (IDC) is the most common breast cancer worldwide. Nowadays, due to IDC heterogeneity and its high capacity for metastasis, it is necessary to discover novel diagnostic and prognostic biomarkers. Thus, this study aimed to identify new prognostic genes of IDC using an integrated bioinformatics approach. Methods: Using the Gene Expression Omnibus (GEO) database, we downloaded publicly available data of the whole-genome mRNA expression profile from the first three stages of IDC in two expression profiling datasets, GSE29044 and GSE32291; intra-group data repeatability tests were conducted using Pearson’s correlation test, and the differentially expressed genes (DEGs) were identified using the online tool GEO2R, followed by the construction of a protein‑protein interaction network (PPI-net) with the common DEGs identified in the three analyzed stages using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software, from these PPI-net we identify the hub genes (prognostic genes). Results: We found seven genes [WW domain-containing E3 ubiquitin-protein ligase 1 (WWP1), STIP1 homology and U-box containing protein 1 (STUB1), F-box and WD repeat domain containing 7 (FBXW7), kelch like family member 13 (KLHL13), ubiquitin-conjugating enzyme E2 Q1 (UBE2Q1), tripartite motif-containing 11 (TRIM11), and the beta-transducin repeat containing E3 ubiquitin-protein ligase (BTRC)] as potential candidates for IDC prognostic biomarkers, which were mainly enriched in the Ubiquitin-specific protease activity, cytoskeletal protein binding, and ligase activity. The role of these genes in the pathophysiology of IDC is not yet well characterized, representing a way to improve our understanding of the process of tumorigenesis and the underlying molecular events of IDC. Conclusions: Genes identified may lead to the discovery of new prognostic targets and precise therapeutics for IDC.

P53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

ABSTRACTp53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection.IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

WWP1 Deficiency Alleviates Cardiac Remodeling Induced by Simulated Microgravity.

Cardiac muscle is extremely sensitive to changes in loading conditions; the microgravity during space flight can cause cardiac remodeling and function decline. At present, the mechanism of microgravity-induced cardiac remodeling remains to be revealed. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is an important activator of pressure overload-induced cardiac remodeling by stabilizing disheveled segment polarity proteins 2 (DVL2) and activating the calcium-calmodulin-dependent protein kinase II (CaMKII)/histone deacetylase 4 (HDAC4)/myocyte-specific enhancer factor 2C (MEF2C) axis. However, the role of WWP1 in cardiac remodeling induced by microgravity is unknown. The purpose of this study was to determine whether WWP1 was also involved in the regulation of cardiac remodeling caused by microgravity. Firstly, we detected the expression of WWP1 and DVL2 in the heart from mice and monkeys after simulated microgravity using western blotting and immunohistochemistry. Secondly, WWP1 knockout (KO) and wild-type (WT) mice were subjected to tail suspension (TS) to simulate microgravity effect. We assessed the cardiac remodeling in morphology and function through a histological analysis and echocardiography. Finally, we detected the phosphorylation levels of CaMKII and HDAC4 in the hearts from WT and WWP1 KO mice after TS. The results revealed the increased expression of WWP1 and DVL2 in the hearts both from mice and monkeys after simulated microgravity. WWP1 deficiency alleviated simulated microgravity-induced cardiac atrophy and function decline. The histological analysis demonstrated WWP1 KO inhibited the decreases in the size of individual cardiomyocytes of mice after tail suspension. WWP1 KO can inhibit the activation of the DVL2/CaMKII/HDAC4 pathway in the hearts of mice induced by simulated microgravity. These results demonstrated WWP1 as a potential therapeutic target for cardiac remodeling and function decline induced by simulated microgravity.

WW Domain-Containing E3 Ubiquitin Protein Ligase 1: A Self-Disciplined Oncoprotein.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a member of C2-WW-HECT E3 ligase family. Although it may execute carcinostatic actions in some scenarios, WWP1 functions as an oncoprotein under most circumstances. Here, we comprehensively review reports on regulation of WWP1 and its roles in tumorigenesis. We summarize the WWP1-mediated ubiquitinations of diverse proteins and the signaling pathways they involved, as well as the mechanisms how they affect cancer formation and progression. According to our analysis of database, in combination with previous reports, we come to a conclusion that WWP1 expression is augmented in various cancers. Gene amplification, as well as expression regulation mediated by molecules such as non-coding RNAs, may account for the increased mRNA level of WWP1. Regulation of enzymatic activity is another important facet to upregulate WWP1-mediated ubiquitinations. Based on the published data, we conclude that WWP1 employs interactions between multiple domains to autoinhibit its polyubiquitination activity in a steady state. Association of some substrates can partially release certain autoinhibition-related domains and make WWP1 have a moderate activity of polyubiquitination. Some cancer-related mutations can fully disrupt the inhibitory interactions and make WWP1 hyperactive. High expression level or hyperactivation of WWP1 may abnormally enhance polyubiquitinations of some oncoproteins or tumor suppressors, such as ΔNp63α, PTEN and p27, and ultimately promote cell proliferation, survival, migration and invasion in tumorigenesis. Given the dysregulation and oncogenic functions of WWP1 in some cancer types, it is promising to explore some therapeutic inhibitors to tune down its activity.

