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NOX5 (NADPH oxidase 5) is a homolog of the gp91 phox subunit of the phagocyte NOX, which generates reactive oxygen species. NOX5 is involved in sperm motility and vascular contraction and has been implicated in diabetic nephropathy, atherosclerosis, and stroke. The function of NOX5 in the cardiac hypertrophy is unknown. Because NOX5 is a Ca 2+ -sensitive, procontractile NOX isoform, we questioned whether it plays a role in cardiac hypertrophy. Studies were performed in (1) cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure, (2) NOX5-expressing rat cardiomyocytes, and (3) mice expressing human NOX5 in a cardiomyocyte-specific manner. Cardiac hypertrophy was induced in mice by transverse aorta coarctation and Ang II (angiotensin II) infusion. NOX5 expression was increased in human failing hearts. Rat cardiomyocytes infected with adenoviral vector encoding human NOX5 cDNA exhibited elevated reactive oxygen species levels with significant enlargement and associated increased expression of ANP (atrial natriuretic peptides) and β-MHC (β-myosin heavy chain) and prohypertrophic genes ( Nppa , Nppb , and Myh7 ) under Ang II stimulation. These effects were reduced by N-acetylcysteine and diltiazem. Pressure overload and Ang II infusion induced left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction, responses that were exaggerated in cardiac-specific NOX5 trangenic mice. These phenomena were associated with increased reactive oxygen species levels and activation of redox-sensitive MAPK (mitogen-activated protein kinase). N-acetylcysteine treatment reduced cardiac oxidative stress and attenuated cardiac hypertrophy in NOX5 trangenic. Our study defines Ca 2+ -regulated NOX5 as an important NOX isoform involved in oxidative stress- and MAPK-mediated cardiac hypertrophy and contractile dysfunction.

Inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation has emerged as a promising target for the treatment of nonalcoholic steatohepatitis (NASH). Multiple forms of posttranslational modifications determine the activity of ASK1. In addition to phosphorylation, recent studies revealed that ubiquitination is essential for ASK1 activation. However, the endogenous factor that regulates ASK1 ubiquitination and activation remains poorly defined. In this study, we identified the E3 ligase Skp1-Cul1-F-box (SCF) protein F-box/WD repeat-containing protein 5 (FBXW5) as a key endogenous activator of ASK1 ubiquitination. FBXW5 is the central component of the SCF complex (SCFFbxw5 ) that directly interacts with and ubiquitinates ASK1 in hepatocytes during NASH development. An in vivo study showed that hepatocyte-specific overexpression of FBXW5 exacerbated diet-induced systemic and hepatic metabolic disorders, as well as the activation of ASK1-related mitogen-activated protein kinase (MAPK) signaling in the liver. Conversely, hepatocyte-specific deletion of FBXW5 significantly prevented the progression of these abnormalities. Mechanically, FBXW5 facilitated the addition of Lys63-linked ubiquitin to ASK1 and thus exacerbated ASK1-c-Jun N-terminal kinase/p38 MAPK signaling, inflammation, and lipid accumulation. Furthermore, we demonstrated that the N-terminus (S1) and C-terminus (S3) of FBXW5 respectively and competitively ablate the function of FBXW5 on ASK1 activation and served as effective inhibitors of NASH progression. Conclusion: This evidence strongly suggests that SCFFbxw5 is an important activator of ASK1 ubiquitination in the context of NASH. The development of FBXW5(S1) or FBXW5(S3)-mimicking drugs and screening of small-molecular inhibitors specifically abrogating ASK1 ubiquitination-dependent activation are viable approaches for NASH treatment.

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2., Public antibody clonotypes that recognize SARS-CoV-2 spike protein are important for protection against COVID-19. Here, the authors characterize sequence motifs in the heavy chain complementarity-determining region (CDR) H3s of two public clonotypes and their association with light chain identity.

