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Poly(ADP-ribose) polymerase 1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes but also enhanced suppressive function of regulatory T cells. Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the posttranslational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications for developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.

Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1–3 upon binding of growth factor neuregulin-1β (NRG1β). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1β-bound near full-length HER2–HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2–HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. Both HER2–HER3 and HER2(S310F)–HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. Our structure of the HER2(S310F)–HER3–NRG1β–trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2–HER3 heterodimer. The unique features of a singly liganded HER2–HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface. Cryo-electron microscopy structures of the HER2–HER3–NRG1β complex reveal a dynamic HER2–HER3 interface and explain the mechanism for the activating HER2 cancer mutations.

Myeloid-derived suppressor cells (MDSCs) are derived from bone marrow progenitor cells commonly, which is a heterogeneous cell group composed of immature granulocytes, dendritic cells, macrophages and early undifferentiated bone marrow precursor cells. Its differentiation and immunosuppressive function are regulated by complex network signals, but the specific regulation mechanisms are not yet fully understood. In this study, we found that in mouse of Lewis lung cancer xenograft, long non-coding RNA Snhg6 (lncRNA Snhg6) was highly expressed in tumor-derived MDSCs compared with spleen-derived MDSCs. LncRNA Snhg6 facilitated the differentiation of CD11b+ Ly6G− Ly6Chigh monocytic MDSCs (Mo-MDSCs) rather than CD11b+ Ly6G+ Ly6Clow polymorphonuclear MDSCs (PMN-MDSCs), but did not affect the immunosuppressive function of MDSCs. Notably, lncRNA Snhg6 could inhibit the expression of EZH2 by ubiquitination pathway at protein level rather than mRNA level during the differentiation of mouse bone marrow cells into MDSCs in vitro. EZH2 may be an important factor in the regulation of lncRNA Snhg6 to promote the differentiation of Mo-MDSCs. So what we found may provide new ideas and targets for anti-tumor immunotherapy targeting MDSCs.

The autosomal dominant monogenetic disease neurofibromatosis type 1 (NF1) affects approximately one in 3,000 individuals and is caused by mutations in the NF1 tumour suppressor gene, leading to dysfunction in the protein neurofibromin (Nf1)1,2. As a GTPase-activating protein, a key function of Nf1 is repression of the Ras oncogene signalling cascade. We determined the human Nf1 dimer structure at an overall resolution of 3.3 Å. The cryo-electron microscopy structure reveals domain organization and structural details of the Nf1 exon 23a splicing3 isoform 2 in a closed, self-inhibited, Zn-stabilized state and an open state. In the closed conformation, HEAT/ARM core domains shield the GTPase-activating protein-related domain (GRD) so that Ras binding is sterically inhibited. In a distinctly different, open conformation of one protomer, a large-scale movement of the GRD occurs, which is necessary to access Ras, whereas Sec14-PH reorients to allow interaction with the cellular membrane4. Zn incubation of Nf1 leads to reduced Ras-GAP activity with both protomers in the self-inhibited, closed conformation stabilized by a Zn binding site between the N-HEAT/ARM domain and the GRD–Sec14-PH linker. The transition between closed, self-inhibited states of Nf1 and open states provides guidance for targeted studies deciphering the complex molecular mechanism behind the widespread neurofibromatosis syndrome and Nf1 dysfunction in carcinogenesis., Cryo-EM structure of Nf1 protein is reported, revealing closed and open conformations that regulate interaction with Ras oncogene, setting the stage for understanding the mechanistic action of Nf1 and how disease mutations lead to dysfunction.

