A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks.

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

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.

Ubiquitin-assisted phase separation of dishevelled-2 promotes Wnt signalling.

Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development, such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in the cell that are critical for relaying the signal from upstream Wnt ligand-frizzled receptor binding to downstream effector ß-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signalling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) at the N-terminus is essential for Dvl2 phase separation. Importantly, we identified the HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitylation of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase-separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving ß-catenin. Together, our results reveal a ubiquitylation-dependent liquid-liquid phase separation as a new process through which Dvl2 forms condensates, which is necessary for transduction of Wnt signalling. This article has an associated First Person interview with the first author of the paper.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

The role of WWP1 and WWP2 in bone/cartilage development and diseases.

Bone and cartilage diseases are often associated with trauma and senescence, manifested as pain and limited mobility. The repair of bone and cartilage lesion by mesenchymal stem cells is regulated by various transcription factors. WW domain-containing protein 1 (WWP1) and WW domain-containing protein 2 (WWP2) are named for WW domain which recognizes PPXY (phono Ser Pro and Pro Arg) motifs of substrate. WWP1and WWP2 are prominent components of the homologous to the E6-AP carboxyl terminus (HECT) subfamily, a group of the ubiquitin ligase. Recently, some studies have found that WWP1 and WWP2 play an important role in the pathogenesis of bone and cartilage diseases and regulate the level and the transactivation of various transcription factors through ubiquitination. Therefore, this review summarizes the distribution and effects of WWP1 and WWP2 in the development of bone and cartilage, discusses the potential mechanism and therapeutic drugs in bone and cartilage diseases such as osteoarthritis, fracture, and osteoporosis.

Expanding the inhibitor space of the WWP1 and WWP2 HECT E3 ligases.

The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC50 of 158.3 µM (95% CI = 128.7, 195.1 µM). A structure-activity relationship by synthesis approach aided by molecular docking led to compound 11 which displayed increased potency with an IC50 of 32.7 µM (95% CI = 24.6, 44.3 µM) for WWP1 and 269.2 µM (95% CI = 209.4, 347.9 µM) for WWP2. Molecular docking yielded active site-bound poses suggesting that the heterocyclic imidazo[4,5-b]pyrazine scaffold undertakes a π-stacking interaction with the phenolic group of tyrosine, and the ethyl ester enables strong ion-dipole interactions. Given the therapeutic potential of WWP1 and WWP2, we propose that compound 11 may provide a basis for future lead compound development.

Activation of E3 ubiquitin ligase WWP2 by non-receptor tyrosine kinase ACK1.

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.

WWP2 confers risk to osteoarthritis by affecting cartilage matrix deposition via hypoxia associated genes.

OBJECTIVE: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. METHOD: Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N = 35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N = 8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. RESULTS: Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ| > 0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. CONCLUSIONS: Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

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

BACKGROUND: Endothelial 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). METHODS: Single-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. RESULTS: The 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. CONCLUSION: Our 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.

WWP2 drives the progression of gastric cancer by facilitating the ubiquitination and degradation of LATS1 protein.

BACKGROUND: Large tumor suppressor kinase 1 (LATS1), one of the predominant components of the Hippo pathway, has been characterized as a key player controlling the proliferation and invasion of cancer cells, including gastric cancer (GC) cells. However, the mechanism by which the functional stability of LATS1 is modulated has yet to be elucidated. METHODS: Online prediction tools, immunohistochemistry and western blotting assays were used to explore the expression of WW domain-containing E3 ubiquitin ligase 2 (WWP2) in GC cells and tissues. Gain- and loss-of-function assays, as well as rescue experiments were performed to determine the role of the WWP2-LATS1 axis in cell proliferation and invasion. Additionally, the mechanisms involving WWP2 and LATS1 were assessed by coimmunoprecipitation (Co-IP), immunofluorescence, cycloheximide and in vivo ubiquitination assays. RESULTS: Our results demonstrate a specific interaction between LATS1 and WWP2. WWP2 was markedly upregulated and correlated with disease progression and a poor prognosis in GC patients. Moreover, ectopic WWP2 expression facilitated the proliferation, migration and invasion of GC cells. Mechanistically, WWP2 interacts with LATS1, resulting in its ubiquitination and subsequent degradation, leading to increased transcriptional activity of YAP1. Importantly, LATS1 depletion abolished the suppressive effects of WWP2 knockdown on GC cells. Furthermore, WWP2 silencing attenuated tumor growth by regulating the Hippo-YAP1 pathway in vivo. CONCLUSIONS: Our results define the WWP2-LATS1 axis as a critical regulatory mechanism of the Hippo-YAP1 pathway that promotes GC development and progression. Video Abstract.

