Deactylation by SIRT1 enables liquid-liquid phase separation of IRF3/IRF7 in innate antiviral immunity.

Innate antiviral immunity deteriorates with aging but how this occurs is not entirely clear. Here we identified SIRT1-mediated DNA-binding domain (DBD) deacetylation as a critical step for IRF3/7 activation that is inhibited during aging. Viral-stimulated IRF3 underwent liquid-liquid phase separation (LLPS) with interferon (IFN)-stimulated response element DNA and compartmentalized IRF7 in the nucleus, thereby stimulating type I IFN (IFN-I) expression. SIRT1 deficiency resulted in IRF3/IRF7 hyperacetylation in the DBD, which inhibited LLPS and innate immunity, resulting in increased viral load and mortality in mice. By developing a genetic code expansion orthogonal system, we demonstrated the presence of an acetyl moiety at specific IRF3/IRF7 DBD site/s abolish IRF3/IRF7 LLPS and IFN-I induction. SIRT1 agonists rescued SIRT1 activity in aged mice, restored IFN signaling and thus antagonized viral replication. These findings not only identify a mechanism by which SIRT1 regulates IFN production by affecting IRF3/IRF7 LLPS, but also provide information on the drivers of innate immunosenescence.

Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

Acetylation-Dependent Deubiquitinase OTUD3 Controls MAVS Activation in Innate Antiviral Immunity.

Accurate regulation of innate immunity is necessary for the host to efficiently respond to invading pathogens and avoid excessive harmful immune pathology. Here we identified OTUD3 as an acetylation-dependent deubiquitinase that restricts innate antiviral immune signaling. OTUD3 deficiency in mice results in enhanced innate immunity, a diminished viral load, and morbidity. OTUD3 directly hydrolyzes lysine 63 (Lys63)-linked polyubiquitination of MAVS and thus shuts off innate antiviral immune response. Notably, the catalytic activity of OTUD3 relies on acetylation of its Lys129 residue. In response to virus infection, the acetylated Lys129 is removed by SIRT1, which promptly inactivates OTUD3 and thus allows timely induction of innate antiviral immunity. Importantly, acetyl-OTUD3 levels are inversely correlated with IFN-ß expression in influenza patients. These findings establish OTUD3 as a repressor of MAVS and uncover a previously unknown regulatory mechanism by which the catalytic activity of OTUD3 is tightly controlled to ensure timely activation of antiviral defense.

iProPhos: A Web-Based Interactive Platform for Integrated Proteome and Phosphoproteome Analysis.

Large-scale omics studies have generated a wealth of mass spectrometry-based proteomics data, which provide additional insights into disease biology spanning genomic boundaries. However, there is a notable lack of web-based analysis and visualization tools that facilitate the reutilization of these data. Given this challenge, we present iProPhos, a user-friendly web server to deliver interactive and customizable functionalities. iProPhos incorporates a large number of samples, including 1444 tumor samples and 746 normal samples across 12 cancer types, sourced from the Clinical Proteomic Tumor Analysis Consortium. Additionally, users can also upload their own proteomics/phosphoproteomics data for analysis and visualization. In iProPhos, users can perform profiling plotting and differential expression, patient survival, clinical feature-related, and correlation analyses, including protein-protein, mRNA-protein, and kinase-substrate correlations. Furthermore, functional enrichment, protein-protein interaction network, and kinase-substrate enrichment analyses are accessible. iProPhos displays the analytical results in interactive figures and tables with various selectable parameters. It is freely accessible at http://longlab-zju.cn/iProPhos without login requirement. We present two case studies to demonstrate that iProPhos can identify potential drug targets and upstream kinases contributing to site-specific phosphorylation. Ultimately, iProPhos allows end-users to leverage the value of big data in cancer proteomics more effectively and accelerates the discovery of novel therapeutic targets.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation.

BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. METHODS: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Oxidative Stress and Endoplasmic Reticulum Stress Contributes to Arecoline and Its Secondary Metabolites-Induced Dyskinesia in Zebrafish Embryos.