Long non‐coding RNA (lncRNA) MIAT (myocardial infarction associated transcript) has been characterized as a functional lncRNA modulating cerebral ischaemic/reperfusion (I/R) injury. However, the underlying mechanisms remain poorly understood. This study explored the functional partners of MIAT in primary rat neurons and their regulation on I/R injury. Sprague‐Dawley rats were used to construct middle cerebral artery occlusion (MCAO) models. Their cerebral cortical neurons were used for in vitro oxygen‐glucose deprivation/reoxygenation (OGD/R) models. Results showed that MIAT interacted with EGLN2 in rat cortical neurons. MIAT overexpression or knockdown did not alter EGLN2 transcription. In contrast, MIAT overexpression increased EGLN2 stability after I/R injury via reducing its ubiquitin‐mediated degradation. EGLN2 was a substrate of MDM2, a ubiquitin E3 ligase. MDM2 interacted with the N‐terminal of EGLN2 and mediated its K48‐linked poly‐ubiquitination, thereby facilitating its proteasomal degradation. MIAT knockdown enhanced the interaction and reduced EGLN2 stability. MIAT overexpression enhanced infarct volume and induced a higher ratio of neuronal apoptosis. EGLN2 knockdown significantly reversed the injury. MIAT overexpression reduced oxidative pentose phosphate pathway flux and increased oxidized/reduced glutathione ratio, the effects of which were abrogated by EGLN2 knockdown. In conclusion, MIAT might act as a stabilizer of EGLN2 via reducing MDM2 mediated K48 poly‐ubiquitination. MIAT‐EGLN2 axis exacerbates I/R injury via altering redox homeostasis in neurons.

G protein pathway suppressor 2 (GPS2) is expressed in most human tissues, including the stomach. However, the biological functions of GPS2 in cancer, as well as the underlying molecular mechanisms, remain poorly understood. Here, we report that GPS2 expression was aberrantly downregulated in gastric cancer (GC) tissues compared with control tissues. Clinicopathologic analysis showed that low GPS2 expression was significantly correlated with pathological grade, lymph node stage, and invasive depth. Kaplan‐Meier analysis indicated that patients with low GPS2 expression showed poorer overall survival rates than those with high GPS2 expression. Moreover, GPS2 overexpression decreased GC cell proliferation, colony formation, tumorigenesis, and invasion. Overexpression of GPS2 reduced the protein expression of epidermal growth factor receptor (EGFR) and inhibited its downstream signaling in GC cells. Interestingly, GPS2 decreased EGFR protein expression, which was reversed by a lysosome inhibitor. Furthermore, GPS2 reduced EGFR protein stability by enhancing the binding of EGFR and an E3 ligase, c‐Cbl, which promoted the ubiquitination of EGFR, ultimately leading to its degradation through the lysosomal pathway. Further analysis indicated that GPS2 activated autophagy and promoted the autophagic flux by destabilizing EGFR. Taken together, these results suggest that low GPS2 expression is associated with GC progression and provide insights into the applicability of the GPS2‐EGFR axis as a potential therapeutic target in GC., G protein pathway suppressor 2 (GPS2) is expressed at low levels in gastric cancer and low GPS2 expression is associated with poor prognosis. GPS2 reduces epidermal growth factor receptor (EGFR) protein stability through c‐Cbl‐mediated ubiquitination and then lysosomal degradation. GPS2 activates autophagy and promotes the autophagic flux by destabilizing EGFR.

RAN binding protein 10 (RANBP10), a ubiquitously expressed and evolutionarily conserved protein, as a RAN-GTP exchange factor (GEF) to regulate several factors involved in cellular progression. Previous studies showed that RANBP10 was overexpressed in prostate cancer cells and was responsible for androgen receptor (AR) activation. However, the biological function of RANBP10 in glioblastoma (GBM) has not been studied. Here, we found that RANBP10 was overexpressed in GBM, and high RANBP10 expression was closely linked to poor survival of patients with GBM. Downregulation of RANBP10 significantly inhibited cell proliferation, migration, invasion, and tumor growth of GBM cells. In addition, we revealed that RANBP10 could suppress the promoter activity of FBXW7, and thereby increase the protein stability of c-Myc in GBM cells. Silencing of FBXW7 in RANBP10-knockdown GBM cells could partly negate the effects induced by RANBP10 downregulation. Taken together, our findings established that RANBP10 significantly promoted GBM progression by control of the FBXW7–c-Myc axis, and suggest that RANBP10 may be a potential target in GBM.