WWP2 ameliorates oxidative stress and inflammation in atherosclerotic mice through regulation of PDCD4/HO-1 pathway.

WWP2 is a HECT-type E3 ubiquitin ligase that regulates various physiological and pathological activities by binding to different substrates, but its role in atherosclerosis (AS) remains largely unknown. The objective of the present study is to investigate the role and underlying molecular mechanisms of WWP2 in endothelial injury. We found that WWP2 expression is significantly decreased in Apolipoprotein E (ApoE) -/- mice. Overexpression of WWP2 attenuates oxidative stress and inflammation in AS mice, while knockdown of WWP2 has opposite effects. WWP2 overexpression alleviates oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) injury, evidenced by the decreased oxidative stress levels and the secretion of inflammatory cytokines. Programmed cell death 4 (PDCD4) is identified as a potential substrate of WWP2. Co-immunoprecipitation (Co-IP) further demonstrates that WWP2 interacts with PDCD4, which is enhanced by ox-LDL treatment. Furthermore, the level of PDCD4 ubiquitination is significantly increased by WWP2 overexpression under the condition of MG132 treatment, while WWP2 knockdown shows opposite results. Subsequently, rescue experiments demonstrate that WWP2 knockdown further aggravates oxidative stress and inflammation in ox-LDL-treated HUVECs, while knockdown of PDCD4 alleviates this effect. Moreover, the use of sn-protoporphyrin (SnPP), an inhibitor of HO-1 pathway, confirms that PDCD4 enhances endothelial injury induced by ox-LDL through inhibiting HO-1 pathway. In conclusion, our results suggest that WWP2 protects against atherosclerosis progression via the PDCD4/HO-1 pathway, which may provide a novel treatment strategy for atherosclerosis.

LINC01588 regulates WWP2-mediated cardiomyocyte injury by interacting with HNRNPL.

Cardiomyocyte dysfunction and apoptosis induced by ischemia-hypoxia are common features of many acute and chronic heart diseases. WW domain-containing E3 ubiquitin ligase (WWP2) has been identified as an important regulator in pathogenesis of some health-threatening diseases. Although a couple of recent reports prompted the potential role of WWP2 in heart dysfunction, however, its exact role and how its expression was regulated in ischemic-hypoxic cardiomyocytes are still elusive. Here, we found that WWP2 protein level was induced in anoxia/reoxygenation (A/R) treated cardiomyocytes in a time-dependent manner, accompanied by synchronous expression of LINC01588 and HNRNPL. Knockdown of LINC01588 increased cardiomyocyte apoptosis, the level of oxidative stress, and expression of pro-inflammatory cytokine genes, down-regulated the expression of WWP2 and promoted expression of SEPT4 gene that contributed to cardiomyocyte dysfunction and was a target gene of WWP2. LINC01588 overexpression improved the functions of A/R treated cardiomyocytes, up-regulated WWP2 and reduced SEPT4 expression. In the mechanism exploration, we found that LINC01588 could directly bind with HNRNPL protein that could interact with WWP2, suggesting that WWP2 was involved in the regulation of LINC01588 in A/R treated cardiomyocytes. Moreover, WWP2 inhibition declined the protective role of LINC01588 in cardiomyocyte dysfunction induced by A/R. Finally, we demonstrated that LINC01588 overexpression improved acute myocardial infarction in mice in vivo. In conclusion, LINC01588 improved A/R-induced cardiomyocyte dysfunction by interacting with HNRNPL and promoting WWP2-mediated degradation of SEPT4.

WWP2 regulates SIRT1-STAT3 acetylation and phosphorylation involved in hypertensive angiopathy.