High Expression of WWP1 Associates with Tumor Progression in Papillary Thyroid Cancer.

Background: WWP1 (WW domain-containing E3 ubiquitin protein ligase 1) is increased in several kinds of carcinomas, but the influence of WWP1 in papillary thyroid cancer (PTC) is not well understood. Materials and Methods: The expression of WWP1 in PTC tissues and cells was detected by real-time reverse transcription PCR. The biological role of WWP1 on PTC cell growth, apoptosis, migration, and invasion ability was assessed with the Cell Counting Kit-8, colony forming, flow cytometry, wound healing, and transwell assays, respectively. Results: The expression of WWP1 mRNA and protein is increased in PTC tissue samples and cells. There is close correlation between the up expression of WWP1 and clinical parameters, such as tumor size, TNM, and distant metastasis. Knockdown of WWP1 blocks cell proliferation, migration, and invasion, causes cell cycle arrest, and induces apoptosis in PTC cells. Knockdown of WWP1 increases PTEN level and reduces p-PI3K and p-Akt level in PTC cells. Conclusions: Knockdown of WWP1 suppressed cell proliferation, migration, and invasion of PTC cell by downregulating the expression of p-PI3K and p-Akt, contributing to their understanding the pathogenesis of PTC.

Integrating HECW1 expression into the clinical indicators exhibits high accuracy in assessing the prognosis of patients with clear cell renal cell carcinoma

sBackgroundAlthough many intratumoral biomarkers have been reported to predict clear cell renal cell carcinoma (ccRCC) patient prognosis, combining intratumoral and clinical indicators could predict ccRCC prognosis more accurately than any of these markers alone. This study mainly examined the prognostic value of HECT, C2 and WW domain-containing E3 ubiquitin protein ligase 1 (HECW1) expression in ccRCC patients in combination with established clinical indicators.MethodsThe expression level of HECW1 was screened out by data-independent acquisition mass spectrometry (DIA-MS) and analyzed in ccRCC patients from the The Cancer Genome Atlas (TCGA) database and our cohort. A total of 300 ccRCC patients were stochastically divided into a training cohort and a validation cohort, and real-time PCR, immunohistochemistry (IHC) and statistical analyses were employed to examine the prognostic value of HECW1 in ccRCC patients.ResultsThe expression level of HECW1 usually decreased in human ccRCC specimens relative to control specimens in TCGA (p < 0.001). DIA-MS, Real-time PCR, and IHC analyses also showed that the majority of ccRCCs harbored decreased HECW1 expression compared with that in normal adjacent tissues (p < 0.001). Additionally, HECW1 expression was reduced in ccRCC cell lines compared with the normal renal cell line HK-2 (p < 0.001). Moreover, lower HECW1 expression was found in ccRCC patients with a higher tumor node metastasis (TNM) stage, bone metastasis, or first-line targeted drug resistance (p < 0.001). Low HECW1 expression indicated higher TNM stage, SSIGN (Stage, Size, Grade, and Necrosis) score and WHO/ISUP grade and poor prognosis in ccRCC patients (p < 0.05). Even after multivariable adjustment, HECW1, TNM stage, and SSIGN score served as independent risk factors. The c-index analysis showed that integrating intratumoral HECW1 expression into TNM stage or SSIGN score resulted in a higher c-index value than these indicators alone for predicting ccRCC patient prognosis.ConclusionHECW1 is a novel prognostic biomarker and therapeutic target in ccRCC, and integrating intratumoral HECW1 expression with established clinical indicators yields higher accuracy in assessing the postoperative prognosis of ccRCC patients.

WWP1 knockout in mice exacerbates obesity-related phenotypes in white adipose tissue but improves whole-body glucose metabolism.

White adipose tissue (WAT) is important for maintenance of homeostasis, because it stores energy and secretes adipokines. The WAT of obese people demonstrates mitochondrial dysfunction, accompanied by oxidative stress, which leads to insulin resistance. WW domain‐containing E3 ubiquitin protein ligase 1 (WWP1) is a member of the HECT‐type E3 family of ubiquitin ligases and is associated with several diseases. Recently, we demonstrated that WWP1 is induced specifically in the WAT of obese mice, where it protects against oxidative stress. Here, we investigated the function of WWP1 in WAT of obese mice by analyzing the phenotype of Wwp1 knockout (KO) mice fed a high‐fat diet. The levels of oxidative stress markers were higher in obese WAT from Wwp1 KO mice. Moreover, Wwp1 KO mice had lower activity of citrate synthase, a mitochondrial enzyme. We also measured AKT phosphorylation in obese WAT and found lower levels in Wwp1 KO mice. However, plasma insulin level was low and glucose level was unchanged in obese Wwp1 KO mice. Moreover, both glucose tolerance test and insulin tolerance test were improved in obese Wwp1 KO mice. These findings indicate that WWP1 participates in the antioxidative response and mitochondrial function in WAT, but knockdown of WWP1 improves whole‐body glucose metabolism.

The structure and regulation of the E3 ubiquitin ligase HUWE1 and its biological functions in cancer.