A double punch against SARS-CoV-2 Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science, this issue p. eabe6230; see also p. 681, SARS-CoV-2–neutralizing nanobodies were combined to design potent multivalent nanobodies., INTRODUCTION The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems. RATIONALE We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular membranes catalyzed by the spike. RESULTS Nanobodies specific for the receptor binding domain (RBD) of SARS-CoV-2 spike were identified by phage display using nanobody libraries from an alpaca and a llama immunized with the RBD and inactivated virus. Four of the resulting nanobodies—VHHs E, U, V, and W—potently neutralize SARS-CoV-2 and SARS-CoV-2–pseudotyped vesicular stomatitis virus. X-ray crystallography revealed that the nanobodies bind to two distinct epitopes on the RBD, interfaces “E” and “UVW,” which can be synergistically targeted by combinations of nanobodies to inhibit infection. Cryo–electron microscopy (cryo-EM) of trimeric spike in complex with VHH E and VHH V revealed that VHH E stabilizes a conformation of the spike with all three RBDs in the “up” conformation (3-up), a state that is typically associated with activation by receptor binding. In line with this observation, we found that VHH E triggers the fusion activity of spike in the absence of the cognate receptor ACE2. VHH V, by contrast, stabilizes spike in a 2-up conformation and does not induce fusion. On the basis of the structural information, we designed bi- and trivalent nanobodies with improved neutralizing properties. VHH EEE most potently inhibited infection, did not activate fusion, and likely inactivated virions by outcompeting interaction of the virus with its receptor. Yet evolution experiments revealed emergence of escape mutants in the spike with single–amino acid changes that were completely insensitive to inhibition by VHH EEE. VHH VE also neutralized more efficiently than VHH E or VHH V alone; stabilized the 3-up conformation of spike, as determined by cryo-EM; and more strongly induced the spike fusogenic activity. We conclude that the premature activation of the fusion machinery on virions was an unexpected mechanism of neutralization, as enhanced neutralization could not be attributed simply to better blocking of virus-receptor interactions. Activation of spike in the absence of target membranes likely induces irreversible conformational changes to assume the energetically favorable postfusion conformation without catalyzing fusion per se. Simultaneous targeting of two independent epitopes by VHH VE largely prevented the emergence of resistant escape mutants in evolution experiments. CONCLUSION Our results demonstrate the strength of the modular combination of nanobodies for neutralization. Premature activation of spike by nanobodies reveals an unusual mode of neutralization and yields insights into the mechanism of fusion. Bivalent nanobodies neutralize by inducing postfusion conformation of the SARS-CoV-2 spike. On virions, SARS-CoV-2 spike trimers are mostly in an inactive configuration with all RBDs in the down conformation (left). Binding of bivalent nanobody VE stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the postfusion conformation, which irreversibly inactivates the spike protein (right)., The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Most antibodies isolated from individuals with coronavirus disease 2019 (COVID-19) are specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here, we determined a crystal structure of the COVA1-16 antibody fragment (Fab) with the SARS-CoV-2 receptor-binding domain (RBD) and negative-stain electron microscopy reconstructions with the spike glycoprotein trimer to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long complementarity-determining region (CDR) H3, and competes with the angiotensin-converting enzyme 2 (ACE2) receptor because of steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with the structural and functional rationale for epitope conservation, provide insights for development of more universal SARS-like coronavirus vaccines and therapies., Graphical Abstract, Highlights • X-ray and EM structures of cross-neutralizing antibody COVA1-16 with SARS-CoV-2 RBD • COVA1-16 binding to SARS-CoV-2 RBD is dominated by CDR H3 • COVA1-16 binds to a highly conserved non-RBS epitope but still competes with ACE2 • IgG avidity is the key for the cross-neutralization activity of COVA1-16, COVA1-16 is a SARS-CoV-2 antibody from an individual with COVID-19 that cross-neutralizes SARS-CoV. Liu et al. reveal that COVA1-16 binds to a highly conserved epitope using a long CDR H3, where its approach angle sterically blocks ACE2 from engaging the RBS. Virus neutralization by COVA1-16 is driven by IgG avidity.

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization, Graphical Abstract, Highlights • Crystal structures of IGHV3-53 antibodies that frequently bind SARS-CoV-2 RBD • Binding modes (A and B) of these IGHV3-53 antibodies depend on CDR H3 length • Germline-encoded CDR H1 and H2 motifs dominate the two binding poses • CDR H3 length of IGHV3-53 antibodies is associated with light chain preference, Antibodies to the SARS-CoV-2 receptor-binding domain are commonly encoded by IGHV3-53 and most have a short CDR H3. Wu et al. show that IGHV3-53 antibodies with a long CDR H3 adopt an alternative binding mode, demonstrating that IGHV3-53 is even more versatile than previously thought in targeting SARS-CoV-2.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation, and the pathogenic mechanism of NAFLD is poorly understood. Ubiquitin-specific peptidase 10 (USP10), a member of the ubiquitin-specific protease family, is involved in environmental stress responses, tumor growth, inflammation, and cellular metabolism. However, the role of USP10 in hepatic steatosis, insulin resistance, and inflammation remains largely unexplored. USP10 expression was detected in livers of patients with NAFLD, mice with high-fat diet (HFD)-induced obesity, and genetically obese (ob/ob) mice, as well as in palmitate-induced hepatocytes. The function of USP10 in hepatic steatosis, insulin resistance, and inflammation was investigated using hepatocyte-specific USP10 deficiency or overexpression in mice induced by HFD treatment or genetic defect. The molecular mechanisms underlying USP10-regulated hepatic steatosis were further investigated in HFD-treated mice. USP10 expression was significantly decreased in the fatty livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. USP10 deficiency exacerbated the metabolic dysfunction induced by HFD treatment for 12 weeks. Conversely, USP10 overexpression significantly suppressed metabolic dysfunction in mice after HFD treatment and inhibited the development of NAFLD in ob/ob mice. Further investigation indicated that USP10 regulates hepatic steatosis by interacting with Sirt6 and inhibiting its ubiquitination and degradation. Sirt6 overexpression markedly ameliorated the effects of USP10 deficiency in hepatic steatosis, insulin resistance, and inflammation. Conversely, Sirt6 deficiency decreased the ameliorative effects of USP10 overexpression in response to HFD treatment. Conclusion: USP10 inhibits hepatic steatosis, insulin resistance, and inflammation through Sirt6.

