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In this issue, Pang and colleagues 1 identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl 3 ) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl 3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases., Competing Interests: None declared., (Thieme. All rights reserved.)

Human adipose-derived stem cells (hADSCs) are adult mesenchymal cells that have attracted the interest of clinical scientists and surgeons due to their large number of advantages including ease of access and expansion, abundance in cell culture, high proliferative rates, and lower senescence. SUMO/sentrin specific protease 1 (SENP1) is a critical protease that is required during the process of SUMOylation and deSUMOylation, which are dynamic mechanisms that influence cell cycle progression, cell proliferation, and apoptotic status. However, the contribution of SENP1 to these important cellular processes in hADSCs is largely uncharacterized and further studies in this area are required. Here, we show for the first time that after knock out SENP1 in hADSCs, their capacity to migrate and proliferate were inhibited, while apoptosis was enhanced. However, SENP1 did not significantly influence the morphology and MSC-related phenotypes of the hADSCs. These results highlight a role for SENP1 during hADSC growth, and its potential as a therapeutic target to improve the efficacy and safety of hADSCs in the clinic.

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1 del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Aims: Chronic cerebral hypoperfusion (CCH) is a state of chronic cerebral blood flow reduction, and it is the main cause of cognitive impairment and neurodegenerative diseases. The abnormal upregulation of legumain, a lysosomal cysteine protease, trigger synaptic plasticity impairment and neuroinflammation, which are involved in the underlying pathophysiology of CCH. At present, few studies have reported the role of legumain in cognitive impairment caused by CCH. In our study, we aimed to investigate the involvement of legumain knockout in cognitive function and neuroinflammation in a CCH mouse model., Main Methods: In this study, right unilateral common carotid artery occlusion (rUCCAO) was used to simulate the pathological state of cerebral ischemic injury. Various behavioural tests were executed to assess cognitive performance. In vivo electrophysiological recordings were used to measure synaptic functions. Western blotting, Golgi staining, haematoxylin/eosin staining, and immunofluorescence assays were conducted to examine pathological changes and molecular mechanisms., Key Findings: The data showed that the level of legumain was significantly increased in the hippocampus of mice subjected to rUCCAO. Legumain knockout significantly improved cognitive function and synaptic plasticity induced by rUCCAO, suggesting that legumain knockout-regulation effectively protected against CCH-induced behavioural dysfunctions. Moreover, legumain knockout suppressed rUCCAO-induced microglial activation, reduced the abnormal expression of inflammatory cytokines and the inflammasome complex, and impeded the activation of P65 and pyroptosis., Significance: These findings suggest that legumain is an effective regulator of CCH, and may be an ideal target for the development of cerebral ischemia treatments in the future., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Potyviridae is the largest family of plant RNA viruses. Their genomes are expressed through long polyproteins that are usually headed by the leader endopeptidase P1. This protein can be classified as type A or type B based on host proteolytic requirements and RNA silencing suppression (RSS) capacity. The main Potyviridae genus is Potyvirus , and a group of potyviruses infecting sweet potato presents an enlarged P1 protein with a polymerase slippage motif that produces an extra product termed P1N-PISPO. These two proteins display some RSS activity and are expressed followed by HCPro, which appears to be the main RNA silencing suppressor in these viruses. Here, we studied the behavior of the P1 protein of Sweet potato feathery mottle virus (SPFMV) using a viral system based on a canonical potyvirus, Plum pox virus (PPV), and discovered that this protein is able to replace both PPV P1 and HCPro. We also found that P1N-PISPO, produced after polymerase slippage, provides extra RNA silencing suppression capacity to SPFMV P1 in this viral context. In addition, the results showed that presence of two type A P1 proteins was detrimental for viral viability. The ample recombination spectrum that we found in the recovered viruses supports the strong adaptation capacity of P1 proteins and signals the N-terminal part of SPFMV P1 as essential for RSS activity. Further analyses provided data to add extra layers to the evolutionary history of sweet potato-infecting potyvirids. IMPORTANCE Plant viruses represent a major challenge for agriculture worldwide and Potyviridae , being the largest family of plant RNA viruses, is one of the primary players. P1, the leader endopeptidase, is a multifunctional protein that contributes to the successful spread of these viruses over a wide host range. Understanding how P1 proteins work, their dynamic interplay during viral infection, and their evolutionary path is critical for the development of strategic tools to fight the multiple diseases these viruses cause. We focused our efforts on the P1 protein of Sweet potato feathery mottle virus , which is coresponsible for the most devastating disease in sweet potato. The significance of our research is in understanding the capacity of this protein to perform several independent functions, using this knowledge to learn more about P1 proteins in general and the potyvirids infecting this host.