Areca nut has been listed as one of the most addictive substances, along with tobacco, alcohol and caffeine. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on embryonic development and motor behavior are rarely addressed in zebrafish embryo-larvae. Herein, we investigated the effects of exposure to three alkaloids (arecoline and secondary metabolites-arecaidine and arecoline N-oxide) on the developmental parameters, locomotive behavior, oxidative stress and transcriptome of zebrafish embryos. Zebrafish embryos exposed to different concentrations (0, 0.1, 1, 10, 100 and 1000 µM) of arecoline, arecaidine and arecoline N-oxide showed no changes in mortality and hatchability rates, but the malformation rate of zebrafish larvae was significantly increased in a dose-dependent manner and accompanied by changes in body length. Moreover, the swimming activity of zebrafish larvae decreased, which may be due to the increase in reactive oxygen species and the imbalance between oxidation and antioxidation. Meanwhile, transcriptome analysis showed that endoplasmic reticulum stress and the apoptosis p53 signaling pathway were significantly enriched after exposure to arecoline and arecoline N-oxide. However, arecaidine exposure focuses on protein synthesis and transport. These findings provide an important reference for risk assessment and early warning of areca nut alkaloid exposure.

Tri-n-butyl phosphate delays tissue repair by dysregulating neutrophil function in zebrafish.

Tri-n-butyl phosphate (TnBP) is a widely used organophosphate ester, but its effects on the regenerative process under damaging circumstances remain unknown. In the present study, zebrafish larvae were exposed to 0, 50, 100, 200 and 1000 µg/L TnBP, and the caudal fins were cut at 72 hours post fertilization (hpf). First, after exposure to TnBP, the number of total neutrophils decreased together with decreased neutrophils in the tail, and TnBP inhibited chemotaxis. Second, reactive oxygen species (ROS) levels in the zebrafish decreased greatly. Following exposure to TnBP, transcription levels of many genes regulating fin regeneration, such as fgf20a, fgfr1a, bmp2a and bmp4, were significantly downregulated, while inflammatory factors such as cxcl8a, cxcl18b, il-6, and tnfa were abnormally upregulated. In addition, TnBP inhibited the regenerative area after caudal fin amputation. The inflammatory state was adverse during the regenerative process. In summary, TnBP exposure is immunotoxic and decreases oxidative stress in injured zebrafish larvae.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.

Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.

Berberine Protects Against Palmitate-Induced Apoptosis in Tubular Epithelial Cells by Promoting Fatty Acid Oxidation.

BACKGROUND Increased lipid accumulation in renal tubular epithelial cells (TECs) contributes to their injury and dysfunction and progression of tubulointerstitial fibrosis. Berberine (BBR), a natural plant alkaloid isolated from traditional medicine herbs, is effective in lowing serum lipid, and has a protective effect on chronic kidney disease (CKD) with dyslipidemia, including diabetic nephropathy. The aim of this study was to investigate the effect of BBR on palmitate (PA)-induced lipid accumulation and apoptosis in TECs. MATERIAL AND METHODS Human kidney proximal tubular epithelial cell line (HK-2) cells were treated with PA, BBR, and/or palmitoyltransferase 1A (CPT1A) inhibitor Etomoxir. Intracellular lipid content was assessed by Oil Red O and Nile Red staining. Cell apoptosis rate was evaluated by flow cytometry assay. The expression of apoptosis-related protein cleaved-caspase3 and fatty acid oxidation (FAO)-regulating proteins, including CPT1A, peroxisome proliferator-activated receptor α (PPARα), and PPARγ co-activator-1α (PGC1α), was measured by Western blot analysis and immunofluorescence. RESULTS In the present study, PA treatment increased intracellular lipid deposition accompanied by elevated apoptosis in TECs compared with control group, whereas the protein expression of CPT1A, PPARα, and PGC1α, did not correspondingly increase in TECs. BBR significantly up-regulated the protein expression of CPT1A, PPARα, and PGC1α in TECs treated with or without PA, and reversed PA-induced intracellular lipid accumulation and apoptosis. Moreover, the CPT1A inhibitor Etomoxir counteracted the protective effect of BBR in TECs. CONCLUSIONS These in vitro findings suggest that PA can induce intracellular lipid accumulation and apoptosis in TECs, and the mechanism may be associated with inducing defective FAO, whereas BBR can protect TECs against PA-induced intracellular lipid accumulation and apoptosis by promoting FAO.

Exomic and Epigenomic Analyses in a Pair of Monozygotic Twins Discordant for Cryptorchidism.

Cryptorchidism represents one of the most common human congenital anomalies. In most cases, its etiology remains unclear and seems to be multifactorial. In the present study, a pair of monozygotic twins discordant for cryptorchidism was identified. Twin zygosity was confirmed by microsatellite genotyping. Whole exome sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) of DNA extract from leucocytes were performed to, respectively, evaluate their exomes and epigenomes. No differences in exome sequencing data were found between the twins after validation. MeDIP-Seq analysis detected 5,410 differentially hypermethylated genes and 2,383 differentially hypomethylated genes. Bioinformatic analysis showed that these genes belonged to several biological processes and signaling pathways, including regulation of actin cytoskeleton, which has been previously implicated in the etiology of cryptorchidism. The findings of the present study suggest that non-genetic factors might contribute to the pathogenesis of cryptorchidism.

