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Toll-like receptor 4 (TLR4) is a key pattern recognition receptor that can be activated by bacterial lipopolysaccharide to elicit inflammatory response. Proper activation of TLR4 is critical for the host defense against microbial infections. Since overactivation of TLR4 causes deleterious effects and inflammatory diseases, its activation needs to be tightly controlled by negative regulatory mechanisms, among which the most pivotal could be deubiquitination of key signaling molecules mediated by deubiquitinating enzymes (DUBs). CYLD is a member of the USP family of DUBs that acts as a critical negative regulator of TLR4-depedent inflammatory responses by deconjugating polyubiquitin chains from signaling molecules, such as TRAF6 and TAK1. Dysregulation of CYLD is implicated in inflammatory diseases. However, how the function of CYLD is regulated during inflammatory response remains largely unclear. Recently, we and other authors have shown that Spata2 functions as an important CYLD partner to regulate enzymatic activity of CYLD and substrate binding by this protein. Here, we show that a Spata2-like protein, Spata2L, can also form a complex with CYLD to inhibit the TLR4-dependent inflammatory response. We found that Spata2L constitutively interacts with CYLD and that the deficiency of Spata2L enhances the LPS-induced NF-κB activation and proinflammatory cytokine gene expression. Mechanistically, Spata2L potentiated CYLD-mediated deubiquitination of TRAF6 and TAK1 likely by promoting CYLD enzymatic activity. These findings identify Spata2L as a novel CYLD regulator, provide new insights into regulatory mechanisms underlying CYLD role in TLR4 signaling, and suggest potential targets for modulating TLR4-induced inflammation.

CYLD was a novel causative gene for frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. Given the clinical and pathological overlap of FTD and Alzheimer's disease (AD), it is necessary to screen CYLD in AD patients and FTD patients in the Chinese population.In our study, using a targeted sequencing panel, we sequenced the CYLD gene in a large cohort of 2485 participants in the Chinese population, including 1008 AD patients, 105 FTD patients, and 1372 controls.In the present study, the average onset age of AD and FTD patients was 66.84 ± 30.42 years old and 60 ± 10.00 years old, respectively. Our study reported three novel CYLD variants: p.Phe288Leu (patient No. 1, AD), p.Tyr485Phe (patients No. 6-9, all AD) and p.Thr951Ala (patient No. 10, AD), plus a previously reported variant: p.Arg397Ser (patient No. 2-5, AD and No. 11, FTD). These variants were absent in our in-house controls and predicted to be deleterious according to the MutationTaster. The variant carriers were composed of 10 AD patients and one FTD patient, and the average onset age was 61.2 ± 10.9 years. The frequency of CYLD variants in AD was similar to that in FTD, which was 0.99% (10/1008) and 0.95% (1/105), respectively.Our finding extended the genotype and phenotype of the CYLD gene and demonstrated that CYLD rare damaging variants may be implicated in AD and FTD pathogenesis.

Proneural (PN) to mesenchymal (MES) transition (PMT) is a crucial phenotypic shift in glioblastoma stem cells (GSCs). However, the mechanisms driving this process remain poorly understood. Here, we report that Fos-like antigen 1 (FOSL1), a component of AP1 transcription factor complexes, is a key player in regulating PMT. FOSL1 is predominantly expressed in the MES subtype, but not PN subtype, of GSCs. Knocking down FOSL1 expression in MES GSCs leads to the loss of MES features and tumor-initiating ability, whereas ectopic expression of FOSL1 in PN GSCs is able to induce PMT and maintain MES features. Moreover, FOSL1 facilitates ionizing radiation (IR)-induced PMT and radioresistance of PN GSCs. Inhibition of FOSL1 enhances the anti-tumor effects of IR by preventing IR-induced PMT. Mechanistically, we find that FOSL1 promotes UBC9-dependent CYLD SUMOylation, thereby inducing K63-linked polyubiquitination of major nuclear factor κB (NF-κB) intermediaries and subsequent NF-κB activation, which results in PMT induction in GSCs. Our study underscores the importance of FOSL1 in the regulation of PMT and suggests that therapeutic targeting of FOSL1 holds promise to attenuate molecular subtype switching in patients with glioblastomas.

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.