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human VH1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines., Broadly neutralizing antibodies are potential therapeutics and can aid rational vaccine development. Here, the authors show that the human monoclonal antibody H3v-47 recognizes a highly conserved epitope in HA of H3N2 viruses, inhibits virus replication by blocking egress and other mechanisms, and protects mice from disease.

Background - Mindin, which is a highly conserved ECM protein, has been documented to play pivotal roles in regulating angiogenesis, inflammatory processes and immune responses. The aim of this study was to assess whether mindin contributes to the development of atherosclerosis. Methods and Results - A significant upregulation of Mindin expression was observed in the serum, arteries and atheromatous plaques of ApoE-/- mice after HFD treatment. Mindin-/-ApoE-/- mice and macrophage-specific mindin overexpression in ApoE-/- mice (Lyz2-mindin-TG) were generated to evaluate the effect of mindin on the development of atherosclerosis. The Mindin-/-ApoE-/- mice exhibited significantly ameliorated atherosclerotic burdens in the entire aorta and aortic root and increased atherosclerotic plaque stability. Moreover, bone marrow transplantation further demonstrated that mindin deficiency in macrophages was largely responsible for the alleviated atherogenesis. The Lyz2-mindin-TG mice exhibited the opposite phenotype. Mindin deficiency enhanced foam cell formation by increasing the expression of cholesterol effectors, including ABCA1 and ABCG1. The mechanistic study indicated that mindin ablation promoted LXR-β expression via a direct interaction. Importantly, LXR-β inhibition largely reversed the ameliorating effect of mindin deficiency on foam cell formation and ABCA1 and ABCG1 expression. Conclusions -The present study demonstrated that mindin deficiency serves as a novel mediator that protects against foam cell formation and atherosclerosis by directly interacting with LXR-β.

Tumor progression locus 2 (TPL2), a serine/threonine kinase, has been regarded as a potentially interesting target for the treatment of various diseases with an inflammatory component. However, the function of TPL2 in regulating hepatocyte metabolism and liver inflammation during the progression of nonalcoholic fatty liver disease (NAFLD) is poorly understood. Here, we report that TPL2 protein expression was significantly increased in fatty liver from diverse species, including humans, monkeys, and mice. Further investigations revealed that compared to wild-type (WT) littermates, hepatocyte-specific TPL2 knockout (HKO) mice exhibited improved lipid and glucose imbalance, reserved insulin sensitivity, and alleviated inflammation in response to high-fat diet (HFD) feeding. Overexpression of TPL2 in hepatocytes led to the opposite phenotype. Regarding the mechanism, we found that mitogen-activated protein kinase kinase 7 (MKK7) was the specific substrate of TPL2 for c-Jun N-terminal kinase (JNK) activation. TPL2-MKK7-JNK signaling in hepatocytes represents a promising drugable target for treating NAFLD and associated metabolic disorders. Conclusion: In hepatocytes, TPL2 acts as a key mediator that promotes both liver and systemic metabolic disturbances by specifically increasing MKK7-JNK activation.