GPR126 protein regulates developmental and pathological angiogenesis through modulation of VEGFR2 receptor signaling.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.

Characterization of Bbx, a member of a novel subfamily of the HMG-box superfamily together with Cic.

High mobility group (HMG)-box proteins, a large and functionally diverse superfamily of architectural protein, are involved in the regulation of DNA-dependent processes such as transcription, replication and DNA repair via the HMG-box domain. Bobby sox homolog (BBX), a newly identified HMG-Box protein, may function as a sequence-specific transcription factor. However, its expression pattern and biological functions are largely unknown. In this work, phylogenetic analysis showed that BBX is highly conserved and belongs to a novel subfamily of HMG-Box superfamily together with CIC (capicua homolog). Real time RT-PCR and whole-mount in situ hybridization in zebrafish embryo revealed that bbx, cica, and cicb were maternally highly expressed from 4 cell to 1K cell stage, and the zygote expression was primarily distributed in the central nervous system (CNS) from 24 to 60 h post-fertilization (hpf). Immunohistochemistry analysis in mouse brain revealed that BBX was weakly expressed in the cerebellum and highly expressed in the cortex and hippocampus. These findings indicate that as a novel HMG-box protein, BBX maybe associated with CNS development and provides useful clues to further study of its biological functions.

Caspase-8 dependent apoptosis contributes to dyskinesia caused by muscle defects and neurotoxicity in zebrafish exposed to zearalenone.

Zearalenone (ZEA), one of the usual mycotoxins, has been recognized in many areas and crops, posing a significant threat to the living organisms even to human beings. However, the mechanisms of locomotive defects remain unknown. Herein, zebrafish larvae was employed to investigate ZEA effects on developmental indexes, muscle and neural toxicity, apoptosis, transcriptome and motor behaviors of zebrafish larvae. Zebrafish larvae exposed to ZEA (0, 0.5, 1, 2 and 4 µM) showed no change in survival rate, but the malformation rate of zebrafish larvae increased dramatically manifesting with severe body bending and accomplished with adverse effects on hatching rate and body length. Moreover, the larvae manifested with defective muscle and abnormal neural development, resulting in decreased swimming ability, which probably due to the abnormal overactivation of apoptosis. And this was confirmed by enriched caspase 8-mediated apoptosis signaling pathway in the following transcriptome analysis. Meanwhile, there was a recovery in swimming behaviors in the larvae co-exposed in ZEA and caspase 8 inhibitor. These findings provide an important evidence for risk assessment and potential treatment target of ZEA exposure.

Integrated Network Analysis of Symptom Clusters Across Monkeypox Epidemics From 1970 to 2023: Systematic Review and Meta-Analysis.

BACKGROUND: The worldwide spread of monkeypox (mpox) has witnessed a significant increase, particularly in nonendemic countries. OBJECTIVE: We aimed to investigate the changing clinical symptoms associated with mpox from 1970 to 2023 and explore their interrelations. METHODS: In this systematic review and meta-analysis, 3 electronic databases were searched for English peer-reviewed studies conducted from January 1970 to April 2023 that reported any symptoms among confirmed mpox cases. We categorized the mpox epidemics into 3 periods: 1970-2002 (period 1, within the African region), 2003-2021(period 2, epidemics outside Africa), and 2022-2023 (period 3, worldwide outbreak). Following PRISMA guidelines, a meta-analysis was performed to estimate the pooled prevalence for each symptom. The correlation among symptoms was analyzed and visualized using network analysis. RESULTS: The meta-analysis included 61 studies that reported 21 symptoms in 720 patients from period 1, 39 symptoms in 1756 patients from period 2, and 37 symptoms in 12,277 patients from period 3. The most common symptom among patients from all 3 periods was rash (period 1: 92.6%, 95% CI 78.2%-100%; period 2: 100%, 95% CI 99.9%-100%; and period 3: 94.8%, 95% CI 90.9%-98.8%), followed by lymphadenopathy (period 1: 59.8%, 95% CI 50.3%-69.2%; period 2: 74.1%, 95% CI 64.2%-84.1%; and period 3: 61.1%, 95% CI 54.2%-68.1%). Fever (99%, 95% CI 97%-100%), enlarged lymph nodes (80.5%, 95% CI 75.4%-85.0%), and headache (69.1%, 95% CI 4%-100%) were the main symptoms in period 1, with a significant decrease in period 3: 37.9%, 31.2%, and 28.7%, respectively. Chills/rigors (73.3%, 95% CI 60.9%-85.7%), fatigue (68.2%, 95% CI 51.6%-84.8%), and dysphagia/swallowing difficulty (61.2%, 95% CI 10.5%-100%) emerged as primary new symptoms in period 2 and decreased significantly in period 3. Most other symptoms remained unchanged or decreased in period 3 compared to the former 2 periods. Nausea/vomiting had the highest degree of correlation (with 13 symptoms) and was highly positively correlated with lymphadenopathy (r=0.908) and conjunctivitis (r=0.900) in period 2. In contrast, rash and headache were 2 symptoms with the highest degree of correlation (with 21 and 21 symptoms, respectively) in period 3 and were highly positively correlated with fever (r=0.918 and 0.789, respectively). CONCLUSIONS: The manifestation of symptoms in patients with mpox has become more diverse, leading to an increase in their correlation. Although the prevalence of rash remains steady, other symptoms have decreased. It is necessary to surveil the evolving nature of mpox and the consequential changes in clinical characteristics. Epidemic countries may shift their focus on the potential association among symptoms and the high synergy risk. TRIAL REGISTRATION: PROSPERO Registration: CRD42023403282; http://tinyurl.com/yruuas5n.