Lung cancer is one of the most common types of carcinoma worldwide. Cigarette smoking is considered the leading cause of lung cancer. Aberrant expression of several YT521-B homology (YTH) family proteins has been reported to be closely associated with multiple cancer types. The present study aims to evaluate the function and regulatory mechanisms of the N6-methyladenosine (m6A) reader protein YTH domain containing 2 (YTHDC2) by in vitro, in vivo and bioinformatics analyses. The results revealed that YTHDC2 was reduced in lung cancer and cigarette smoke-exposed cells. Notably, bioinformatics and tissue arrays analysis demonstrated that decreased YTHDC2 was highly associated with smoking history, pathological stage, invasion depth, lymph node metastasis and poor outcomes. The in vivo and in vitro studies revealed that YTHDC2 overexpression inhibited the proliferation and migration of lung cancer cells as well as tumor growth in nude mice. Furthermore, YTHDC2 decreased expression was modulated by copy number deletion in lung cancer. Importantly, the cylindromatosis (CYLD)/NF-κB pathways were confirmed as the downstream signaling of YTHDC2, and this axis was mediated by m6A modification. The present results indicated that smoking-related downregulation of YTHDC2 was associated with enhanced proliferation and migration in lung cancer cells, and appeared to be regulated by DNA copy number variation. Importantly, YTHDC2 functions as a tumor suppressor through the CYLD/NF-κB signaling pathway, which is mediated by m6A modification.

Oxidative stress-induced damage to and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD); however, the underlying molecular mechanism is not fully understood. Long noncoding RNAs (lncRNAs) have important roles in various biological processes. In this study, using an oxidative damage model in RPE cells, we identified a novel oxidation-related lncRNA named CYLD-AS1. We further revealed that the expression of CYLD-AS1 was increased in RPEs during oxidative stress. Depletion of CYLD-AS1 promoted cell proliferation and mitochondrial function and protected RPE cells against hydrogen peroxide (H

Ferroptosis is an iron-dependent cell death and contributes to doxorubicin-induced cardiotoxicity, but the mechanisms behind intracellular iron overload in cardiomyocyte after administration of doxorubicin remain largely unknown. Ferritinophagy is a selective type of autophagy and could be a novel source for intracellular free iron. Spermatogenesis-associated protein 2 (SPATA2), a member of the TNF signaling pathway, can recruit cylindromatosis (CYLD, a deubiquitinating enzyme) to regulate cell death. This study aims to explore whether ferritinophagy is the source for intracellular iron overload in cardiomyocyte upon doxorubicin treatment and whether the SPATA2/CYLD pathway is involved in regulation of nuclear receptor coactivator 4 (NCOA4) level, the selective cargo receptor for ferritinophagy. The C57BL/6J mice were subjected to a single injection of doxorubicin, which showed the compromised cardiac functions, accompanied by the upregulation of SPATA2 and CYLD and the enhanced interaction between them, the increases in ferritinophagy (reflecting by increases in NCOA4 and ratio of LC3Ⅱ/LC3Ⅰ while decreases in NCOA4 ubiquitination and ferritin) and ferroptosis (reflecting by intracellular iron overload and increase of acyl-CoA synthetase long chain family member 4). Consistently, similar results were achieved in the cultured cardiomyocytes after incubation with doxorubicin. Knocked down of SPATA2 notably reduced doxorubicin-induced cardiomyocyte injury concomitant with the attenuated ferritinophagy and the decreased ferroptosis. Based on these observations, we conclude that a novel pathway of SPATA2/CYLD has been identified, which contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy through a mechanism involving the deubiquitination of NCOA4.

Background The cylindromatosis (CYLD) tumor suppressor is a microtubule‐associated deubiquitinase that plays a critical role in the regulation of cell signaling and contributes to a variety of physiological and pathological processes. However, the functions of CYLD in zebrafish are less well known, particularly with regard to their development and physiology. In this context, we investigated the loss of function of CYLD in zebrafish via transcription activator‐like effector nuclease (TALEN)‐based gene deletion. Methods Semi‐quantitative RT‐PCR was used to quantify CYLD mRNA expression in zebrafish embryos at various developmental stages. We also performed whole‐mount in situ hybridization to further assess the dynamic expression and distribution of CYLD in the entire zebrafish embryos at different stages. In addition, we deleted CYLD in zebrafish with TALENs to investigate its potential impact on embryonic development. Results The expression of CYLD mRNA varied during early embryonic development. The CYLD mRNA localized to the brain and notochord of developing zebrafish embryos. Homozygous deletion of CYLD resulted in embryonic death before 8 h post‐fertilization. Conclusions CYLD appears to play an important role in central nervous system development in zebrafish. Although severe embryonic death restricted analysis of homozygous mutants, further research into the role of CYLD in central nervous system development is warranted., Cylindromatosis (CYLD) has important pathophysiological roles in humans. More than just a tumor suppressor, CYLD has many potential functions. We exploited the zebrafish model to investigate dynamic changes in CYLD expression during embryonic development. In doing so, we discovered a potential role for CYLD in brain and notochord development, providing experimental evidence for a novel pathophysiological role in central nervous system development.