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG + memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated V H 5-51/V L 4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of V H 5-51 and V L 4-1 recognizes a common Pro-X n -Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent and complex disease that confers a high risk of severe liver disorders. Despite such public and clinical health importance, very few effective therapies are currently available for NAFLD. We report a protective function and the underlying mechanism of dual-specificity phosphatase 14 (DUSP14) in NAFLD and related metabolic disorders. Insulin resistance, hepatic lipid accumulation, and concomitant inflammatory responses, key pathological processes involved in NAFLD development, were significantly ameliorated by hepatocyte-specific DUSP14 overexpression (DUSP14-HTG) in high-fat diet (HFD)-induced or genetically obese mouse models. By contrast, specific DUSP14 deficiency in hepatocytes (DUSP14-HKO) aggravated these pathological alterations. We provided mechanistic evidence that DUSP14 directly binds to and dephosphorylates transforming growth factor β-activated kinase 1 (TAK1), resulting in the reduced activation of TAK1 and its downstream signaling molecules c-Jun N-terminal kinase 1 (JNK), p38, and nuclear factor kappa B NF-κB. This effect was further evidenced by the finding that inhibiting TAK1 activity effectively attenuated the deterioration of glucolipid metabolic phenotype in DUSP14-HKO mice challenged by HFD administration. Furthermore, we identified that both the binding domain and the phosphatase activity of DUSP14 are required for its protective role against hepatic steatosis, because interruption of the DUSP14-TAK1 interaction abolished the mitigative effects of DUSP14. CONCLUSION Hepatocyte DUSP14 is required for maintaining hepatic metabolic homeostasis and for suppressing inflammation, a novel function that relies on constraining TAK1 hyperactivation. (Hepatology 2018;67:1320-1338).

Nonalcoholic steatohepatitis (NASH) is a common clinical condition that can lead to advanced liver diseases. Lack of effective pharmacotherapies for NASH is largely attributable to an incomplete understanding of its pathogenesis. The deubiquitinase cylindromatosis (CYLD) plays key roles in inflammation and cancer. Here we identified CYLD as a suppressor of NASH in mice and in monkeys. CYLD is progressively degraded upon interaction with the E3 ligase TRIM47 in proportion to NASH severity. We observed that overexpression of Cyld in hepatocytes concomitantly inhibits lipid accumulation, insulin resistance, inflammation and fibrosis in mice with NASH induced in an experimental setting. Mechanistically, CYLD interacts directly with the kinase TAK1 and removes its K63-linked polyubiquitin chain, which blocks downstream activation of the JNK-p38 cascades. Notably, reconstitution of hepatic CYLD expression effectively reverses disease progression in mice with dietary or genetically induced NASH and in high-fat diet-fed monkeys predisposed to metabolic syndrome. Collectively, our findings demonstrate that CYLD mitigates NASH severity and identify the CYLD-TAK1 axis as a promising therapeutic target for management of the disease.

Activation of apoptosis signal-regulating kinase 1 (ASK1) in hepatocytes is a key process in the progression of nonalcoholic steatohepatitis (NASH) and a promising target for treatment of the condition. However, the mechanism underlying ASK1 activation is still unclear, and thus the endogenous regulators of this kinase remain open to be exploited as potential therapeutic targets. In screening for proteins that interact with ASK1 in the context of NASH, we identified the deubiquitinase tumor necrosis factor alpha-induced protein 3 (TNFAIP3) as a key endogenous suppressor of ASK1 activation, and we found that TNFAIP3 directly interacts with and deubiquitinates ASK1 in hepatocytes. Hepatocyte-specific ablation of Tnfaip3 exacerbated nonalcoholic fatty liver disease- and NASH-related phenotypes in mice, including glucose metabolism disorders, lipid accumulation and enhanced inflammation, in an ASK1-dependent manner. In contrast, transgenic or adeno-associated virus-mediated TNFAIP3 gene delivery in the liver in both mouse and nonhuman primate models of NASH substantially blocked the onset and progression of the disease. These results implicate TNFAIP3 as a functionally important endogenous suppressor of ASK1 hyperactivation in the pathogenesis of NASH and identify it as a potential new molecular target for NASH therapy.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, impaired insulin sensitivity, and chronic low-grade inflammation. However, the pathogenic mechanism of NAFLD is poorly understood, which hinders the exploration of possible treatments. Here, we report that ubiquitin-specific protease 18 (USP18), a member of the deubiquitinating enzyme family, plays regulatory roles in NAFLD progression. Expression of USP18 was down-regulated in the livers of nonalcoholic steatohepatitis patients and high-fat diet (HFD)-induced or genetically obese mice. When challenged with HFD, hepatocyte-specific USP18 transgenic mice exhibited improved lipid metabolism and insulin sensitivity, whereas mice knocked out of USP18 expression showed adverse trends regarding hepatic steatosis and glucose metabolic disorders. Furthermore, the concomitant inflammatory response was suppressed in USP18-hepatocyte-specific transgenic mice and promoted in USP18-hepatocyte-specific knockout mice treated with HFD. Mechanistically, hepatocyte USP18 ameliorates hepatic steatosis by interacting with and deubiquitinating transforming growth factorβ-activated kinase 1 (TAK1), which inhibits TAK1 activation and subsequently suppresses the downstream c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. This is further validated by alleviated steatotic phenotypes and highly activated insulin signaling in HFD-fed USP18-hepatocyte-specific knockout mice administered a TAK1 inhibitor. The therapeutic effect of USP18 on NAFLD relies on its deubiquitinating activity because HFD-fed mice injected with active-site mutant USP18 failed to inhibit hepatic steatosis. Conclusion USP18 associates with and deubiquitinates TAK1 to protect against hepatic steatosis, insulin resistance, and the inflammatory response. (Hepatology 2017;66:1866-1884).