TRIM28-mediated nucleocapsid protein SUMOylation enhances SARS-CoV-2 virulence.

Viruses, as opportunistic intracellular parasites, hijack the cellular machinery of host cells to support their survival and propagation. Numerous viral proteins are subjected to host-mediated post-translational modifications. Here, we demonstrate that the SARS-CoV-2 nucleocapsid protein (SARS2-NP) is SUMOylated on the lysine 65 residue, which efficiently mediates SARS2-NP's ability in homo-oligomerization, RNA association, liquid-liquid phase separation (LLPS). Thereby the innate antiviral immune response is suppressed robustly. These roles can be achieved through intermolecular association between SUMO conjugation and a newly identified SUMO-interacting motif in SARS2-NP. Importantly, the widespread SARS2-NP R203K mutation gains a novel site of SUMOylation which further increases SARS2-NP's LLPS and immunosuppression. Notably, the SUMO E3 ligase TRIM28 is responsible for catalyzing SARS2-NP SUMOylation. An interfering peptide targeting the TRIM28 and SARS2-NP interaction was screened out to block SARS2-NP SUMOylation and LLPS, and consequently inhibit SARS-CoV-2 replication and rescue innate antiviral immunity. Collectively, these data support SARS2-NP SUMOylation is critical for SARS-CoV-2 virulence, and therefore provide a strategy to antagonize SARS-CoV-2.

Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling and regulates innate immune activation.

The Toll-like receptor (TLR)/IL-1 receptor (IL-1R) signaling pathway is essential for innate immune responses and immune homeostasis. Lys-63-polyubiquitinated TRAF6 mediates its downstream signaling activation. In a gain-of-expression screen of 66 different deubiquitinating enzymes, we identified USP4 as a potent negative regulator of TLR/IL-1R signaling and TRAF6-interacting protein. USP4 deubiquitinates TRAF6 and thereby prevents the activation of NF-κB and AP-1 transcription factors and subsequent proinflammatory responses. LPS-treated usp4-depleted zebrafish larvae expressed higher levels of proinflammatory cytokines and were more susceptible to endotoxic challenge. Taken together, our results demonstrate that USP4 plays an essential role in negative regulation of the TLR/IL-1R signaling-mediated innate immune response.

The Amotl2 gene inhibits Wnt/ß-catenin signaling and regulates embryonic development in zebrafish.

The Motin family proteins can regulate cell polarity, cell mobility, and proliferation during embryonic development by controlling distinct signaling pathways. In this study, we demonstrate that amotl2 knockdown in zebrafish wild-type embryos results in embryonic dorsalization, and this effect can be antagonized by co-knockdown of the dorsal inducer ß-catenin2. Overexpression of amotl2 in masterblind (mbl) homozygous embryos, in which canonical Wnt signaling is up-regulated due to an axin1 mutation, transforms eyeless phenotype into smaller eyes, whereas co-knockdown of amot, amotl1, and amotl2 leads to development of smaller eyes in mbl heterozygotes. In cultured mammalian cells, Motin family members all possess the ability to attenuate Wnt/ß-catenin signaling. Focusing on Amotl2, we show that Amotl2 can associate with and trap ß-catenin in the Rab11-positive recycling endosomes, and as a result, the amount of ß-catenin in the cytosol and nucleus is reduced. Thus, our findings provide novel insights into functions of Motin family members and regulation of Wnt/ß-catenin signaling.