Xing Lin,1 Jian Zhan,2 Jin Jiang,3 Yikun Ren3 1Department of Biological Immunotherapy, Chongqing University Cancer Hospital, Chongqing Cancer Institute, Chongqing Cancer Hospital, Shapingba District, Chongqing, 400030, People’s Republic of China; 2Department of Neurology, The Second Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou Province, 563000, People’s Republic of China; 3Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People’s Republic of ChinaCorrespondence: Jin Jiang; Yikun RenThe First Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing, 400016, People’s Republic of ChinaTel +86 177 4992 3714; +86 151 9609 7005Email m17749923714@163.com; renyikunde@163.comBackground: Activated microglia are polarized into the M1 or M2 phenotype. We previously reported that electroacupuncture (EA) effectively prevented nuclear factor-κB (NF-κB) nuclear translocation and improved neuronal C-X-C motif 3 chemokine ligand 1 (CX3CL1) expression, repressing microglial activation by upregulating neuronal cylindromatosis (CYLD) expression in the periischemic cortex. However, the potential mechanisms are unclear. Therefore, we explored whether EA improved CYLD protein expression to regulate microglial polarization-mediated neuroinflammation and the potential mechanisms in an ischemic stroke model.Methods: A middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in male Sprague-Dawley (SD) rats. The rats were treated with EA at the Baihui, Hegu and Taichong acupoints once daily beginning 2 h after focal cerebral ischemia. CYLD gene interference was used to investigate the role of CYLD in microglial polarization. We used neurobehavioral evaluations and TTC staining to examine the neuroprotective effect of EA via CYLD upregulation. Immunofluorescence and RT-qPCR were used to measure NLRP3 activation, M1/M2 microglial activation, pro-/anti-inflammatory gene mRNA expression and crosstalk (CX3CL1/CX3CR1 axis) between neurons and microglia. Western blotting was used to assess the underlying molecular mechanism.Results: CYLD inhibited M1 microglial activation and improved M2 microglial activation after 72 h of reperfusion. CYLD overexpression decreased the NLRP3 mRNA level. CYLD suppressed microglial overactivation by inhibiting NLRP3 activation. CYLD gene silencing partially weakened EA improvement of neurological function deficits and reduction of infarct volumes after 72 h reperfusion. In addition, EA inhibited M1-like phenotypic microglial activation and promoted M2-like phenotypic microglia through upregulating CYLD expression. Finally, EA-mediated modulation of the CX3CL1/CX3CR1 axis and NLRP3 inflammasome was reversed by CYLD gene silencing in the periischemic cortex.Conclusion: EA-induced upregulation of neuronal CYLD expression plays anti-inflammatory and neuroprotective roles and regulates the interaction between neurons and microglia, thereby suppressing M1 and improving M2 microglial activation in the periischemic cortex.Keywords: microglial polarization, cerebral ischemia, neuroinflammation, CYLD, electroacupuncture, NLRP3 inflammasome, CX3CL1/CX3CR1 axis

In this study, we determined the involvement of SOX2 and its downstream signaling molecules in hepatocellular carcinoma (HCC) progression. We carried out lentiviral transfection in HepG2 cells to determine the roles of SOX2, CCAT1, EGFR, miR-222-5p, and CYLD in HepG2 cells. We first determined the interaction between SOX2 and CCAT1 and that between miR-222-5p and CYLD and their effect on tumor growth in vivo was analyzed in HCC-xenograft bearing nude mice xenografts. SOX2 and CCAT1 were highly expressed in HCC tissues and HepG2 cells. SOX2 bound to the regulatory site of CCAT1. Silencing of SOX2 or CCAT1 inhibited HepG2 cell proliferation, migration, and invasion as well as decreased the expression of CCAT1 and EGFR. CCAT1 silencing reduced EGFR expression, but EGFR expression was increased in HCC tissues and HepG2 cells, which promoted proliferation, migration, and invasion in vitro. EGFR upregulated miR-222-5p, leading to downregulation of CYLD. miR-222-5p inhibition or CYLD overexpression repressed cell functions in HepG2 cells. SOX2 silencing decreased CCAT1, EGFR, and miR-222-5p expression but increased CYLD expression. Loss of SOX2 also reduced the growth rate of tumor xenografts. In summary, SOX2-mediated HCC progression through an axis involving CCAT1, EGFR, and miR-222-5p upregulation and CYLD downregulation.

