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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.)

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.

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.

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.

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.

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.

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.)

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.)

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.

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.)

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.)

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.

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.

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.)

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.)

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations., (Copyright© Ferrata Storti Foundation.)

At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.

Disturbed blood flow (d-flow) causes endothelial cell (EC) dysfunction, leading to atherosclerotic plaque formation. We have previously shown that d-flow increases SUMOylation of p53 and ERK5 through downregulation of sentrin/SUMO-specific protease 2 (SENP2) function; however, it is not known how SENP2 itself is regulated by d-flow. Here, we determined that d-flow activated the serine/threonine kinase p90RSK, which subsequently phosphorylated threonine 368 (T368) of SENP2. T368 phosphorylation promoted nuclear export of SENP2, leading to downregulation of eNOS expression and upregulation of proinflammatory adhesion molecule expression and apoptosis. In an LDLR-deficient murine model of atherosclerosis, EC-specific overexpression of p90RSK increased EC dysfunction and lipid accumulation in the aorta compared with control animals; however, these pathologic changes were not observed in atherosclerotic mice overexpressing dominant negative p90RSK (DN-p90RSK). Moreover, depletion of SENP2 in these mice abolished the protective effect of DN-p90RSK overexpression. We propose that p90RSK-mediated SENP2-T368 phosphorylation is a master switch in d-flow-induced signaling, leading to EC dysfunction and atherosclerosis.

Introduction: The link between cardiovascular disease (CVD) and patients with chronic inflammation is not clearly understood. We examined a knock-in mouse expressing a poly-ubiquitin-binding-defective mutant of the protein ABIN1 (ABIN1(D485N)), which develops a systemic lupus erythematosus-like autoimmune disease because of the hyperactivation of IκB kinases (IκKs) and mitogen-activated protein kinases (MAPKs). These mice were used to determine the potential role of these signaling pathways in inflammation-mediated CVD development., Methods: Laser Doppler imaging in combination with the iontophoresis of vasoactive chemicals were used to assess endothelium-dependent vasodilatation in vivo in ABIN1 (D485N)) mutant defective (n=29) and wild-type (WT) control (n=26) mice. Measurements were made at baseline, and animals were subdivided to receive either chow or a proatherogenic diet for 4 weeks, after which, follow-up assessments were made. Paired and unpaired t tests, and ANOVA with post hoc Bonferroni correction were used for statistical significance at P<0.05., Results: Endothelium-dependent vasodilatation to acetylcholine was attenuated at 4 weeks in ABIN1(D485N)-chow-fed mice compared with age-matched WT-chow-fed mice (P<0.05). The magnitude of attenuation was similar to that observed in WT-cholesterol-fed animals (versus WT-chow, P<0.01). ABIN1(D485N)-cholesterol-fed mice had the poorest endothelium-dependent responses compared with other groups (P<0.001). ABIN1(D485N)-chow-fed mice had increased plasma interleukin-6 (IL-6) levels (versus WT-chow, P<0.001), and this was further elevated in ABIN1(D485N)-cholesterol-fed mice (versus ABIN1(D485N)-chow; P<0.05). IL-1α was significantly greater in all groups compared with WT-chow (P<0.01). ABIN1(D485N) mice showed significant cardiac hypertrophy (P<0.05)., Conclusions: The ABIN(D485N) mice display endothelial dysfunction and cardiac hypertrophy, which is possibly mediated through IL-6 and, to a lesser degree, IL-1α. These results suggest that the ABIN1-mediated hyperactivation of IKKs and MAPKs might mediate chronic inflammation and CVD development.

Autophagy is a well-conserved catabolic process essential for cellular homeostasis. First described in yeast as an adaptive response to starvation, this pathway is also present in higher eukaryotes, where it is triggered by stress signals such as damaged organelles or pathogen infection. Autophagy is characterized at the cellular level by the engulfment of portions of the cytoplasm in double-membrane structures called autophagosomes. Autophagosomes fuse with lysosomes, resulting in degradation of the inner autophagosomal membrane and luminal content. This process is coordinated by complex molecular systems, including the ATG8 ubiquitin-like conjugation system and the ATG4 cysteine proteases, which are implicated in the formation, elongation, and fusion of these autophagic vesicles. In this Review, we focus on the diverse functional roles of the autophagins, a protease family formed by the four mammalian orthologs of yeast Atg4. We also address the dysfunctional expression of these proteases in several pathologic conditions such as cancer and inflammation and discuss potential therapies based on their modulation.

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.