E3 ligases are a class of critical enzymes that can catalyse the transfer of ubiquitin (Ub) from an E2 enzyme to the substrate and are essential to cellular processes. The E3 ligase HUWE1 (also known as ARF-BP1, HECTH9, HSPC272, Ib772, LASU1, MULE, URE-B1, UREB1, and HECT, UBA and WWE domain-containing E3 ubiquitin protein ligase 1) is encoded by the huwe1 gene. HUWE1 is a key regulator of the DNA damage response, transcription, autophagy, apoptosis and metabolism in a variety of cancers. Due to its pivotal role in conferring substrate specificity, HUWE1 has attracted enormous attention as a promising anticancer drug target. In this review, we indicate the specific molecular structure of HUWE1 and its role in various cellular signalling pathways and highlight new insights into HUWE1 in cancer. Finally, we discuss outstanding questions regarding HUWE1 in oncology and highlight its limitations in drug development and clinical guidance to better define the role of HUWE1 in multiple cancers.

Structural insights into a HECT-type E3 ligase AREL1 and its ubiquitination activities in vitro

The HECT E3 ligase family comprises three subfamilies: NEDD4 E3 ubiquitin protein ligase (NEDD4), HECT and RLD domain-containing E3 ubiquitin protein ligase (HERC), and "other." Most previous studies have focused on the NEDD4 subfamily. Apoptosis-resistant E3 ligase 1 (AREL1) belongs to "other" subfamily HECT that inhibits apoptosis by ubiquitinating and degrading proapoptotic proteins. Here, we report the crystal structure of the extended HECT domain of AREL1 (amino acids (aa) 436-823) at 2.4 Å resolution and its ubiquitination of the proapoptotic protein second mitochondria-derived activator of caspase (SMAC). We found that the extended HECT domain adopts an inverted, T-shaped, bilobed conformation and harbors an additional loop (aa 567-573) absent in all other HECT members. We also show that the N-terminal extended region (aa 436-482) preceding the HECT domain is indispensable for its stability and activity and that without this region, the HECT domain becomes inactive. AREL1 ubiquitinated SMAC, primarily on Lys62 and Lys191 We solved the crystal structure of the tetrameric form of SMAC to 2.8 Å resolution, revealing the Lys62 and Lys191 locations. The AREL1 HECT domain assembled Lys33-, Lys48-, and Lys63-linked polyubiquitin chains. Moreover, E701A substitution in the AREL1 HECT domain substantially increased its autopolyubiquitination and SMAC ubiquitination activity, whereas deletion of the last three amino acids at the C terminus completely abrogated AREL1 autoubiquitination and reduced SMAC ubiquitination. Finally, an AREL1-specific ubiquitin variant inhibited SMAC ubiquitination in vitro Our findings may assist in the development of AREL1 inhibitors that block its anti-apoptotic activity in cancer.

MiR-129-5p and -3p co-target WWP1 to suppress gastric cancer proliferation and migration.

Gastric cancer (GC) is a worldwide health problem. Uncovering the underlining molecular mechanisms of GC is of vital significance. Here, we identified a novel oncogene WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in GC. WWP1 could promote GC cell proliferation and migration in vitro and expedite GC growth in vivo. We also found out two microRNAs (miRNAs): miR-129-5p and -3p could both target WWP1. Interestingly, miR-129-5p bound to the CDS region of WWP1 mRNA. The miR-129 pairs (miR-129-5p and -3p) play pivotal roles in GC to suppress its proliferation and migration in vitro and slow down GC growth in vivo by repressing WWP1. In summary, we identified two tumor suppressive miRNAs which share the same precursor that could regulate the same oncogene WWP1 in GC. Our finding would add new route for GC research and treatment.

WW domain-containing E3 ubiquitin protein ligase 1 depletion evokes antitumor effect in cutaneous squamous cell carcinoma by inhibiting signal transducer and activator of transcription 3 signaling pathway.

ObjectivesWW domain-containing E3 ubiquitin protein ligase 1 (WWP1) has been implicated in tumor progression. We aimed to investigate the role of WWP1 in cutaneous squamous cell carcinoma (CSCC).MethodsWWP1 gene and protein levels were detected using semi-quantitative reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The effects of WWP1 on cell cycle, apoptosis, cell migration and invasion were examined by flow cytometry, wound healing and Transwell assays, respectively. The antitumor efficacy of WWP1 small interfering RNA was determined in CSCC tumor xenografts in mice.ResultsWWP1 expression was significantly higher in CSCC tissues and cells than in normal skin and cells, respectively. WWP1 expression was significantly associated with histological grade, invasion depth and lymph node metastasis in patients with CSCC. High expression predicted metastatic potential and an unfavorable prognosis. WWP1 downregulation suppressed tumor growth in vitro and in vivo, reduced cell migration and invasion, arrested the cell cycle in G0/G1 and induced apoptosis in A431 cells. WWP1 depletion also decreased phosphorylated signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-2, cyclin D1 and Bcl-2, but did not affect total STAT3.ConclusionsWWP1 is a potential target for the diagnosis, prognosis and therapy of patients with CSCC.