Mutations in the tumor suppressor CYLD, known to be causative of cylindromas, were recently described in a subset of high-risk (hr) HPV-positive head and neck squamous cell carcinomas (HNSCC). Pathologic and genetic characterization of these CYLD-mutant carcinomas, however, remains limited. Here, we investigated whether CYLD mutations characterize a histopathologically and genomically distinct subset of hrHPV-positive HNSCC. Comprehensive genomic profiling via hybrid capture-based DNA sequencing was performed on 703 consecutive head and neck carcinomas with hrHPV sequences, identifying 148 unique cases (21%) harboring CYLD mutations. Clinical data, pathology reports, and histopathology were reviewed. CYLD mutations included homozygous deletions (n = 61/148; 41%), truncations (n = 52; 35%), missense (n = 26; 18%) and splice-site (n = 9; 6%) mutations, and in-frame deletion (n = 1; 1%). Among hrHPV-positive HNSCC, the CYLD-mutant cohort showed substantially lower tumor mutational burden than CYLD-wildtype cases (n = 555) (median 2.6 vs. 4.4 mut/Mb, p

Mutations in PINK1 and parkin highlight the mitochondrial axis of Parkinson’s disease (PD) pathogenesis. PINK1/parkin regulation of the transcriptional repressor PARIS bears direct relevance to dopamine neuron survival through augmentation of PGC-1α–dependent mitochondrial biogenesis. Notably, knockout of PARIS attenuates dopaminergic neurodegeneration in mouse models, indicating that interventions that prevent dopaminergic accumulation of PARIS could have therapeutic potential in PD. To this end, we have identified the deubiquitinase cylindromatosis (CYLD) to be a regulator of PARIS protein stability and proteasomal degradation via the PINK1/parkin pathway. Knockdown of CYLD in multiple models of PINK1 or parkin inactivation attenuates PARIS accumulation by modulating its ubiquitination levels and relieving its repressive effect on PGC-1α to promote mitochondrial biogenesis. Together, our studies identify CYLD as a negative regulator of dopamine neuron survival, and inhibition of CYLD may potentially be beneficial in PD by lowering PARIS levels and promoting mitochondrial biogenesis.

Evolving understanding of head and neck squamous cell carcinoma (HNSCC) is leading to more specific diagnostic disease classifications. Among HNSCC caused by the human papilloma virus (HPV), tumors harboring defects in

Yang et al. found that mutation of the nonsense‐mediated decay factor SMG7 enhanced resistance to tumor necrosis factor (TNF)‐induced apoptosis via two oncogenic pathways. Downregulation of CYLD and overexpression of the long noncoding RNA Pvt1 converge to promote NF‐κB activation. The results indicate a composite tumor suppressor role for SMG7 in response to TNF., Nonsense‐mediated decay (NMD) proteins are responsible for the surveillance and degradation of aberrant RNAs. Suppressor with morphogenetic effect on genitalia 7 (SMG7) is an NMD complex protein and a regulator of tumor necrosis factor (TNF)‐induced extrinsic apoptosis; however, this unique function has not been explored in detail. In this study, we show that loss of Smg7 leads to unrestricted expression of long noncoding RNAs (lncRNAs) in addition to NMD targets. Functional analysis of Smg7−/− cells showed downregulation of the tumor suppressor cylindromatosis (CYLD) and diminished caspase activity, thereby switching cells to nuclear factor‐κB (NF‐κB)‐mediated protection. This positive relationship between SMG7 and CYLD was found to be widely conserved in human cancer cell lines and renal carcinoma samples from The Cancer Genome Atlas. In addition to CYLD suppression, upregulation of lncRNAs Pvt1 and Adapt33 rendered cells resistant to TNF, while pharmacologic inhibition of NF‐κB in Pvt1‐overexpressing TNF‐resistant cells and Smg7‐deficient spheroids re‐established TNF‐induced lethality. Thus, loss of SMG7 decouples regulation of two separate oncogenic factors with cumulative downstream effects on the NF‐κB pathway. The data highlight a novel and specific regulation of oncogenic factors by SMG7 and pinpoint a composite tumor suppressor role in response to TNF.

The deubiquitinase cylindromatosis (CYLD) functions as a tumor suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although short hairpin RNA (shRNA) knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigations demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3-untranslated region (3-UTR) of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.