Silicosis is an irreversible occupational pulmonary disease that is characterized as progressed pulmonary fibrosis. In this study, we investigated the changes of UFSP2 and the related UFMylation in silica-induced pulmonary injury mice models. The experimental silicosis models were prepared by intratracheal injection of silica particles, and the lung samples were harvested at the first or the seventh day after treatment. We found that the UFSP2 expression in the 1-day models was comparable, whereas it was upregulated in the 7-day models. Consistently, the UFMylation in the lung tissues of the 7-day models was activated. In addition, we observed the CADM2, an adhesion molecule, was reported to associate with epithelial-mesenchymal transition, was upregulated in the lungs of 7-day models. In contrast, it remained comparable in the 1-day models. Our data indicated that the UFSP2/UFMylation pathway and the CADM2 might be involved in the silica-induced pulmonary injury.

Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified pro-inflammation form of programmed cell death, causing cell lysis and IL-1β release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1β levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1β levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

Purpose: Various sources of radiation including radiofrequency, electromagnetic radiation (EMR), low- dose X-radiation, low-level microwave radiation and ionizing radiation (IR) are indispensable parts of modern life. In the current review, we discussed the adaptive responses of biological systems to radiation with a focus on the impacts of radiation-induced oxidative stress (RIOS) and its molecular downstream signaling pathways. Materials and methods: A comprehensive search was conducted in Web of Sciences, PubMed, Scopus, Google Scholar, Embase, and Cochrane Library. Keywords included Mesh terms of "radiation," "electromagnetic radiation," "adaptive immunity," "oxidative stress," and "immune checkpoints." Manuscripts published up until December 2019 were included. Results: RIOS induces various molecular adaptors connected with adaptive responses in radiation exposed cells. One of these adaptors includes p53 which promotes various cellular signaling pathways. RIOS also activates the intrinsic apoptotic pathway by depolarization of the mitochondrial membrane potential and activating the caspase apoptotic cascade. RIOS is also involved in radiation-induced proliferative responses through interaction with mitogen-activated protein kinases (MAPks) including p38 MAPK, ERK, and c-Jun N-terminal kinase (JNK). Protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) signaling pathway has also been reported to be involved in RIOS-induced proliferative responses. Furthermore, RIOS promotes genetic instability by introducing DNA structural and epigenetic alterations, as well as attenuating DNA repair mechanisms. Inflammatory transcription factors including macrophage migration inhibitory factor (MIF), nuclear factor κB (NF-κB), and signal transducer and activator of transcription-3 (STAT-3) paly major role in RIOS-induced inflammation. Conclusion: In conclusion, RIOS considerably contributes to radiation induced adaptive responses. Other possible molecular adaptors modulating RIOS-induced responses are yet to be divulged in future studies.

Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1 flox /Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1 C134A , was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Sortase A contributes to adhesion and biofilm formation of Streptococcus mutans by anchoring surface proteins like P1 onto the cell wall, and few other functional characterization has been annotated to this protein and its coding gene srtA. In this study we investigated that whether srtA deletion would affect S. mutans virulence determinants in addition to adhesion and further explored whether these effects were caused due to changes in S. mutans genomic transcription. We used acid-killing assays, glycolytic rate assessments, and exopolysaccharide (EPS) formation tests to detect whether srtA deletion influenced S. mutans acid tolerance/production and glucan formation. Comparisons between RNA-sequencing data from both the exponential and stationary phases of UA159 and the srtA-deleted strain were made to determine the impact of srtA knockout on S. mutans genomic transcription. Results of our assays indicated that S. mutans aciduricity was enhanced in the srtA deleted strain when bacterial cells were directly subjected to pH 2.8, but the enhancement was repressed when the acid tolerance response was induced in advance. The srtA mutation strain exhibited reduced EPS formation in mature biofilms. SrtA deletion led to pleiotropic changes in the S. mutans transcriptome with a growth phase-dependent pattern. The affected genes mainly included those involved in aciduricity, carbohydrate transport, and EPS formation. It was concluded that S. mutans srtA exhibited multiple effects on the virulence traits of this pathogen, including acid tolerance and glucan formation, and that these alterations could be partially due to transcriptional changes upon loss of srtA., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Blood coagulation involves extrinsic and intrinsic pathways, which merge at the activation step of blood coagulation factor X to factor Xa. This step is catalysed by the extrinsic or intrinsic Xase, which consists of a complex of factor VIIa and its cofactor tissue factor or factor IXa (FIXa) and its cofactor coagulation factor VIIIa (FVIIIa). Upon complex formation with FVIIIa, FIXa is conformationally activated to the Xase complex. However, the mechanistic understanding of this molecular recognition is limited. Here, we examined FVIIIa-FIXa binding in the context of FIXa's activation status. Given the complexity and the labile nature of FVIIIa, we decided to employ two FVIII-derived peptides (558-loop, a2 peptide) to model the cofactor binding of FIX(a) using biosensor chip technology. These two FVIII peptides are known to mediate the key interactions between FVIIIa and FIXa. We found both of these cofactor mimetics as well as full-length FVIIIa bind more tightly to zymogenic FIX than to proteolytically activated FIXa. Consequently and surprisingly, we observed that the catalytically inactive FIX zymogen can outcompete the activated FIXa from the complex with FVIIIa, resulting in an inactive, zymogenic Xase complex. By contrast, the thrombophilic Padua mutant FIXa-R170 in complex with the protein-substrate analogue BPTI bound tighter to FVIIIa than to the zymogen form FIX-R170L, suggesting that the active Xase complex preferentially forms in the Padua variant. Together, these results provide a mechanistic basis for the thrombophilic nature of the FIX-R170L mutant and suggest the existence of a newly discovered safety measure within the coagulation cascade., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Although numerous studies have demonstrated that neuronal mechanisms regulate alcohol-related behaviors, very few have investigated the direct role of glia in behavioral responses to alcohol. The results described here begin to fill this gap in the alcohol behavior and gliobiology fields. Since Drosophila exhibit conserved behavioral responses to alcohol and their CNS glia are similar to mammalian CNS glia, we used Drosophila to begin exploring the role of glia in alcohol behavior. We found that knockdown of Cysteine proteinase-1 ( Cp1 ) in glia increased Drosophila alcohol sedation and that this effect was specific to cortex glia and adulthood. These data implicate Cp1 and cortex glia in alcohol-related behaviors. Cortex glia are functionally homologous to mammalian astrocytes and Cp1 is orthologous to mammalian Cathepsin L. Our studies raise the possibility that cathepsins may influence behavioral responses to alcohol in mammals via roles in astrocytes., Competing Interests: Competing interestsThe authors declare no competing interests.

Nur77 (NR4A1) plays an important role in various inflammatory responses. Nur77 is rapidly degraded in cells and its protein level is critically controlled. Although few E3 ligases regulating the Nur77 protein have been defined, the deubiquitinase (DUB) responsible for Nur77 stability has not been reported to date. We identified ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) as a DUB that stabilizes Nur77 by preventing its proteasomal degradation. We found that OTUB1 interacted with Nur77 to deubiquitinate it, thereby stabilizing Nur77 in an Asp88-dependent manner. This suggests that OTUB1 targets Nur77 for deubiquitination via a non-canonical mechanism. Functionally, OTUB1 inhibited TNFα-induced IL-6 production by promoting Nur77 protein stability. OTUB1 modulated the stability of Nur77 as a counterpart of tripartite motif 13 (Trim13). That is, OTUB1 reduced the ubiquitination and degradation of Nur77 potentiated by Trim13. In addition, this DUB also inhibited IL-6 production, which was further amplified by Trim13 in TNFα-induced responses. These findings suggest that OTUB1 is an important regulator of Nur77 stability and plays a role in controlling the inflammatory response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad array of anionic drugs from kidney, thereby avoiding their possibly toxic side effects in the body. We earlier demonstrated that OAT3 is subjected to a specific type of post-translational modification called SUMOylation. SUMOylation is a dynamic event, where de-SUMOylation is catalyzed by a class of SUMO-specific proteases. In the present investigation, we assessed the role of SUMO-specific protease Senp2 in OAT3 SUMOylation, expression and function. We report here that overexpression of Senp2 in COS-7 cells led to a reduced OAT3 SUMOylation, which correlated well with a decreased OAT3 expression and transport activity. Such phenomenon was not observed in cells overexpressing an inactive mutant of Senp2. Furthermore, transfection of cells with Senp2-specific siRNA to knockdown the endogenous Senp2 resulted in an increased OAT3 SUMOylation, which correlated well with an enhanced OAT3 expression and transport activity. Coimmunoprecipitation experiments showed that Senp2 directly interacted with OAT3 in the kidneys of rats. Together these results provided first demonstration that Senp2 is a significant regulator for OAT3-mediated organic anion/drug transport., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2 nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity., (© 2019 The Authors.)