The ubiquitin-like SUMO system regulates gene expression, but the molecular insights into this process are incomplete. We show that the SUMO-specific isopeptidase SENP3 controls H3K4 methylation by regulating histone-modifying SET1/MLL complexes. SET1/MLL complexes are composed of a histone methyltransferase and the regulatory components WDR5, RbBP5, Ash2L, and DPY-30. MLL1/MLL2 complexes contain menin as additional component and are particularly important for the activation of HOX genes. We demonstrate that SENP3 is associated with MLL1/MLL2 complexes and catalyzes deSUMOylation of RbBP5. This is required for activation of a subset of HOX genes, including the developmental regulator DLX3. In the absence of SENP3, the association of menin and Ash2L with the DLX3 gene is impaired, leading to decreased H3K4 methylation and reduced recruitment of active RNA polymerase II. Importantly, the SENP3-DLX3 pathway dictates osteogenic differentiation of human stem cells, thus delineating the importance of balanced SUMOylation for epigenetic control of gene expression programs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion.

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.

Autophagy complements the ubiquitin-proteasome system in mediating protein turnover. Whereas the proteasome degrades individual proteins modified with ubiquitin chains, autophagy degrades many proteins and organelles en masse. Macromolecules destined for autophagic degradation are 'selected' through sequestration within a specialized double-membrane compartment termed the phagophore, the precursor to an autophagosome, and then are hydrolyzed in a lysosome- or vacuole-dependent manner. Notably, a pair of distinctive ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate degradation by autophagy in unique ways by controlling autophagosome biogenesis and recruitment of specific cargos during selective autophagy. Here we review structural mechanisms underlying the functions and conjugation of these UBLs that are specialized to provide interaction platforms linked to phagophore membranes.

Estrogen receptors (ERs) are ligand-activated transcription factors involved in many physiological and pathological processes, including breast cancer. Their activity is fine-tuned by posttranslational modifications, notably sumoylation. In the present study, we investigated the role of the small ubiquitin-related modifier (SUMO) protease, SUMO1/sentrin/suppressor of Mif 2-specific peptidase 2 (SENP2), in the regulation of ERα activity. We first found SENP2 to significantly repress estradiol-induced transcriptional activity in breast cancer cells (MCF7 and T47D). This effect was observed with a reporter plasmid and on endogenous genes such as TFF1 and CTSD, which were shown to recruit SENP2 in chromatin immunoprecipitation experiments. Using glutathione S-transferase pull-down, coimmunoprecipitation and proximity ligation assays, SENP2 was found to interact with ERα and this interaction to be mediated by the amino-terminal region of the protease and the hinge region of the receptor. Interestingly, we demonstrated that ERα repression by SENP2 is independent of its SUMO protease activity and requires a transcriptional repressive domain located in the amino-terminal end of the protease. Using small interfering RNA assays, we evidenced that this domain recruits the histone deacetylase 3 (HDAC3), to be fully active. Furthermore, using both overexpression and knockdown strategies, we showed that SENP2 robustly represses estrogen-dependent and independent proliferation of MCF7 cells. We provided evidence that this effect requires both the proteolytic and transcriptional activities of SENP2. Altogether, our study unravels a new property for a SUMO protease and identifies SENP2 as a classical transcription coregulator.

Posttranslational modification of different proteins via direct ubiquitin attachment is vital for mediating various cellular processes. Cylindromatosis (CYLD), a deubiquitination enzyme, is able to cleave the polyubiquitin chains from the substrate and to regulate different signaling pathways. Loss, or reduced expression, of CYLD is observed in different types of human cancer, such as hepatocellular carcinoma (HCC). However, the molecular mechanism by which CYLD affects cancerogenesis has to date not been unveiled. The aim of the present study was to examine how CYLD regulates cellular functions and signaling pathways during hepatocancerogenesis. We found that mice lacking CYLD were highly susceptible to chemically induced liver cancer. The mechanism behind proved to be an elevated proliferation rate of hepatocytes, owing to sustained c-Jun N-terminal kinase 1 (JNK1)-mediated signaling via ubiquitination of TNF receptor-associated factor 2 and expression of c-MYC. Overexpression of wild-type CYLD in HCC cell lines prevented cell proliferation, without affecting apoptosis, adhesion and migration. A combined immunohistochemical and tissue microarray analysis of 81 human HCC tissues revealed that CYLD expression is negatively correlated with expression of proliferation markers Ki-67 and c-MYC. To conclude, we found that downregulation of CYLD induces tumor cell proliferation, consequently contributing to the aggressive growth of HCC. Our findings suggest that CYLD holds potential to serve as a marker for HCC progression, and its link to c-MYC via JNK1 may provide the foundation for new therapeutic strategies for HCC patients.