It has been reported that microRNA (miRNA/miR)-181b plays an important role in regulating cellular proliferation, invasion and apoptosis in various tumors. However, the role of miR-181b and its molecular mechanisms in colon cancer cells have not yet been elucidated. The present study thus aimed to investigate the mechanisms of miR-181b targeting cylindromatosis (CYLD) to regulate the nuclear factor-κB (NF-κB) signaling pathway, and to determine its role in colon cancer cell proliferation and apoptosis. For this purpose, miR-181b was overexpressed and silenced in the SW480 cell line. The cell proliferation and apoptotic rates were determined using a Cell Counting kit and colony formation assays, and Annexin V-FITC staining, respectively. The expression levels of proteins associated with the NF-κB signaling pathway and apoptosis were detected by western blot analysis. Furthermore, a dual luciferase assay was applied to confirm the interaction between miR-181b and CYLD. CYLD was also overexpressed and silenced in the SW480 cell line using a CYLD overexpression plasmid and siRNA technology, respectively. Transfected cells were used for subsequent experiments. In addition, a nude mouse model was established to measure tumor volume and weight. Immunohistochemistry and a TUNEL assay were performed to detect the Ki67 levels and the cell apoptotic rate, respectively. Compared with the control group, miR-181 silencing or CYLD overexpression significantly attenuated cell proliferation, invasion and migration, and notably increased the proportion of apoptotic cells. Furthermore, the expression levels of Bax and cleaved caspase-3 were markedly increased, whereas those of Bcl-2 were significantly decresaed (P

Objectives Recent genomic studies suggest the biological significance of the cylindromatosis (CYLD) gene in thymic epithelial tumors (TETs). CYLD is a crucial regulator of immune response, and we previously reported that CYLD mutation is associated with high PD-L1 expression in thymic carcinoma. Therefore, we wanted to explore the role and mechanism of CYLD in regulating PD-L1 expression in TETs. Materials and methods The role of CYLD in PD-L1 expression was assessed by knockdown of CYLD in TET cells upon stimulation with interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α) or polyinosinic-polycytidylic acid (poly I:C). The molecular mechanism was investigated through analysis of downstream molecules in the STAT1/IRF1 pathway. Moreover, the clinical correlation between low CYLD and high PD-L1 expression, and the clinical impact of CYLD expression were evaluated in tissue microarrays of 105 TET cases. Results CYLD knockdown significantly enhanced the expression of PD-L1 in presence of IFN-γ stimulation in most TET cell lines. However, this phenomenon was not observed in presence of TNF-α stimulation. CYLD knockdown upregulated IFN-γ mediated activation of the STAT1/IRF1 axis, which in turn induced PD-L1 expression. Interestingly, we found a significant association between low CYLD expression and ≥ 50 % PD-L1 expression (p = 0.001). In addition, the average proportion of tumor cells exhibiting PD-L1 staining was significantly higher in the low CYLD expression group (24.7 %) than in the high CYLD expression group (5.2 %) (p = 0.005). There was no correlation between CYLD expression and the frequency of pre-existing paraneoplastic auto-immune diseases. In advanced stages (III/IV), the low CYLD expressing group had numerically worse survival than the high CYLD group (log-rank p = 0.089). Conclusions Our findings provide insight into the mechanism of regulation of PD-L1 expression by CYLD in TET cells. Tumors with low CYLD expression could be potential targets for PD-1/PD-L1 inhibitors.

Deubiquitinase cylindromatosis (CYLD) inhibits MAPK and NF-κB activation pathways by deubiquitinating upstream regulatory factors. Although CYLD has been identified and actively studied in mammals, nothing is known about its putative function in fish. In this study, we identified the gene encoding CYLD (OmCYLD) from rainbow trout, Oncorhynchus mykiss, and examined its role during pathogenic infections. The deduced amino acid sequence of OmCYLD contains conserved CAP-Gly and USP domains. In RTH-149 cells, the expression of OmCYLD was increased by stimulation with Edwardsiella tarda and Streptococcus iniae. Gain-of-function and loss-of-function experiments showed that OmCYLD down-regulates the activation of MAPK and NF-κB and the expression of pro-inflammatory cytokines in E. tarda-stimulated RTH-149 cells. These findings suggest that OmCYLD might function like those of mammals to negatively regulate bacteria-triggered signaling pathway in fish.

The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.