Snail is an important transcription factor of epithelial-mesenchymal transition (EMT) and related to poor prognosis and distant metastasis of tumor patients. Snail is a liable protein and degraded by ubiquitin-proteasome system. There are various E3 ligases mediating its degradation, but the deubiquitinating enzyme reversed Snail degradation is not fully understood. In this study, we screened a DUB library and found USP26 is a potent deubiquitinase mediating Snail stabilization. We also identified that USP26 is a booster of esophageal squamous cell carcinoma (ESCC) cell migration and invasion, and it is highly expressed in ESCC samples. Our observation demonstrates that USP26 is a novel deubiquitinating enzyme of Snail and it provides a potential target for the therapy of esophageal cancer metastasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis ., (Copyright © 2019 American Society for Microbiology.)

Objective- Macrophages participate in the pathogenesis of pulmonary arterial hypertension (PAH). Lgmn (Legumain), a newly discovered cysteine proteinase belonging to the C13 peptidase family, is primarily expressed in macrophages; however, its roles in PAH remain unknown. Approach and Results- Herein, Lgmn was upregulated in lung tissues of PAH mice subjected to hypoxia plus SU5416 and PAH rats challenged with monocrotaline. Global Lgmn ablation and macrophage-specific ablation alleviated PAH compared with wild-type mice, evident from a reduction in right ventricular systolic pressure, the ratio of the right ventricular wall to the left ventricular wall plus the septum, the pulmonary vascular media thickness, and pulmonary vascular muscularization. Increased expression of ECM (extracellular matrix) proteins was correlated with MMP (matrix metalloproteinase)-2 activation and TGF (transforming growth factor)-β1 signaling in the PAs. Although Lgmn did not affect inflammatory cell infiltration and PA smooth muscle cell proliferation, it drove increased the synthesis of ECM proteins via MMP-2 activation. MMP-2 hydrolyzed the TGF-β1 precursor to the active form. An Lgmn-specific inhibitor markedly ameliorated PAH. Clinically, serum Lgmn levels were closely associated with the severity of idiopathic PAH. Conclusions- Our results indicate that Lgmn inhibition could be an effective strategy for preventing or delaying PAH.

Background and Aims: pathological angiogenesis plays an important role in the progression of chronic liver diseases. Asparaginyl endopeptidase (AEP) participates in tumor angiogenesis and was recently shown to be associated with liver fibrosis. This study aimed to explore the effect of AEP on liver sinusoidal endothelial cell (LSECs) angiogenesis and determine the underlying mechanism., Methods: cultured LSECs were infected with lentiviruses in order to suppress AEP expression (AEP-KD1, AEP-KD2). The effect of AEP on LSECs proliferation, apoptosis and migration were subsequently determined by a CCK8 assay, flow cytometry and wound-healing and Transwell assays, respectively, in AEP knocked-down and control LSECs. The expression of the endothelial cell surface markers CD31, CD34 and von Willebrand factor (vWF) were detected by immunofluorescence assay and western blot. The angiogenic factors, vascular endothelial growth factor receptor 2 (VEGFR2) and interleukin 8 (IL 8) were detected by real-time PCR and western blot. The effect of AEP on vessel tube formation by LSECs was examined by Matrigel™ tube-formation assay. Phosphoinositide 3-kinase (PI3K)/Akt expression and phosphorylation were detected by western blot., Results: AEP was effectively knocked down by lentivirus infection in LSECs. Down-regulation of AEP expression significantly decreased proliferation and migration and increased apoptosis of LSECs. Moreover, expression levels of the endothelial cell surface markers CD31, CD34 and vWF, as well as angiogenic factors VEGFR2 and IL 8, were also reduced after AEP was knocked-down. The vessel tube formation abilities of AEP-KD1 and AEP-KD2 LSECs were significantly inhibited compared with LSECs without AEP knocked-down. Down-regulation of AEP also inhibited the phosphorylation of PI3K and Akt., Conclusion: AEP promotes LSECs angiogenesis in vitro, possibly via the PI3K/Akt pathway. AEP may therefore be a potential therapeutic target for preventing the progression of liver fibrosis.

Post‑translational modification via small ubiquitin‑like modifier (SUMO) is involved in the regulation of various important cellular processes. SUMO modification can be regulated at the level of conjugation, and can also be reversed by the SUMO‑specific proteases (SENPs). However, current studies of the regulation and function of SENP in lung development remain limited. In this study, the expression levels of SENP1 and SUMO1 were assessed during lung development in rats. SUMO1 modification occurred during lung development and changes in SENP1 expression were consistent with the changes in the presence of free SUMO1. In order to investigate the function of SENP1, alveolar type (AT) 2 cells were transfected with SENP1‑targeting small interfering RNA, and the proliferation, apoptosis and differentiation function of AT2 cells was subsequently evaluated. Marked upregulation of conjugated SUMO1 was observed following SENP1 inhibition. Furthermore, depletion of SENP1 resulted in increased apoptosis, decreased proliferation and impaired differentiation status of AT2 cells. Thus, the results support that SENP1 is an essential regulator of the balance between SUMOylation and deSUMOylation during lung development, specifically affecting the proliferation and differentiation status of AT2 cells.