Oncogenic c-Myc is a master regulator of G1/S transition. Long non-coding RNAs (lncRNAs) emerge as new regulators of various cell activities. Here, we found that lncRNA SnoRNA Host Gene 17 (SNHG17) was elevated at the early G1-phase of cell cycle. Both gain- and loss-of function studies disclosed that SNHG17 increased c-Myc protein level, accelerated G1/S transition and cell proliferation, and consequently promoted tumor cell growth in vitro and in vivo. Mechanistically, the 1-150-nt of SNHG17 physically interacted with the 1035-1369-aa of leucine rich pentatricopeptide repeat containing (LRPPRC) protein, and disrupting this interaction abrogated the promoting role of SNHG17 in c-Myc expression, G1/S transition, and cell proliferation. The effect of SNHG17 in stimulating cell proliferation was attenuated by silencing c-Myc or LRPPRC. Furthermore, silencing SNHG17 or LRPPRC increased the level of ubiquitylated c-Myc and reduced the stability of c-Myc protein. Analysis of human hepatocellular carcinoma (HCC) tissues revealed that SNHG17, LRPPRC, and c-Myc were significantly upregulated in HCC, and they showed a positive correlation with each other. High level of SNHG17 or LRPPRC was associated with worse survival of HCC patients. These data suggest that SNHG17 may inhibit c-Myc ubiquitination and thus enhance c-Myc level and facilitate proliferation by interacting with LRPPRC. Our findings identify a novel SNHG17-LRPPRC-c-Myc regulatory axis and elucidate its roles in G1/S transition and tumor growth, which may provide potential targets for cancer therapy.

Innate immune sensing of cytosolic DNA via absent in melanoma 2 (AIM2) is a key mechanism leading to inflammatory responses. As aberrant immune responses by dysregulated AIM2 are associated with autoinflammatory diseases, activation of the AIM2 inflammasome should be tightly controlled. In this study, we discovered that ubiquitination and deubiquitination of AIM2 are critical events that regulate AIM2 inflammasome activation. In resting human macrophage cells, AIM2 is constitutively ubiquitinated and undergoes proteasomal degradation to avoid autoinflammation. Upon DNA stimulation, USP21 binds to AIM2 and deubiquitinates it, thereby increasing its protein stability. In addition to the role of USP21 in regulating AIM2 turnover, we uncovered that USP21-mediated deubiquitination of AIM2 is required for the assembly of the AIM2 inflammasome. Depletion of USP21 does not affect the DNA-binding ability of AIM2 but inhibits the formation of the AIM2–ASC complex. Our findings establish that fine-tuning of AIM2 by the ubiquitin system is important for regulating AIM2 inflammasome activation.

Colorectal tumorigenesis is a heterogeneous disease driven by multiple genetic and epigenetic alterations. F-box and WD repeat domain containing 11 (FBXW11) is a member of the F-box protein family that regulates the ubiquitination of key factors associated with tumor growth and aggressiveness. Our study aimed to explore the role of FBXW11 in the development and metastasis of colorectal cancer (CRC). FBXW11 was overexpressed in colorectal tumor tissues and its overexpression was associated with a poor prognosis of CRC patients. The upregulation of FBXW11 not only promoted cell proliferation, invasion, and migration, but also contributed to maintaining stem-cell features in colorectal tumor cells. Further analysis revealed that FBXW11 targeted hypermethylated in cancer 1 (HIC1) and reduced its stability in CRC cells through ubiquitination. Moreover, the expression of sirtuin 1 (SIRT1), a deacetylase in tumor cells was upregulated by FBXW11 via regulating HIC1 expression. The mouse xenograft models of CRC confirmed that FBXW11 knockdown impeded colorectal tumor growth and liver metastasis in vivo. In summary, our study identified FBXW11 as an oncogenic factor that contributed to stem-cell-like properties and liver metastasis in CRC via regulating HIC1-mediated SIRT1 expression. These results provide a rationale for the development of FBXW11-targeting drugs for CRC patients.