Background: The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown. Methods: IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)–specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)–TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated. Results: IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation. Conclusions: IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.

Antibodies that target both group 1 and group 2 influenza A viruses are valuable for therapeutic and vaccine development, but only a few have been reported to date. Here, we describe a new VH1-69 antibody 27F3 that broadly recognizes heterosubtypic hemagglutinins (HAs) from both group 1 and group 2 influenza A viruses. Structural characterization of 27F3 Fab with A/California/04/2009 (H1N1) hemagglutinin illustrates that 27F3 shares the key binding features observed in other VH1-69 antibodies to the HA stem. Compared to other VH1-69 antibodies, the 27F3 VH domain interacts with the HA stem in a distinct orientation, which alters its epitope and may have influenced its breadth. The diverse rotations of VH1-69 antibodies on the HA stem epitope highlight the different ways that this antibody family can evolve to broadly neutralize influenza A viruses. These results have important implications for understanding how to elicit broad antibody responses against influenza virus.

Atherosclerosis is a chronic inflammatory disease. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is associated with the pathological processes of various inflammatory diseases. However, the potential function and underlying mechanisms of LILRB4 in atherogenesis remain to be investigated. In the present study, LILRB4 expression was examined in both human and mouse atherosclerotic plaques. The effects and possible mechanisms of LILRB4 in atherogenesis and plaque instability were evaluated in LILRB4-/-ApoE-/- and ApoE-/- mice fed a high-fat diet (HFD). We found that LILRB4 was located primarily in macrophages, and its expression was up-regulated in atherosclerotic lesions from human coronary arteries and mouse aortic roots. LILRB4 deficiency significantly accelerated the development of atherosclerotic lesions and increased the instability of plaques, as evident by the increased infiltration of lipids, decreased amount of collagen components and smooth muscle cells. Moreover, LILRB4 deficiency in bone marrow derived cells promoted the development of atherosclerosis. In vivo and in vitro analyses revealed that the proinflammatory effects of LILRB4 deficiency were mediated by the increased activation of NF-κB signaling due to decreased src homolog 2 domain containing phosphatase (Shp) 1 phosphorylation. In conclusion, the present study indicates that LILRB4 deficiency promotes atherogenesis, at least partly, through reduced Shp1 phosphorylation, which subsequently enhances the NF-κB-mediated inflammatory response. Thus, targetting the ‘LILRB4-Shp1’ axis may be a novel therapeutic approach for atherosclerosis.

Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase/tRNA pair that allows the site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Non-alcoholic steatohepatitis (NASH) is an increasingly prevalent liver pathology that can progress from non-alcoholic fatty liver disease (NAFLD), and it is a leading cause of cirrhosis and hepatocellular carcinoma. There is currently no pharmacological therapy for NASH. Defective lysosome-mediated protein degradation is a key process that underlies steatohepatitis and a well-recognized drug target in a variety of diseases; however, whether it can serve as a therapeutic target for NAFLD and NASH remains unknown. Here we report that transmembrane BAX inhibitor motif-containing 1 (TMBIM1) is an effective suppressor of steatohepatitis and a previously unknown regulator of the multivesicular body (MVB)-lysosomal pathway. Tmbim1 expression in hepatocytes substantially inhibited high-fat diet-induced insulin resistance, hepatic steatosis and inflammation in mice. Mechanistically, Tmbim1 promoted the lysosomal degradation of toll-like receptor 4 by cooperating with the ESCRT endosomal sorting complex to facilitate MVB formation, and the ubiquitination of Tmbim1 by the E3 ubiquitin ligase Nedd4l was required for this process. We also found that overexpression of Tmbim1 in the liver effectively inhibited a severe form of NAFLD in mice and NASH progression in monkeys. Taken together, these findings could lead to the development of promising strategies to treat NASH by targeting MVB regulators to properly orchestrate the lysosome-mediated protein degradation of key mediators of the disease.