SARS-coronavirus (CoV) is a zoonotic agent derived from rhinolophid bats, in which a plethora of SARS-related, conspecific viral lineages exist. Whereas the variability of virulence among reservoir-borne viruses is unknown, it is generally assumed that the emergence of epidemic viruses from animal reservoirs requires human adaptation. To understand the influence of a viral factor in relation to interspecies spillover, we studied the papain-like protease (PLP) of SARS-CoV. This key enzyme drives the early stages of infection as it cleaves the viral polyprotein, deubiquitinates viral and cellular proteins, and antagonizes the interferon (IFN) response. We identified a bat SARS-CoV PLP, which shared 86% amino acid identity with SARS-CoV PLP, and used reverse genetics to insert it into the SARS-CoV genome. The resulting virus replicated like SARS-CoV in Vero cells but was suppressed in IFN competent MA-104 (3.7-fold), Calu-3 (2.6-fold) and human airway epithelial cells (10.3-fold). Using ectopically-expressed PLP variants as well as full SARS-CoV infectious clones chimerized for PLP, we found that a protease-independent, anti-IFN function exists in SARS-CoV, but not in a SARS-related, bat-borne virus. This PLP-mediated anti-IFN difference was seen in primate, human as well as bat cells, thus independent of the host context. The results of this study revealed that coronavirus PLP confers a variable virulence trait among members of the species SARS-CoV, and that a SARS-CoV lineage with virulent PLPs may have pre-existed in the reservoir before onset of the epidemic., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Ocular trauma is common in civilian and military populations. Among other injuries, closed globe blunt ocular trauma causes acute disruption of photoreceptor outer segments (commotio retinae) and retinal ganglion cell (RGC) death (traumatic optic neuropathy [TON]), both of which permanently impair vision. Caspase-2-dependent cell death is important and evidenced in models of RGC degeneration. We assessed the role of caspase-2 as a mediator of RGC and photoreceptor death in a rat blunt ocular trauma model., Methods: Bilateral ballistic closed globe blunt ocular trauma was induced in female Lister-hooded rats and caspase-2 cleavage and localization assessed by Western blotting and immunohistochemistry. Retinal caspase-2 was knocked down by intravitreal injection of caspase-2 small interfering RNA (siCASP2). In retinal sections, RGC survival was assessed by BRN3A-positive cell counts and photoreceptor survival by outer nuclear layer (ONL) thickness, respectively. Retinal function was assessed by electroretinography (ERG)., Results: Raised levels of cleaved caspase-2 were detected in the retina at 5, 24, and 48 hours after injury and localized to RGC but not photoreceptors. Small interfering RNA-mediated caspase-2 knockdown neuroprotected RGC around but not in the center of the injury site. In addition, caspase-2 knockdown increased the amplitude of the ERG photopic negative response (PhNR) at 2 weeks after injury. However, siCASP2 was not protective for photoreceptors, suggesting that photoreceptor degeneration in this model is not mediated by caspase-2., Conclusions: Caspase-2 mediates death in a proportion of RGC but not photoreceptors at the site of blunt ocular trauma. Thus, intravitreally delivered siCASP2 is a possible therapeutic for the effective treatment of RGC death to prevent TON.

Small ubiquitin-like modifier (SUMOylation) is a reversible post-translational modification, which plays important roles in numerous biological processes. SUMO could be covalently attached to target proteins in an isopeptide bond manner that occurs via a lysine ε-amino group on the target proteins and the glycine on SUMO C-terminus. This covalent binding could affect the subcellular localization and stability of target proteins. SUMO modification can be reversed by members of the Sentrin/SUMO-specific proteases (SENPs) family, which are highly evolutionarily conserved from yeast to human. SENP2, a member of the SENPs family, mainly plays a physiological function in the nucleus. SENP2 can promote maturity of the SUMO and deSUMOylate for single-SUMO modified or poly-SUMO modified proteins. SENP2 can affect the related biological processes through its peptidase activity or the amino terminal transcriptional repression domain. It plays important roles by inhibiting or activating some molecular functions. Therefore, the research achievements of SENP2 are reviewed in order to understand its related functions and the underlying molecular mechanisms and provide a clue for future research on SENP2., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH-rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH-rate profile of k cat / K m for Cbz-Arg-Arg-AMC indicated p K 1 = 6.6 (unprotonated) and p K 2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the p K = 6.2-6.6 group exhibited a Δ H ion value of 8.4 kcal/mol, typical of histidine. The pH-rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a p K a of 9.8. Normal solvent kinetic isotope effects were observed for both D 2 O k cat (1.6-2.1) and D 2 O k cat / K m (1.1-1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on k cat can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.