WWP2 is a HECT-type E3 ubiquitin ligase that regulates various physiological and pathological activities by binding to different substrates, but its function and regulatory mechanism in vascular smooth muscle cells (VSMCs) are still unknown. Here, we clarified the role of WWP2 in the regulation of SIRT1-STAT3 and the impact of this regulatory process in VSMCs. We demonstrated that WWP2 expression was significantly increased in angiotensin II-induced VSMCs model. Knockdown of WWP2 significantly inhibited angiotensin II-induced VSMCs proliferation, migration and phenotypic transformation, whereas overexpression of WWP2 had opposite effects. In vivo experiments showed that vascular smooth muscle-specific WWP2 knockout mice significantly relieved angiotensin II-induced hypertensive angiopathy. Mechanistically, mass spectrometry and co-immunoprecipitation assays identified that WWP2 is a novel interacting protein of SIRT1 and STAT3. Moreover, WWP2 formed a complex with SIRT1-STAT3, inhibiting the interaction between SIRT1 and STAT3, then reducing the inhibitory effect of SIRT1 on STAT3, ensuing promoting STAT3-K685 acetylation and STAT3-Y705 phosphorylation in angiotensin II-induced VSMCs and mice. In conclusion, WWP2 modulates hypertensive angiopathy by regulating SIRT1-STAT3 and WWP2 suppression in VSMCs can alleviate hypertensive angiopathy vitro and vivo. These findings provide new insights into the treatment of hypertensive vascular diseases.

Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases.

NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1-related ubiquitin ligases.

WWP2 regulates proliferation of gastric cancer cells in a PTEN-dependent manner.

WW domain containing E3 Ub-protein ligase 2 (WWP2) plays an important role in tumor progression as an E3 ligase of PTEN. Here, we investigated the role of WWP2 in gastric cancer (GC). We found that WWP2 is overexpressed in GC tissues, which is closely related to poor prognosis of GC patients. Using a WWP2-shRNA lentivirus expressing system, we established WWP2 stable-knockdown GC cell lines and found that knockdown of WWP2 inhibits the proliferation of GC cells both in vitro and in vivo. Also, WWP2 silencing induced the up-regulation of PTEN protein level and down-regulation of AKT phosphorylation level. We further investigated the role of PTEN in this regulating process by performing rescue assay and found that PTEN is essential for WWP2-mediated regulation of GC cells proliferation. Taken together, our results demonstrated that WWP2 promotes proliferation of GC cells by downregulating PTEN, which may provide new therapeutic targets for GC.

WWP2 ameliorates acute kidney injury by mediating p53 ubiquitylation and degradation.

E3 ubiquitin ligase gene, WWP2, is associated with acute kidney injury (AKI). This research was conducted to explore the role of WWP2 in AKI. AKI cell model was produced in human renal proximal tubular epithelial cell line (HK-2) by ischemia-reperfusion (IR) injury. CCK8 and flow cytometry assay were performed to explore the influence of WWP2 overexpression on cell proliferation and apoptosis of IR-induced HK-2 cells. Quantitative real-time PCR and immunoblotting (IB) were performed to assess the gene and protein expression. Then, the influence of WWP2 on p53 ubiquitylation and degradation was estimated by immunoprecipitation assay. Our data indicated that WWP2 was down-regulated and p53 was up-regulated in IR-induced HK-2 cells. WWP2 overexpression promoted proliferation and inhibited apoptosis of IR-induced HK-2 cells. And WWP2 interacted with p53 and regulated p53 ubiquitylation and degradation. Furthermore, the influence of WWP2 on cell proliferation and apoptosis was rescued by MG132 (proteasome inhibitor) treatment. In conclusion, our work described for the first time the role of WWP2 in AKI, showing that WWP2 ameliorated AKI by mediating p53 ubiquitylation and degradation. Moreover, the study offers some important insights into the occurrence of AKI and WWP2 may be a novel target of AKI treatment. SIGNIFICANCE OF THE STUDY: Our data elaborates that WWP2 has protective effect against AKI by mediating p53 ubiquitylation and degradation. Thus, WWP2 might be a therapeutic target for AKI.

WWP2 is a physiological ubiquitin ligase for phosphatase and tensin homolog (PTEN) in mice.