Tripartite motif 8 (TRIM8), an E3 ligase ubiquitously expressed in various cells, is closely involved in innate immunity. However, its role in nonalcoholic steatohepatitis is largely unknown. Here, we report evidence that TRIM8 is a robust enhancer of steatohepatitis and its complications induced by a high-fat diet or a genetic deficiency (ob/ob). Using gain-of-function and loss-of-function approaches, we observed dramatic exacerbation of insulin resistance, hepatic steatosis, inflammation, and fibrosis by hepatocyte-specific TRIM8 overexpression, whereas deletion or down-regulation of TRIM8 in hepatocytes led to a completely opposite phenotype. Furthermore, investigations of the underlying mechanisms revealed that TRIM8 directly binds to and ubiquitinates transforming growth factor-beta-activated kinase 1, thus promoting its phosphorylation and the activation of downstream c-Jun N-terminal kinase/p38 and nuclear factor κB signaling. Importantly, the participation of TRIM8 in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis was verified on the basis of its dramatically increased expression in the livers of these patients, suggesting a promising development of TRIM8 disturbance for the treatment of nonalcoholic steatohepatitis-related metabolic disorders. Conclusion The E3 ligase TRIM8 is a potent regulator that exacerbates steatohepatitis and metabolic disorders dependent on its binding and ubiquitinating capacity on transforming growth factor-beta-activated kinase 1. (Hepatology 2017;65:1492-1511).

Nonalcoholic steatohepatitis (NASH) is a progressive disease that is often accompanied by metabolic syndrome and poses a high risk of severe liver damage. However, no effective pharmacological treatment is currently available for NASH. Here we report that CASP8 and FADD-like apoptosis regulator (CFLAR) is a key suppressor of steatohepatitis and its metabolic disorders. We provide mechanistic evidence that CFLAR directly targets the kinase MAP3K5 (also known as ASK1) and interrupts its N-terminus-mediated dimerization, thereby blocking signaling involving ASK1 and the kinase MAPK8 (also known as JNK1). Furthermore, we identified a small peptide segment in CFLAR that effectively attenuates the progression of steatohepatitis and metabolic disorders in both mice and monkeys by disrupting the N-terminus-mediated dimerization of ASK1 when the peptide is expressed from an injected adenovirus-associated virus 8-based vector. Taken together, these findings establish CFLAR as a key suppressor of steatohepatitis and indicate that the development of CFLAR-peptide-mimicking drugs and the screening of small-molecular inhibitors that specifically block ASK1 dimerization are new and feasible approaches for NASH treatment.

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.

Background & Aims Dickkopf-3 (DKK3), a protein belonging to the DKK family, has been extensively investigated in the context of cancer, including liver cancer. However, the role of DKK3 in hepatic steatosis and related metabolic disorders remains largely unexplored. Methods We detected the expression of DKK3 in the fatty livers of NAFLD patients and of obese mice and investigated the function of DKK3 in hepatic steatosis and related metabolic disorders by using hepatocyte-specific DKK3 deficiency or overexpression obese mice induced by high fat diet (HFD) or genetic defect ( ob/ob ). The molecular mechanisms underlying DKK3-regulated hepatic steatosis were further explored and verified in mice. Results DKK3 expression was significantly decreased in the livers of NAFLD patients and of obese mice as well as in cultured hepatocytes stimulated with palmitate. Further investigation indicated that specific overexpression of DKK3 in hepatocytes enhanced insulin sensitivity and glucose tolerance, reduced the inflammatory response, and ameliorated the imbalance of lipid metabolism in response to HFD or genetic defects. In contrast, DKK3 deficiency in hepatocytes led to an almost complete reversal of these pathologies. Mechanistically, DKK3 combined with Apoptosis signal-regulating kinase 1 (ASK1) under palmitate stimulation, and thus inhibited the activation of the downstream P38/JNK pathway. Importantly, dominant-negative ASK1 blocked the accelerated effects of DKK3 deficiency, while the constitutively active form of ASK1 overcame the inhibitory effects of DKK3 overexpression on HFD-induced metabolic disorders in vivo . Conclusion DKK3 functions as a negative regulator of insulin resistance, hepatic steatosis, and associated inflammatory responses, which depends on its inhibitory regulation of ASK1 activity. Lay summary DKK3 expression is decreased in the non-alcoholic fatty liver of humans and mice. Adding DKK3 expression alleviates fatty liver in mice by inhibiting ASK1 activity.