The sessile nature of plants requires them to cope with an ever-changing environment. Effective adaptive responses require sophisticated cellular mechanisms at the post-transcriptional and post-translational levels. Post-translational modification by small ubiquitin-like modifier (SUMO) proteins is emerging as a key player in these adaptive responses. SUMO conjugation can rapidly change the overall fate of target proteins by altering their stability or interaction with partner proteins or DNA. SUMOylation entails an enzyme cascade that leads to the activation, conjugation and ligation of SUMO to lysine residues of target proteins. In addition to their SUMO processing activities, SUMO proteases also possess de-conjugative activity capable of cleaving SUMO from target proteins, providing reversibility and buffering to the pathway. These proteases play critical roles in the maintenance of the SUMO machinery in equilibrium. We hypothesize that SUMO proteases provide the all-important substrate specificity within the SUMO system. Furthermore, we provide an overview of the role of SUMO in plant innate immunity. SUMOylation also overlaps with multiple growth-promoting and defence-related hormone signalling pathways, and hence is pivotal for the maintenance of the growth-defence balance. This review aims to highlight the intricate molecular mechanisms utilized by SUMO to regulate plant defence and to stabilize the growth-defence equilibrium., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)

Background: The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated., Methods: Flow cytometry, caspase activity assay and western blotting were used for cell death rate evaluation. Western blotting, gel-filtration, siRNA approach and qRT-PCR were used to elucidate the mechanism underlying cell death potentiation upon SD., Results: We demonstrated that SD sensitizes cancer cells to treatment with chemotherapeutic agent cisplatin. This effect is independent on activation of caspases-2 and -8, apical caspases triggering apoptosis in response to genotoxic stress. SD potentiates cell death via downregulation of the anti-apoptotic protein Mcl-1. In fact, SD reduces the Mcl-1 mRNA level, which consequently decreases the Mcl-1 protein level and renders cells more susceptible to apoptosis induction via the formation of apoptosome., Conclusions: Mcl-1 protein is an important regulator of sensitivity of cancer cells to apoptotic stimuli upon SD., General Significance: This study identifies Mcl-1 as a new target for the sensitization of human cancer cells to cell death by SD, which is of great significance for the development of efficient anti-cancer therapies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation.

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, leading us to explore a connection between this pathway and the splicing machinery. We show here that addition of a recombinant SUMO-protease decreases the efficiency of pre-mRNA splicing in vitro. By mass spectrometry analysis of anti-SUMO immunoprecipitated proteins obtained from purified splicing complexes formed along the splicing reaction, we identified spliceosome-associated SUMO substrates. After corroborating SUMOylation of Prp3 in cultured cells, we defined Lys 289 and Lys 559 as bona fide SUMO attachment sites within this spliceosomal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is unable to co-precipitate U2 and U5 snRNA and the spliceosomal proteins U2-SF3a120 and U5-Snu114. This SUMOylation-deficient mutant fails to restore the splicing of different pre-mRNAs to the levels achieved by the wild type protein, when transfected into Prp3-depleted cultured cells. This mutant also shows a diminished recruitment to active spliceosomes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involvement of Prp3 SUMOylation in U4/U6•U5 tri-snRNP formation and/or recruitment., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sensing of viral RNA by the cytosolic receptors RIG-I and melanoma differentiation-associated gene 5 (MDA5) leads to innate antiviral response. How RIG-I and MDA5 are dynamically regulated in innate antiviral response is not well understood. Here, we show that TRIM38 positively regulates MDA5- and RIG-I-mediated induction of downstream genes and acts as a SUMO E3 ligase for their dynamic sumoylation at K43/K865 and K96/K888, respectively, before and after viral infection. The sumoylation of MDA5 and RIG-I suppresses their K48-linked polyubiquitination and degradation in uninfected or early-infected cells. Sumoylation of the caspase recruitment domains of MDA5 and RIG-I is also required for their dephosphorylation by PP1 and activation upon viral infection. At the late phase of viral infection, both MDA5 and RIG-I are desumoylated by SENP2, resulting in their K48-linked polyubiquitination and degradation. These findings suggest that dynamic sumoylation and desumoylation of MDA5 and RIG-I modulate efficient innate immunity to RNA virus and its timely termination., (© 2017 Hu et al.)