Nuclear factor (NF)-κB signaling in cancer cells has been reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Here, we aimed to identify and characterize noncanonical activation of NF-κB signaling by ubiquitin-conjugating enzyme E2S (UBE2S) in lung adenocarcinoma cells. TCGA and the Human Atlas Protein Database were used to analyze the survival rate of lung adenocarcinoma patients in conjunction with UBE2S expression. In addition, PC9, H460, H441 and A549 lung adenocarcinoma cells were used in this study. PC9 and H460 cells were selected for further analysis because they expressed different UBE2S protein levels. Specific IKK inhibitors, PS1145 and SC514, were used to assess IκBα phosphorylation. Western blot analysis was used to assess protein levels in PC9 and H460 cells. A scratch wound-healing assay was used to analyze the migrative abilities of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze their effects on downstream protein levels. Immunoprecipitation, immunofluorescent staining, glutathione S transferase (GST) pull-down and in vitro binding assays were used to analyze the interaction between UBE2S and IκBα. A luciferase assay was used to analyze activation of NF-κB signaling regulated by UBE2S. An in vivo zebrafish xenograft model was used to assess metastasis of PC9 cells regulated by UBE2S. We found that UBE2S expression in lung adenocarcinoma patients was negatively related to survival rate. The protein level of UBE2S was higher in PC9 cells than in H460 cells, which was opposite to that observed for IκBα. PC9 cells showed a higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with the IKK-specific inhibitors PS1145 and SC514 in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells revealed that the protein levels of IκBα were inversely regulated. Immunoprecipitation, immunofluorescent staining, GST pull-down and in vitro binding assays revealed direct binding of UBE2S with IκBα. Nuclear P65 protein levels and luciferase assays showed that NF-κB signaling was regulated by UBE2S. The expression of epithelial-to-mesenchymal (EMT) markers and the migrative ability of lung adenocarcinoma cells were also regulated by UBE2S. A zebrafish xenograft tumor model showed a reduction in the metastasis of PC9 cells that was induced by UBE2S knockdown. Higher UBE2S expression in lung adenocarcinomas may lead to increased binding with IκBα to activate NF-κB signaling and promote adenocarcinoma cell metastasis. UBE2S may serve as a potential therapeutic target for lung adenocarcinomas.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1–80 mM l-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s−1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s−1) value obtained by assaying at 1–50 mM l-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs.

Introduction/Objective: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. Materials and Methods: Sediments of cultured RAJI cells (derived from Burkitt’s lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. Results: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. Conclusions: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics., The molecular mechanism underlying the DNA hypermethylation phenotype observed in the SPOP-mutant prostate cancers is unclear. Here, the authors show that mutant SPOP induces global aberrant DNA methylation patterns through GLP/G9a and renders prostate cancer cells susceptible to DNA demethylating agents.

Kinesin Family Member 15 (KIF15) is a plus end-directed microtubule motor, which exerts complex regulations in cancer biology. This study aimed to explore the functional role of KIF15 in leiomyosarcoma (LMS). Bioinformatic analysis was carried out using data from The Cancer Genome Atlas (TCGA)-Sarcoma (SARC). LMS cell lines SK-UT-1 and SK-LMS-1 were used as in vitro cell models. Results showed that LMS patients with high KIF15 expression had significantly worse survival than the low KIF15 expression counterparts. KIF15 knockdown slowed, while KIF15 overexpression increased the proliferation of SK-UT-1 and SK-LMS-1 cells. Co-IP assay confirmed mutual interaction between endogenous KIF15 and DEK (encoded by DEK proto-oncogene). KIF15 knockdown facilitated DEK degradation, while KIF15 overexpression slowed DEK degradation. In ubiquitination assay, a significant increase in DEK polyubiquitylation was observed when KIF15 expression was suppressed. USP15 physically interacted with both DEK and KIF15 in the cells. USP15 knockdown decreased DEK protein stability and canceled KIF15-mediated DEK stabilization. USP15 overexpression enhanced DEK stability, the effect of which was impaired by KIF15 knockdown. USP15 overexpression reduced DEK polyubiquitination. USP15 knockdown increased DEK polyubiquitination and canceled the effect of KIF15 overexpression on reducing DEK polyubiquitination. DEK overexpression enhanced the proliferation of SK-UT-1 and SK-LMS-1 cells. DEK knockdown decreased cell proliferation and canceled the effect of KIF15 overexpression on cell proliferation. In conclusion, this study revealed a novel mechanism that KIF15 enhances LMS cell proliferation via preventing DEK protein from degradation by increasing USP15 mediated deubiquitylation.