The liver is an essential insulin-responsive organ that is critical for maintaining glucose homeostasis and lipid metabolism. Oncostatin M receptor β chain (OSMRβ) is implicated in adipose tissue- and immune cell-mediated metabolic regulation. However, the role of hepatocyte-derived OSMRβ in metabolic disorders remains unclear. Here, we report on the central role of OSMRβ in the protection against obesity and deregulation of glucose and lipids. We observed significantly varied expression levels of OSMRβ in hepatic tissues in both human samples and mouse models of nonalcoholic fatty liver disease. Mice lacking either whole-body or hepatic OSMRβ displayed exacerbated diet-induced insulin resistance, hepatic steatosis, and inflammation, both in diet-induced and genetically (ob/ob) obese mice. These adverse effects were markedly attenuated by hepatocyte-specific overexpression of OSMRβ. Mechanistically, we showed that OSMRβ phosphorylates and activates the Janus kinase 2 (JAK2)/STAT3 signaling pathway in the liver. More importantly, the liver-restricted overexpression of STAT3 rescued glucose tolerance and ameliorated hepatic steatosis and inflammation in OSMRβ knockout mice, whereas OSMRβ overexpression failed to protect against hepatic steatosis, insulin resistance, and hepatic inflammation in STAT3-deficient mice. Thus, activation of STAT3 is both sufficient and required to produce OSMRβ-mediated beneficial effects. In conclusion, hepatic OSMRβ expression alleviates obesity-induced hepatic insulin resistance and steatosis through the activation of JAK2/STAT3 signaling cascades.

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and a systemic pro-inflammatory response. Here we show that tumour necrosis factor receptor-associated factor 3 (TRAF3) is upregulated in mouse and human livers with hepatic steatosis. After 24 weeks on a high-fat diet (HFD), obesity, insulin resistance, hepatic steatosis and inflammatory responses are significantly ameliorated in liver-specific TRAF3-knockout mice, but exacerbated in transgenic mice overexpressing TRAF3 in hepatocytes. The detrimental effects of TRAF3 on hepatic steatosis and related pathologies are confirmed in ob/ob mice. We further show that in response to HFD, hepatocyte TRAF3 binds to TGF-β-activated kinase 1 (TAK1) to induce TAK1 ubiquitination and subsequent autophosphorylation, thereby enhancing the activation of downstream IKKβ–NF-κB and MKK–JNK–IRS1307 signalling cascades, while disrupting AKT–GSK3β/FOXO1 signalling. The TRAF3–TAK1 interaction and TAK1 ubiquitination are indispensable for TRAF3-regulated hepatic steatosis. In conclusion, hepatocyte TRAF3 promotes HFD-induced or genetic hepatic steatosis in a TAK1-dependent manner., TRAF family proteins regulate immune signalling cascades. Here, the authors show that TRAF3 is upregulated in the liver in non-alcoholic fatty liver disease, promoting insulin resistance, inflammation and hepatic steatosis via its interaction with the kinase TAK1.

Background Carboxyl‐terminal modulator protein ( CTMP ) has been implicated in cancer, brain injury, and obesity. However, the role of CTMP in pathological cardiac hypertrophy has not been identified. Methods and Results In this study, decreased expression of CTMP was observed in both human failing hearts and murine hypertrophied hearts. To further explore the potential involvement of CTMP in pathological cardiac hypertrophy, cardiac‐specific CTMP knockout and overexpression mice were generated. In vivo experiments revealed that CTMP deficiency exacerbated the cardiac hypertrophy, fibrosis, and function induced by pressure overload, whereas CTMP overexpression alleviated the response to hypertrophic stimuli. Consistent with the in vivo results, adenovirus‐mediated gain‐of‐function or loss‐of‐function experiments showed that CTMP also exerted a protective effect against hypertrophic responses to angiotensin II in vitro. Mechanistically, CTMP ameliorated pathological cardiac hypertrophy through the blockade of the protein kinase B signaling pathway. Moreover, inhibition of protein kinase B activation with LY 294002 rescued the deteriorated effect in aortic banding–treated cardiac‐specific CTMP knockout mice. Conclusions Taken together, these findings imply, for the first time, that increasing the cardiac expression of CTMP may be a novel therapeutic strategy for pathological cardiac hypertrophy.