Porphyromonas gingivalis is a bacterium associated with chronic periodontitis that possesses a family of genes encoding hemagglutinins required for heme acquisition. In this study we generated ΔhagB and ΔhagC mutants in strain W83 and demonstrate that both hagB and hagC are required for adherence to oral epithelial cells. Unexpectedly, a double ΔhagB/ΔhagC mutant had less severe adherence defects than either of the single mutants, but was found to exhibit increased expression of the gingipain-encoding genes rgpA and kgp, suggesting that a ΔhagB/ΔhagC mutant is only viable in populations of cells that exhibit increased expression of genes involved in heme acquisition. Disruption of hagB in the fimbriated strain ATCC33277 demonstrated that HagB is also required for stable attachment of fimbriated bacteria to oral epithelial cells. Mutants of hagC were also found to form defective single and multi-species biofilms that had reduced biomass relative to biofilms formed by the wild-type strain. This study highlights the hitherto unappreciated importance of these genes in oral colonization and biofilm formation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution., (Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.)

Increased post-translational modification of proteins by SUMO-2/3 is a cytoprotective response against cell stress induced by ischaemia and reperfusion. However, it is still unclear what other cell stressors trigger protein SUMOylation, what mechanisms enhance and maintain the enhanced SUMOylation, and if it is a general protective mediator against other cytotoxic stresses. Here, we show increased levels of SUMOylation and decreased levels of the SUMO deconjugating enzyme SENP3 in PC12 cells treated with the toxic heavy metal cadmium. In addition, SENP3 knockdown reduced cadmium-induced caspase 3 cleavage and cell death in PC12 cells, while SENP3 overexpression enhanced cell death. These results suggest that SENP3 is an important regulator of the cellular response to cadmium stress in PC12 cells. Our findings are consistent with previous reports of decreased SENP3 and increased SUMOylation in ischaemia, and imply that the regulation of SENP3 levels and subsequent changes in SUMOylation could be a cytoprotective mechanism against caspase 3-mediated cell death.

In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

Many bacteria symbiotic and parasitic in humans are included in the genera Bacteroides, Prevotella, Porphyromonas and others, which belong to the phylum Bacteroidetes. We have been studying gingipain, a major secretory protease of Porphyromonas gingivalis which is a periodontopathogenic bacterium belonging to the genus Porphyromonas, and pili which contribute to host colonization in the bacterium. In the process, it was found that gingipain was secreted by a system not reported previously. Furthermore, this secretion system was found to exist widely in the Bacteroidetes phylum bacteria and closely related to the gliding motility of bacteroidete bacteria, and it was named the Por secretion system (later renamed the type IX secretion system). Regarding P. gingivalis pili, it was found that the pilus protein is transported as a lipoprotein to the cell surface, and the pilus formation occurs due to degradation by arginine-gingipain. Pili with this novel formation mechanism was found to be widely present in bacteria belonging to the class Bacteroidia in the phylum Bacteroidetes and was named the type V pili.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. The role of SENP3 in lipid metabolism, particularly NAFLD, is unclear. Our results showed that hepatic SENP3 was up-regulated in NAFLD patients and an animal model in vivo and after loading hepatocytes with free fatty acids (FFA) in vitro. Intracellular lipid accumulation was determined in SENP3 silenced or overexpressed hepatocytes with/without FFA in vitro. Confirming a role for SENP3, gene silencing was associated in vitro with amelioration of lipid accumulation and overexpression with enhancement of lipid accumulation. SENP3 related genes in NAFLD were determined in vitro using RNA-Seq. Eleven unique genes closely associated with lipid metabolism were generated using bioinformatics. Three selected genes (apoe, a2m and tnfrsf11b) were verified in vitro, showing apoe, a2m and tnfrsf11b were regulated by SENP3 with FFA stimulation. Intrahepatic and circulating APOE, A2M and TNFRSF11B were elevated in NAFLD compared with controls. These data demonstrate the important role of SENP3 in lipid metabolism during the development of NAFLD via downstream genes, which may be useful information in the development of NAFLD. The precise role of SENP3 in NAFLD will be investigated using liver-specific conditional knockout mice in future studies.

Retinoic acid-inducible gene I (RIG-I) plays important roles in pathogen recognition and antiviral signalling transduction. Here we show that syndecan-4 (SDC4) is a RIG-I-interacting partner identified in a yeast two-hybrid screen. We find that SDC4 negatively regulates the RIG-I-mediated antiviral signalling in a feedback-loop control manner. The genetic evidence obtained by using knockout mice further emphasizes this biological role of SDC4 in antiviral signalling. Mechanistically, we show that SDC4 interacts with both RIG-I and deubiquitinase CYLD via its carboxyl-terminal intracellular region. SDC4 likely promotes redistribution of RIG-I and CYLD in a perinuclear pattern post viral infection, and thus enhances the RIG-I-CYLD interaction and potentiates the K63-linked deubiquitination of RIG-I. Collectively, our findings uncover a mechanism by which SDC4 antagonizes the activation of RIG-I in a CYLD-mediated deubiquitination-dependent process, thereby balancing antiviral signalling to avoid deleterious effects on host cells.