Genetic polymorphisms in DNA damage repair and tumor suppressor genes have been associated with increasing the risk of several types of cancer. Analyses of putative functional single nucleotide polymorphisms (SNP) in such genes can greatly improve human health by guiding choice of therapeutics. In this study, we selected nine genes responsible for various cancer types for gene enrichment analysis and found that BRCA1, ATM, and TP53 were more enriched in connectivity. Therefore, we used different computational algorithms to classify the nonsynonymous SNPs which are deleterious to the structure and/or function of these three proteins. Our study demonstrated that V1687G and V1736G variants of BRCA1, I2865T and V2906A variants of ATM, V216G and L194H variants of TP53 are major mutations with pathogenic impact and are likely to have a greater impact on destabilizing the proteins. To stabilize the high-risk SNPs, we performed mutation site-specific molecular docking analysis and validated using molecular dynamics (MD) simulation and molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) studies. Additionally, SNPs of untranslated regions of these genes affecting miRNA binding were characterized. Hence, this study will assist in developing precision medicines for cancer types related to these polymorphisms.

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro. Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases.

Background: Without adequate treatment, pathological cardiac hypertrophy induced by sustained pressure overload eventually leads to heart failure. WWP1 (WW domain–containing E3 ubiquitin protein ligase 1) is an important regulator of aging-related pathologies, including cancer and cardiovascular diseases. However, the role of WWP1 in pressure overload–induced cardiac remodeling and heart failure is yet to be determined. Methods: To examine the correlation of WWP1 with hypertrophy, we analyzed WWP1 expression in patients with heart failure and mice subjected to transverse aortic constriction (TAC) by Western blotting and immunohistochemical staining. TAC surgery was performed on WWP1 knockout mice to assess the role of WWP1 in cardiac hypertrophy, heart function was examined by echocardiography, and related cellular and molecular markers were examined. Mass spectrometry and coimmunoprecipitation assays were conducted to identify the proteins that interacted with WWP1. Pulse-chase assay, ubiquitination assay, reporter gene assay, and an in vivo mouse model via AAV9 (adeno-associated virus serotype 9) were used to explore the mechanisms by which WWP1 regulates cardiac remodeling. AAV9 carrying cardiac troponin T (cTnT) promoter–driven small hairpin RNA targeting WWP1 (AAV9-cTnT-shWWP1) was administered to investigate its rescue role in TAC-induced cardiac dysfunction. Results: The WWP1 level was significantly increased in the hypertrophic hearts from patients with heart failure and mice subjected to TAC. The results of echocardiography and histology demonstrated that WWP1 knockout protected the heart from TAC-induced hypertrophy. There was a direct interaction between WWP1 and DVL2 (disheveled segment polarity protein 2). DVL2 was stabilized by WWP1-mediated K27-linked polyubiquitination. The role of WWP1 in pressure overload–induced cardiac hypertrophy was mediated by the DVL2/CaMKII/HDAC4/MEF2C signaling pathway. Therapeutic targeting WWP1 almost abolished TAC induced heart dysfunction, suggesting WWP1 as a potential target for treating cardiac hypertrophy and failure. Conclusions: We identified WWP1 as a key therapeutic target for pressure overload induced cardiac remodeling. We also found a novel mechanism regulated by WWP1. WWP1 promotes atypical K27-linked ubiquitin multichain assembly on DVL2 and exacerbates cardiac hypertrophy by the DVL2/CaMKII/HDAC4/MEF2C pathway.

The promoting roles of faciogenital dysplasia 5 (FGD5) in tumor progression have been identified in various tumors, however, its roles in gastric cancer progression are still confusing. Currently, it was found that FGD5 was highly expressed in gastric cancer tissues and negatively correlated with different types of survival of gastric cancer patients via online dataset analysis. In vitro analysis of different types of gastric cancer cell lines and normal gastric epithelial cells obtained a consistent result. Then FGD5 was knocked down in gastric cancer cell lines through two independent siRNAs against FGD5 and it was identified that FGD5 knockdown suppressed the cancer stem cell (CSC)-like traits of gastric cancer cells through analyzing the expression of CSC markers, ALDH1 activity and spheroid-formation ability. Further mechanistic studies revealed that FGD5 interacted with Sox2 protein, a critical regulator of CSC progression, enhanced Sox2 protein stability and decreased its ubquitination. Additionally, FGD5 supported the CSC-like traits dependent on Sox2 expression. Taken together, this work identified a novel FGD5/Sox2 axis responsible for the CSC-like traits of gastric cancer cells.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis., p53 regulates signalling pathways involved in metabolic homeostasis. Here the authors show that O-GlcNAcylation of p53 in the liver plays a key role in the physiological regulation of glucose homeostasis, potentially via controlling the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase.