Chengjiang Qiu,1 Kairui Liu,1 Sheng Zhang,1 Simin Gao,2 Weirun Chen,1 Dateng Li,3 Youxing Huang1 1Department of Abdominal Surgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, People’s Republic of China; 2Department of Breast Surgery, Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China; 3Department of Statistical Science, Southern Methodist University, Dallas, TX 75275, USACorrespondence: Youxing HuangDepartment of Abdominal Surgery, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 111 Dade Road, Guangzhou, Guangdong Province, People’s Republic of ChinaTel +86-13632255441Fax + 86-20-39318790Email waiqike7@163.comBackground: Bisdemethoxycurcumin (BDMC), a stable bioactive ingredient in curcuminoids, is associated with various antitumor functions, such as proliferation inhibition, metastasis suppression and apoptosis induction, in many cancer types. However, the mechanism of BDMC in hepatocellular carcinoma (HCC) remains unclear.Methods: We assessed the toxicity and the inhibitory effect of BDMC in the HepG2 cell line by using CCK-8 and colony formation assays. The regulatory effects of BDMC on Akt and MAPK signaling were investigated by Western blotting and immunoprecipitation.Results: We found that the half-maximum inhibitory concentration (IC50) of BDMC after 48hrs of treatment was 59.13 μM, and BDMC inhibited proliferation in a time- and dose-dependent manner in HepG2 cells. The inhibitory effect was caused by the inactivation of Akt signaling, but not Erk, Jnk or p38 signaling. In addition, the inactivation of Akt signaling was attributed to the inhibition of ubiquitination mediated by K63-Ub but not K48-Ub. Furthermore, we found that BDMC upregulated the expression of CYLD, leading to Akt deubiquitination and inactivation.Conclusion: BDMC inhibited HCC cell proliferation, and that this effect was induced by Akt inactivation via CYLD-mediated deubiquitination.Keywords: bisdemethoxycurcumin, hepatocellular carcinoma, proliferation, deubiquitination

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-κB activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-κB signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-κB signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs.

Endometrial cancer is a threat to women health worldwide. Cylindromatosis (CYLD) enzyme is a tumour suppressor, considered an effective prognostic marker in various malignancies, but its role in endometrial carcinoma is not fully elucidated. Here, we sought to estimate the prognostic value of CYLD expression in endometrial carcinoma.CYLD levels were immunohistochemically evaluated in 65 patients with endometrial carcinoma and inferential statistics were applied.Low or negative CYLD expression significantly correlates with older ages, non-endometrioid and invasive carcinomas, tumours with moderate or poor differentiation and advanced stages. Moreover, non-endometrioid and invasive carcinomas are independent risk factors for weaker CYLD expression. Kaplan-Meier analysis illustrated that negative or low CYLD expression is statistically significantly associated with increased death risk, compared to moderate or high expression.This study demonstrates for the first time a clear correlation between CYLD expression and clinicohistopathological parameters of endometrial carcinoma patients, suggesting its use as a potential prognostic/predictive marker for Endometrial Carcinoma.

Polycythemia vera (PV) is characterized by increased proliferation and accumulation of erythroid and mature myeloid cells and megakaryocyte in the bone marrow and peripheral blood. The JAK2V617F mutation is present in most PV patients. Deubiquitinase (DUB) genes, including TNFAIP3 (A20), CYLD and Cezanne, function as negative regulators of inflammatory reaction through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ęB) signaling.To determine single nucleotide polymorphisms (SNPs) profiling and gene expression of the DUB genes as well as the immunophenotype of PV cells.Seventy-seven patients with PV and 55 healthy individuals with well-characterized clinical profiles were enrolled. Gene expression profile was determined using quantitative real-time polymerase chain reaction (qRT-PCR), the immunophenotype with flow cytometry, secretion of cytokines using enzyme-linked immunosorbent assay (ELISA), and gene polymorphisms using direct DNA sequencing.Inactivation of A20, CYLD and Cezanne, and increases in interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-á) levels, as well as the enhanced number of CD25+CD4 T, Th1 and regulatory T cells were observed in PV patients. The genetic analysis of the CYLD gene identified 11 SNPs, in which a novel W736G nsSNP in exon 15 and a SNP c.2483+6 TG in intron 15 were observed in PV cases with the frequencies of 18.2% and 5.2%, respectively. The W736G non-synonymous SNP (nsSNP) was found to be most likely to exert deleterious effect and the intronic SNP c.2483+6 TG was identified as aberrant splicing. Sequencing of Cezanne gene identified 7 SNPs in intron 10 and PV carriers of the SNPs had at least 2 SNPs in this gene. Importantly, PV carriers of the W736G nsSNP had multiple SNPs in CYLD, but not in A20 or Cezanne gene.Two identified SNPs, including the W736G nsSNP and the SNP c.2483+6 TG, in CYLD gene might be associated with a risk of PV disease, in which the deleterious effect of the W736G nsSNP in CYLD gene could contribute to the pathogenesis of PV.