Nuclear pore complexes are composed of ∼30 different proteins, each present at the pore in multiple copies. Together these proteins create specialized channels that convey cargo between the cytoplasm and the nuclear interior. With the building blocks of nuclear pores identified, one challenge is to decipher how these proteins are coordinately produced and assembled into macromolecular pore structures with each cell division. Specific individual pore proteins and protein cofactors have been probed for their role in the assembly process, as well as certain kinases that add a layer of regulation via the phosphorylation status of nucleoporins. Other posttranslational modifications are candidates for coordinating events of pore assembly as well. In this study of two pore-associated small ubiquitin-like modifier (SUMO) proteases, sentrin/SUMO-specific protease 1 (SENP1) and SENP2, we observe that many nucleoporins are mislocalized and, in some cases, reduced in level when SENP1 and SENP2 are codepleted. The pore complexes present under these conditions are still capable of transport, although the kinetics of specific cargo is altered. These results reveal a new role for the pore-associated SENPs in nucleoporin homeostasis and in achieving proper configuration of the nuclear pore complex.

Heart failure in children and adults is often the consequence of myocarditis associated with Coxsackievirus (CV) infection. Upon CV infection, enteroviral protease 2A cleaves a small number of host proteins including dystrophin, which links actin filaments to the plasma membrane of muscle fiber cells (sarcolemma). It is unknown whether protease 2A-mediated cleavage of dystrophin and subsequent disruption of the sarcolemma play a role in CV-mediated myocarditis. We generated knockin mice harboring a mutation at the protease 2A cleavage site of the dystrophin gene, which prevents dystrophin cleavage following CV infection. Compared with wild-type mice, we found that mice expressing cleavage-resistant dystrophin had a decrease in sarcolemmal disruption and cardiac virus titer following CV infection. In addition, cleavage-resistant dystrophin inhibited the cardiomyopathy induced by cardiomyocyte-restricted expression of the CV protease 2A transgene. These findings indicate that protease 2A-mediated cleavage of dystrophin is critical for viral propagation, enteroviral-mediated cytopathic effects, and the development of cardiomyopathy.

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5-30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1-5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.

The cysteine protease legumain plays important functions in immunity and cancer at different cellular locations, some of which appeared conflicting with its proteolytic activity and stability. Here, we report crystal structures of legumain in the zymogenic and fully activated form in complex with different substrate analogs. We show that the eponymous asparagine-specific endopeptidase activity is electrostatically generated by pH shift. Completely unexpectedly, the structure points toward a hidden carboxypeptidase activity that develops upon proteolytic activation with the release of an activation peptide. These activation routes reconcile the enigmatic pH stability of legumain, e.g., lysosomal, nuclear, and extracellular activities with relevance in immunology and cancer. Substrate access and turnover is controlled by selective protonation of the S1 pocket (KM) and the catalytic nucleophile (kcat), respectively. The multibranched and context-dependent activation process of legumain illustrates how proteases can act not only as signal transducers but as decision makers.

Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²⁺-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); we used proteins with an N-terminal (oligo)glycine as nucleophiles. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein-based nucleophiles in different intracellular compartments., (© 2012 John Wiley & Sons A/S.)

Background: Viral myocarditis, which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. However, efficient therapies targeting inflammation are still lacking and much needed. A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is a key negative regulator of inflammation. But whether A20 may affect cardiac inflammation during acute viral myocarditis remains to be elucidated. The aim of this study was to investigate the potential protective effect of A20 on CVB3-induced myocarditis., Methodology/principal Findings: Mice were intraperitoneally inoculated with CVB3 to establish acute viral myocarditis model. We found that the expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) were markedly and persistently increased during the progression of CVB3-induced myocarditis, and positively correlated with the disease severity. Notably, intravenous injection in vivo with adenovirus expressed A20 (Ad-A20) remarkably reduced CVB3-induced pro-inflammatory cytokines production and alleviated the severity of myocarditis. Further, we observed that nuclear factor-kappaB (NF-κB) signaling which mediates inflammatory response was significantly inhibited in CVB3-infected mice with Ad-A20 treatment. Finally, we revealed that A20 was required to inhibit CVB3-induced NF-κB signaling by restricting TNF receptor associated factor 6 (TRAF6) ubiquitylation., Conclusion/significance: This study demonstrates the protective role of A20 against CVB3-induced myocarditis, which may provide a new therapeutic strategy for the treatment of viral myocarditis.