Failures to treat triple-negative breast cancer (TNBC) are mainly due to chemoresistance or radioresistance. We and others previously discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is a massive driver causing these resistance. However, how to dynamically modulate the intrinsic expression of ZEB1 during cell cycle progression is elusive. Here integrated affinity purification combined with mass spectrometry and TCGA analysis identify a cell cycle-related E3 ubiquitin ligase, checkpoint with forkhead and ring finger domains (CHFR), as a key negative regulator of ZEB1 in TNBC. Functional studies reveal that CHFR associates with and decreases ZEB1 expression in a ubiquitinating-dependent manner and that CHFR represses fatty acid synthase (FASN) expression through ZEB1, leading to significant cell death of TNBC under chemotherapy. Intriguingly, a small-molecule inhibitor of HDAC under clinical trial, Trichostatin A (TSA), increases the expression of CHFR independent of histone acetylation, thereby destabilizes ZEB1 and sensitizes the resistant TNBC cells to conventional chemotherapy. In patients with basal-like breast cancers, CHFR levels significantly correlates with survival. These findings suggest the therapeutic potential for targeting CHFR-ZEB1 signaling in resistant malignant breast cancers.

Head and neck squamous cell carcinoma (HNSCC) is a common cancer with high mortality. Anilin actin-binding protein (ANLN) has been reported to be associated with carcinogenesis in multiple tumors. However, the expression pattern and functional effects of ANLN in HNSCC remain to be unclear. Clinical data and online databases were used to analyze the expression of ANLN and its relationship with HNSCC patient survival. Expression of two major splice variants of ANLN was assessed in HNSCC tissues and cell lines. The functional effects and related mechanisms of ANLN isoforms were investigated in HNSCC in vitro and in vivo. Our study showed that patients with high expression of ANLN had a poor prognosis. The two primary isoforms of ANLN transcripts ANLN-201 and ANLN-210 were highly expressed in HNSCC tissues and cell lines. Knockout of ANLN restrained cell proliferation, migration, and invasion of SCC-9 cells. Mechanically, ANLN-201 could interact with c-Myc to keep its protein stability, thereby playing a oncogenic role in HNSCC. ANLN-210 could be transferred to macrophages via exosomes by binding to RNA-binding protein hnRNPC. Exosomal ANLN-210 promoted macrophage polarization via PTEN/PI3K/Akt signaling pathway, thus stimulating tumor growth of HNSCC. ANLN was an independent prognostic factor in patients with HNSCC. Alternatively spliced ANLN isoforms collaboratively promote HNSCC tumorigenesis in vitro and in vivo, which might provide the in-depth role and mechanism of ANLN in HNSCC development.

Pancreatic cancer (PC) remains a major cause of cancer-related deaths primarily due to its inherent potential of therapy resistance. Checkpoint inhibitors have emerged as promising anti-cancer agents when used in combination with conventional anti-cancer therapies. Recent studies have highlighted a critical role of the Greatwall kinase (MASTL; Microtubule-associated serine/threonine-protein kinase-like) in promoting oncogenic malignancy and resistance to anti-cancer therapies; however, its role in PC remains unknown. Based on a comprehensive investigation involving PC patient samples, murine models of PC progression (Kras;PdxCre-KC and Kras;p53;PdxCre-KPC), and loss and gain of function studies, we report a previously undescribed critical role of MASTL in promoting cancer malignancy and therapy resistance. Mechanistically, MASTL promotes PC by modulating the epidermal growth factor receptor (EGFR) protein stability and, thereupon, kinase signaling. We further demonstrate that combinatorial therapy targeting MASTL promotes the efficacy of the cell-killing effects of Gemcitabine using both genetic and pharmacological inhibitions. Taken together, this study identifies a key role of MASTL in promoting PC progression and its utility as a novel target in promoting sensitivity to the anti-pancreatic cancer therapies.