According to the global cancer statistic, lung cancer is one of the most dangerous tumors, which poses a serious threat to human health. Exploration the mechanism of lung cancer and new targeted therapeutic measures is always the hot topic. Long noncoding RNA (lncRNA) is an important factor affecting the development of tumors. However, the research on the mechanism of lncRNA in the progress of lung cancer needs to be further expanded. In this study, we found that the expression of lncRNA GMDS‐AS1 was significantly reduced in lung adenocarcinoma (LUAD) tissues and cells. Upregulated GMDS‐AS1 can significantly inhibit the proliferation of LUAD cells and promote cell apoptosis in vitro and in vivo. The results indicate that GMDS‐AS1 acts as a tumor suppressor gene to affect the development of LUAD. Further studies revealed that GMDS‐AS1 is a target gene of miR‐96‐5p, and GMDS‐AS1 regulates proliferation and apoptosis of LUAD cells in association with miR‐96‐5p. In addition, we also confirmed that CYLD lysine 63 deubiquitinase (CYLD) is also a target gene of miR‐96‐5p. Through various validations, we confirmed that GMDS‐AS1 can act as a ceRNA to upregulate the expression of CYLD by sponging miR‐96‐5p. Moreover, the intervention of GMDS‐AS1/miR‐96‐5p/CYLD network can regulate the proliferation and apoptosis of LUAD cells. In this study, we revealed that the GMDS‐AS1/miR‐96‐5p/CYLD network based on ceRNA mechanism plays an important role in the development of LUAD and provides a new direction and theoretical basis for targeted therapy of LUAD., GMDS‐AS1/miR‐96‐5p/CYLD network based on ceRNA mechanism plays an important role in the development of lung cancer and provides a new direction and theoretical basis for targeted therapy of lung cancer.

Exposure to high doses of anticancer drugs can induce the emergence of a subpopulation of weakly proliferative and drug-tolerant cells. Drug tolerance can reduce the benefits obtained from canonical treatment and reduce the survival rate of patients. Regulation of SRY-related HMG box transcription factor 4 (SOX4) has been proved to affect drug sensitivity. The current study aimed to explore the role of SOX4 in drug resistance of colorectal cancer (CRC) cells as well as the related molecular mechanisms. Expression patterns of SOX4, microRNA-17 (miR-17), and CYLD in both CRC tissues and cells were determined with their relationship analyzed by bioinformatics analysis, dual-luciferase reporter gene assay, and ChIP. Loss- and gain-function assays were performed to ascertain the effect of SOX4, miR-17, and CYLD on biological cellular processes and drug resistance to 5-FU. SOX4 and miR-17 were found to be highly expressed while CYLD was poorly expressed in CRC tissues and cells. Silencing of SOX4 resulted in the suppression of cellular proliferation, invasion, migration as well as a reduction in CRC drug resistance. Mechanically, CYLD was specifically targeted by miR-17, while SOX4 upregulated the expression of miR-17. Functionally, SOX4 triggered drug resistance of CRC cells to 5-FU through the miR-17/CYLD axis. Taken together, the key findings of the present study provides evidence suggesting that SOX4 elevates miR-17 to decrease CYLD, thus inducing chemotherapy resistance of CRC cells.

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpin(cpdm/cpdm) mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpin(cpdm/cpdm) mice were fully reversed in Sharpin(cpdm/cpdm) Cyld(−/−) mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpin(cpdm/cpdm) cells, a finding dependent on CYLD since we observed reversal in Sharpin(cpdm/cpdm) Cyld(−/−) cells. Enhanced RIPK1 recruitment to Complex II in Sharpin(cpdm/cpdm) cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpin(cpdm/cpdm) mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpin(cpdm/cpdm) phenotype illustrates the pathological consequences when CYLD inhibition fails.

During heart maturation, gap junctions assemble into hemichannels and polarize to the intercalated disc at cell borders to mediate electrical impulse conduction. However, the molecular mechanism underpinning cardiac gap junction assembly remains elusive. Herein, we demonstrate an important role for the deubiquitinating enzyme cylindromatosis (CYLD) in this process. Depletion of CYLD in mice impairs the formation of cardiac gap junctions, accelerates cardiac fibrosis, and increases heart failure. Mechanistically, CYLD interacts with plakoglobin and removes lysine 63-linked polyubiquitin chains from plakoglobin. The deubiquitination of plakoglobin enhances its interaction with the desmoplakin/end-binding protein 1 complex localized at the microtubule plus end, thereby promoting microtubule-dependent transport of connexin 43 (Cx43), a key component of gap junctions, to the cell membrane. These findings establish CYLD as a critical player in regulating gap junction assembly and have important implications in heart development and diseases.