The tumor suppressor phosphatase and tensin homolog (PTEN) plays a central role in regulating phosphatidylinositol 3-kinase (PI3K) signaling, and its gene is very frequently mutated in various human cancers. Numerous studies have revealed that PTEN levels are tightly regulated by both transcriptional and posttranslational modifications, with especially ubiquitylation significantly regulating PTEN protein levels. Although several ubiquitin ligases have been reported to mediate PTEN ubiquitylation in vitro, the ubiquitin ligase that promotes PTEN degradation in vivo has not been reported. Here we took advantage of specific knockout mouse models to demonstrate that WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) promotes PTEN degradation under physiological conditions, whereas another ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), had no such effect. WWP2 knockout mice exhibited reduced body size, elevated PTEN protein levels, and reduced phosphorylation levels of the serine/threonine kinase and PTEN target AKT. In contrast, we observed no elevation of PTEN protein levels in CHIP knockout tissues and mouse embryonic fibroblasts. Furthermore, PTEN protein levels in CHIP/WWP2 double knockout mice were very similar to those in WWP2 single knockout mice and significantly higher than in WT and CHIP knockout mice. Our results demonstrate that WWP2, rather than CHIP, is an ubiquitin ligase that promotes PTEN degradation in vivo Considering PTEN's significant role in tumor development, we propose that WWP2 may be a potential target for fine-tuning PTEN levels in anticancer therapies.

The E3 ubiquitin ligase WWP2 facilitates RUNX2 protein transactivation in a mono-ubiquitination manner during osteogenic differentiation.

Poly-ubiquitination-mediated RUNX2 degradation is an important cause of age- and inflammation-related bone loss. NEDD4 family E3 ubiquitin protein ligases are thought to be the major regulators of RUNX2 poly-ubiquitination. However, we observed a mono-ubiquitination of RUNX2 that was catalyzed by WWP2, a member of the NEDD4 family of E3 ubiquitin ligases. WWP2 has been reported to catalyze the mono-ubiquitination of Goosecoid in chondrocytes, facilitating craniofacial skeleton development. In this study, we found that osteogenic differentiation of mesenchymal stem cells promoted WWP2 expression and nuclear accumulation. Knockdown of Wwp2 in mesenchymal stem cells and osteoblasts led to significant deficiencies of osteogenesis, including decreased mineral deposition and down-regulation of osteogenic marker genes. Co-immunoprecipitation experiments showed the interaction of WWP2 with RUNX2 in vitro and in vivo Mono-ubiquitination by WWP2 leads to RUNX2 transactivation, as evidenced by the wild type of WWP2, but not its ubiquitin ligase-dead mutant, augmenting RUNX2-reponsive reporter activity. Moreover, deletion of WWP2-dependent mono-ubiquitination resulted in striking defects of RUNX2 osteoblastic activity. In addition, ectopic expression of the constitutively active type 1A bone morphogenetic protein receptor enhanced WWP2-dependent RUNX2 ubiquitination and transactivation, demonstrating a regulatory role of bone morphogenetic protein signaling in the WWP2-RUNX2 axis. Taken together, our results provide evidence that WWP2 serves as a positive regulator of osteogenesis by augmenting RUNX2 transactivation in a non-proteolytic mono-ubiquitination manner.

Elevated expression of WWP2 in human lung adenocarcinoma and its effect on migration and invasion.

Lung cancer has been a hot area of research because of its high incidence and mortality. In this study, WWP2, an E3 ubiquitin ligase, is proposed to be an oncoprotein contributing to lung tumorigenesis. We attempted to determine if WWP2 gene expression is correlated with the development of human lung adenocarcinoma. Real-time PCR and western blotting were used to detect the expression of WWP2 in 65 paired lung adenocarcinoma and adjacent normal lung tissues. We found that WWP2 expression was elevated in lung adenocarcinoma tissues and was correlated with the tumor differentiation stage, TNM stage and presence of lymph node metastasis. We performed CCK-8 and colony formation assays and found that down-regulation of WWP2 inhibited proliferation in A549 and SPC-A-1 cells. A wound healing assay and trans-well invasion assays showed that down-regulation of WWP2 inhibited the migration and invasion of lung adenocarcinoma cells. It could be predicted from these data that elevated expression of WWP2 may play a role in facilitating the development of lung adenocarcinoma.