Alzheimer's disease (AD) is a progressive disorder that causes brain cells to degenerate and die. AD is one of the common causes of dementia that leads to a decline in thinking, behavioral and social skills that disrupts a person's ability to function independently. Tau-tubulin kinase1 (TTBK1) is a crucial disease regulating AD protein, which is majorly responsible for the phosphorylation and accumulation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy. TTBK1 involvement in many diseases and the restricted expression of TTBK1 to the central nervous system (CNS) makes TTBK1 an attractive therapeutic target for tauopathies. The genetic variations in TTBK1 are primarily involved in the TTBK1 pathogenesis. This study highlighted the destabilizing, damaging and deleterious effect of the mutation R142Q on TTBK1 structure through computational predictions and molecular dynamics simulations. The protein deviation, fluctuations, conformational dynamics, solvent accessibility, hydrogen bonding, and the residue-residue mapping confirmed the mutant effect to cause structural aberrations, suggesting overall destabilization due to the protein mutation. The presence of well-defined free energy minima was observed in TTBK1-wild type, as opposed to that in the R142Q mutant, reflecting structural deterioration. The overall findings from the study reveal that the presence of R142Q mutation on TTBK1 is responsible for the structural instability, leading to disruption of its biological functions. The mutation could be used as future diagnostic markers in treating AD.

Immunotherapy using checkpoint blockers (antibodies) has been a major advance in recent years in the management of various types of solid cancers including lung cancer. One target of checkpoint blockers is programmed death ligand 1 (PD-L1) expressed by cancer cells, which engages programmed death 1 on T cells and Natural Killer (NK) cells resulting in suppression of their activation and cancer-killing function, respectively. Apart from antibodies, other clinically relevant agents that can inhibit PD-L1 are limited. PD-L1 protein stability depends on its glycosylation. Here we show that l-glutamine:d-fructose-6-phosphate amidotransferase 1 (GFAT1), a rate-limiting enzyme of the hexosamine biosynthesis pathway, which produces uridine diphosphate-N-acetyl-β-glucosamine, a precursor for glycosylation, is required for the stability of PD-L1 protein. Inhibition of GFAT1 activity markedly reduced interferon gamma (IFNγ)-induced PD-L1 levels in various lung cancer cell lines. GFAT1 inhibition suppressed glycosylation of PD-L1 and accelerated its proteasomal degradation. Importantly, inhibition of GFAT1 in IFNγ-treated cancer cells enhanced the activation of T cells and the cancer-killing activity of NK cells. These findings support using GFAT1 inhibitors to manipulate PD-L1 protein level that could augment the efficacy of immunotherapy for lung cancer.

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

Abscisic acid (ABA) is a plant hormone that activates adaptive mechanisms to environmental stress conditions. Plant adaptive mechanisms are complex and highly modulated processes induced by stress-responsive proteins; however, the precise mechanisms by which these processes function under adverse conditions remain unclear. Here, we isolated CaUBP12 (Capsicum annuum ubiquitin-specific protease 12) from pepper (C. annuum) leaves. We show that CaUBP12 expression is significantly induced after exposure to abiotic stress treatments. We conducted loss-of-function and gain-of-function genetic studies to elucidate the biological functions of CaUBP12 in response to ABA and dehydration stress. CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively; these phenotypes were characterized by regulation of transpirational water loss and stomatal aperture. Under dehydration stress conditions, CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants exhibited lower and higher expression levels of stress-related genes, respectively, than the control plants. We isolated a CaUBP12 interaction protein, CaSnRK2.6, which is a homolog of Arabidopsis OST1; degradation of this protein was partially inhibited by CaUBP12. Similar to CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants, CaSnRK2.6-silenced pepper plants and CaSnRK2.6-overexpressing Arabidopsis displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively. Our findings suggest that CaUBP12 positively modulates the dehydration stress response by suppressing CaSnRK2.6 protein degradation.