Background Obesity-related nephropathy (ORN) has become one of the leading causes of end-stage renal disease and has tripled over the past decade. Previous studies have demonstrated that decreased reactive oxygen species production may contribute to improving ORN by ameliorating oxidative stress injury. Here, IκB kinase (IKK) was hypothesized to inactivate the deubiquitination activity of cylindromatosis (CYLD) by activating the phosphorylation of CYLD, thus promoting the ubiquitination of NF-E2-related factor 2 (Nrf2) and further aggravating oxidative stress injury of the kidney in ORN. This study was aimed to confirm this hypothesis. Methods Haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Oil Red O staining were performed to assess histopathology. Dihydroethidium (DHE) staining and MDA, SOD, CAT, and GSH-PX assessments were performed to measure reactive oxygen species (ROS) production. Immunohistochemical (IHC) staining, qRT–PCR and/or western blotting were performed to assess the expression of related genes. JC-1 assays were used to measure the mitochondrial membrane potential (ΔΨm) of treated HK-2 cells. Co-immunoprecipitation experiments (Co-IP) were used to analyse the interaction between CYLD and Nrf2 in ORN. Results ORN in vivo and in vitro models were successfully constructed, and oxidative stress injury was detected in the model tissues and cells. Compared with the control groups, the phosphorylation level of CYLD increased while Nrf2 levels decreased in ORN model cells. An IKK inhibitor reduced lipid deposition, ROS production, CYLD phosphorylation levels and ΔΨm in vitro, which were reversed by knockdown of CYLD. Nrf2 directly bound to CYLD and was ubiquitinated in ORN cells. The proteasome inhibitor MG132 activated the Nrf2/ARE signalling pathway, thereby reversing the promoting effect of CYLD knockdown on oxidative stress. Conclusion IKK inactivates the deubiquitination activity of CYLD by activating the phosphorylation of CYLD, thus promoting the ubiquitination of Nrf2 and further aggravating oxidative stress injury of the kidney in ORN. This observation provided a feasible basis for the treatment of kidney damage caused by ORN.

The tumor suppressive role of CYLD lysine 63 deubiquitinase (

Nondegradative ubiquitin chains attached to specific targets via Lysine 63 (K63) residues have emerged to play a fundamental role in synaptic function. The K63-specific deubiquitinase CYLD has been widely studied in immune cells and lately also in neurons. To better understand if CYLD plays a role in brain and synapse homeostasis, we analyzed the behavioral profile of CYLD-deficient mice. We found that the loss of CYLD results in major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. Furthermore, the absence of CYLD leads to a reduction in hippocampal network excitability, long-term potentiation, and pyramidal neuron spine numbers. By providing evidence that CYLD can modulate mechanistic target of rapamycin (mTOR) signaling and autophagy at the synapse, we propose that synaptic K63-linked ubiquitination processes could be fundamental in understanding the pathomechanisms underlying autism spectrum disorder.

The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α), accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation, moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.

Patients with CYLD cutaneous syndrome (CCS; syn. Brooke-Spiegler syndrome) carry germline mutations in the tumor suppressor CYLD and develop multiple skin tumors with diverse histophenotypes. Here, we comprehensively profile the genomic landscape of 42 benign and malignant tumors across 13 individuals from four multigenerational families and discover recurrent mutations in epigenetic modifiers DNMT3A and BCOR in 29% of benign tumors. Multi-level and microdissected sampling strikingly reveal that many clones with different DNMT3A mutations exist in these benign tumors, suggesting that intra-tumor heterogeneity is common. Integrated genomic, methylation and transcriptomic profiling in selected tumors suggest that isoform-specific DNMT3A2 mutations are associated with dysregulated methylation. Phylogenetic and mutational signature analyses confirm cylindroma pulmonary metastases from primary skin tumors. These findings contribute to existing paradigms of cutaneous tumorigenesis and metastasis., CYLD cutaneous syndrome (also known as Brooke-Spiegler syndrome) is characterised by germline mutations in the tumor suppressor CYLD. Here, the authors highlight recurrent mutations in DNMT3A and BCOR, indicating a role for epigenetic dysregulation in this rare genetic skin disease.