Background: Dermatomyositis (DM) and systemic lupus erythematosus (SLE) have common skin features, including dermal mucin deposition and interferon signature, although their roles are unknown., Objectives: To identify common or specific molecular changes in DM and SLE skin., Methods: Proteomic analysis was performed using DM and healthy skin. Glycosaminoglycans were analysed by high-performance liquid chromatography., Results: The expression of 60 proteins was upregulated or downregulated in DM skin compared with healthy skin in the proteomic analysis. Among those proteins, PSMB9, an immunoproteasome subunit, was upregulated in the epidermis of DM and SLE, but not in other skin diseases. Furthermore, versican V1, a core protein for glycosaminoglycans, was upregulated, while type I collagen was downregulated in the dermis of DM and SLE skin. Interferon stimulated PSMB9 expression in cultured keratinocytes and reduced collagen expression in dermal fibroblasts, but did not affect versican expression. The PSMB9 knock-down in keratinocytes led to significant suppression of transforming growth factor (TGF)-β2 and TGF-β3, inducers of versican synthesis. TGF-β3 expression was upregulated in both DM and SLE, while TGF-β2 expression was increased only in the DM epidermis. ΔDiHS-diS1, a component of heparan sulfate, was significantly increased only in DM. TGF-β2 expression significantly increased the ΔDiHS-diS1 expression in dermal fibroblasts in vitro., Conclusions: The interferon signature in DM and SLE skin reduces collagen in dermal fibroblasts, whereas overexpression of PSMB9 induced by interferon stimulates versican inducers in epidermal keratinocytes. In addition, the TGF-β2-ΔDiHS-diS1 pathway may be responsible for the specific molecular change in DM skin., (© 2015 British Association of Dermatologists.)

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.

In order to study whether Tnfaip3 is related to stress response and further to find it's genetic regulation, we use C57BL/6J (B6) and DBA/2 (D2) mice to built the model of chronic unpredictable mild stress. RT-PCR, Western blotting and immunohistochemistry were used for studying the expression variation of Tnfaip3 in hippocampus tissue of B6 and D2 mice after being stressed. We found that the expression of Tnfaip3 was more remarkably increased in chronic unpredictable stress models than that in untreated mice (P<0.05). It is indicated that Tnfaip3 might take part in the process of stress response. The expression of Tnfaip3 is regulated by a cis-acting quantitative trait locus (cis-eQTL). We identified 5 genes are controlled by Tnfaip3 and the expression of 64 genes highly associated with Tnfaip3, 9 of those have formerly been participate in stress related pathways. In order to estimate the relationship between Tnfaip3 and its downstream genes or network members, we transfected SH-SY5Y cells with Tnfaip3 siRNA leading to down-regulation of Tnfaip3 mRNA. We confirmed a significant influence of Tnfaip3 depletion on the expression of Tsc22d3, Pex7, Rap2a, Slc2a3, and Gap43. These validated downstream genes and members of Tnfaip3 gene network provide us new insight into the biological mechanisms of Tnfaip3 in chronic unpredictable stress., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Small ubiquitin-like modifier (SUMO)-specific proteases (SENPs) that reverse protein modification by SUMO are involved in the control of numerous cellular processes, including transcription, cell division, and cancer development. However, the physiological function of SENPs in energy metabolism remains unclear. Here, we investigated the role of SENP2 in fatty acid metabolism in C2C12 myotubes and in vivo. In C2C12 myotubes, treatment with saturated fatty acids, like palmitate, led to nuclear factor-κB-mediated increase in the expression of SENP2. This increase promoted the recruitment of peroxisome proliferator-activated receptor (PPAR)δ and PPARγ, through desumoylation of PPARs, to the promoters of the genes involved in fatty acid oxidation (FAO), such as carnitine-palmitoyl transferase-1 (CPT1b) and long-chain acyl-CoA synthetase 1 (ACSL1). In addition, SENP2 overexpression substantially increased FAO in C2C12 myotubes. Consistent with the cell culture system, muscle-specific SENP2 overexpression led to a marked increase in the mRNA levels of CPT1b and ACSL1 and thereby in FAO in the skeletal muscle, which ultimately alleviated high-fat diet-induced obesity and insulin resistance. Collectively, these data identify SENP2 as an important regulator of fatty acid metabolism in skeletal muscle and further implicate that muscle SENP2 could be a novel therapeutic target for the treatment of obesity-linked metabolic disorders., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools., (© 2015 FEBS.)