Ubiquitin-specific peptidases7 (USP7) participates in the regulation of various metabolic and immune disorders. However, the role of USP7 in lupus nephritis (LN) remains unknown. The current study set out to elucidate the regulatory role of USP7 in LN together with JMJD3 and NF-κB. SLE MRL/LPR mice and mouse glomerular mesangial cells SV40 MES 13 cells were employed for in vivo or vitro experiments. USP7, JMJD3 and NF-κB expression in MRL/LPR mice were detected, followed by investigation of their functions in the proliferation of mesangial cells and mesangial matrix. Subsequently, the interaction among USP7, JMJD3 and NF-κB was determined by means of ChIP and co-immunoprecipitation assay. The results indicated that USP7, JMJD3, p-NF-κB p65 were all highly-expressed in MRL/LPR mice. USP7 promoted the proliferation of mesangial cells and mesangial matrix, and stabilized the JMJD3 protein via deubiquitination in SV40 MES 13 cells. Meanwhile, silencing of JMJD3 inhibited the promotive effect of USP7 on the proliferation of mesangial cells and mesangial matrix. Furthermore, JMJD3 increased the expression of NF-κB p65 through demethylation, whereas silencing JMJD3 alleviated the proliferation of mesangial cells and mesangial matrix. Lastly, NF-κB p65 was proved to aggravate LN pathogenesis. Altogether, our findings highlighted that USP7 promoted the occurrence of LN by regulating the NF-κB p65 signaling pathway via stabilization of JMJD3.

Neutrophils are significant compositions of solid tumors and exert distinct functions in different types of tumors. However, the precise role of neutrophils in the progression of breast cancer (BC) is presently unclear. In this study, by investigating the single-cell RNA sequencing data, we identify a new neutrophil subset, C5aR1-positive neutrophils, that correlates with tumor progression and poor survival for BC patients. Furthermore, it is discovered that C5aR1-positive neutrophils enhance BC cell glycolysis via upregulating ENO1 expression. Mechanically, C5aR1-positive neutrophil-secreted IL1β and TNFα cooperatively activate ERK1/2 signaling, which phosphorylates WTAP at serine341 and thereby stabilizes WTAP protein. The stabilization of WTAP further promotes RNA m6A methylation of ENO1, impacting the glycolytic activity of BC cells. Importantly, C5aR1-positive neutrophils also promote breast cancer growth in vivo, and this effect is abolished by WTAP silencing. In clinical BC samples, increased C5aR1-positive neutrophils correlate with elevated IL1β, TNFα, and ENO1 expression. A high co-expression of C5aR1-positive neutrophil gene signature and ENO1 predicts worse prognosis of BC patients compared with a low co-expression. Collectively, our study reveals a novel subset of C5aR1-positive neutrophils that induces breast cancer glycolysis via increasing ERK1/2-WTAP-dependent m6A methylation of ENO1. These findings support the potential for exploration of C5aR1-positive neutrophils as a therapeutic target in breast cancer.

Both bench and bedside investigations have challenged the supportive role of Hedgehog (Hh) activity in the progression of colorectal cancers, thus raising a critical need to further deeply determine the contribution of Hh to the growth of colorectal cancer. Combining multiple complementary means, including in vitro and in vivo inflammatory colorectal cancer models, and pathological analysis of clinical colorectal cancer patients samples. We report that colorectal cancer cells hijack prostaglandin E2 (PGE2) to non-canonically promote Hh transcriptional factor Gli activity and Gli-dependent proliferation of colorectal cancer cells in a Smo-independent manner. Mechanistically, PGE2 activates c-Jun N-terminal kinase (JNK), which in turn enables Gli2 to evade ubiquitin-proteasomal degradation by phosphorylating Gli2 at Thr1546. This study not only presents evidence for understanding the contribution of Hh to colorectal cancers, but also provides a novel molecular portrait underlying how PGE2-activated JNK fine-tunes the evasion of Gli2 from ubiquitin-proteasomal degradation. Therefore, it proposes a rationale for the future evaluation of chemopreventive and selective therapeutic strategies for colorectal cancers by targeting PGE2-JNK-Gli signaling route.

Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation, and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.