Background Tollip, a well‐established endogenous modulator of Toll‐like receptor signaling, is involved in cardiovascular diseases. The aim of this study was to investigate the role of Tollip in neointima formation and its associated mechanisms. Methods and Results In this study, transient increases in Tollip expression were observed in platelet‐derived growth factor‐ BB –treated vascular smooth muscle cells and following vascular injury in mice. We then applied loss‐of‐function and gain‐of‐function approaches to elucidate the effects of Tollip on neointima formation. While exaggerated neointima formation was observed in Tollip‐deficient murine neointima formation models, Tollip overexpression alleviated vascular injury–induced neointima formation by preventing vascular smooth muscle cell proliferation, dedifferentiation, and migration. Mechanistically, we demonstrated that Tollip overexpression may exert a protective role in the vasculature by suppressing Akt‐dependent signaling, which was further confirmed in rescue experiments using the Akt‐specific inhibitor ( AKTI ). Conclusions Our findings indicate that Tollip protects against neointima formation by negatively regulating vascular smooth muscle cell proliferation, dedifferentiation, and migration in an Akt‐dependent manner. Upregulation of Tollip may be a promising strategy for treating vascular remodeling–related diseases.

The hemagglutinin (HA) receptor-binding site (RBS) in human influenza A viruses is critical for attachment to host cells, which imposes a functional constraint on its natural evolution. On the other hand, being part of the major antigenic sites, the HA RBS of human H3N2 viruses needs to constantly mutate to evade the immune system. From large-scale mutagenesis experiments, we here show that several of the natural RBS substitutions become integrated into an extensive epistatic network that prevents substitution reversion. X-ray structural analysis reveals the mechanistic consequences as well as changes in the mode of receptor binding. Further studies are necessary to elucidate whether such entrenchment limits future options for immune escape or adversely affect long-term viral fitness., The receptor-binding site (RBS) of influenza A viruses evolves to evade immune pressure, while maintaining efficient attachment to the host receptor. Wu et al. here identify the complex epistatic network in RBS of H3N2 viruses that limits reversibility of naturally occurring mutations to retain infectivity.

Atherosclerosis is considered to be a chronic inflammatory disease that can lead to severe clinically important cardiovascular events. miR-150 is a small noncoding RNA that significantly enhances inflammatory responses by upregulating endothelial cell proliferation and migration, as well as intravascular environmental homeostasis. However, the exact role of miR-150 in atherosclerosis remains unknown. Here, we investigated the effect of miR-150 deficiency on atherosclerosis development. Using double-knockout (miR-150(−/−) and ApoE(−/−)) mice, we measured atherosclerotic lesion size and stability. Meanwhile, we conducted in vivo bone marrow transplantation to identify cellular-level components of the inflammatory response. Compared with mice deficient only in ApoE, the double-knockout mice had significantly smaller atherosclerotic lesions and displayed an attenuated inflammatory response. Moreover, miR-150 ablation promoted plaque stabilization via increases in smooth muscle cell and collagen content and decreased macrophage infiltration and lipid accumulation. The in vitro experiments indicated that an inflammatory response with miR-150 deficiency in atherosclerosis results directly from upregulated expression of the cytoskeletal protein, PDZ and LIM domain 1 (PDLIM1), in macrophages. More importantly, the decreases in phosphorylated p65 expression and inflammatory cytokine secretion induced by miR-150 ablation were reversed by PDLIM1 knockdown. These findings suggest that miR-150 is a promising target for the management of atherosclerosis.

Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury.SIGNIFICANCE STATEMENTStroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting the TRAF6-Rac1 axis may provide a novel therapeutic strategy for stroke recovery.

Background & Aims The hepatic injury caused by ischemia/reperfusion (I/R) insult is predominantly determined by the complex interplay of sterile inflammation and liver cell death. Caspase recruitment domain family member 6 (CARD6) was initially shown to play important roles in NF-κB activation. In our preliminary studies, CARD6 downregulation was closely related to hepatic I/R injury in liver transplantation patients and mouse models. Thus, we hypothesized that CARD6 protects against hepatic I/R injury and investigated the underlying molecular mechanisms. Methods A partial hepatic I/R operation was performed in hepatocyte-specific Card6 knockout mice (HKO), Card6 transgenic mice with CARD6 overexpression specifically in hepatocytes (HTG), and the corresponding control mice. Hepatic histology, serum aminotransferases, inflammatory cytokines/chemokines, cell death, and inflammatory signaling were examined to assess liver damage. The molecular mechanisms of CARD6 function were explored in vivo and in vitro. Results Liver injury was alleviated in Card6-HTG mice compared with control mice as shown by decreased cell death, lower serum aminotransferase levels, and reduced inflammation and infiltration, whereas Card6-HKO mice had the opposite phenotype. Mechanistically, phosphorylation of ASK1 and its downstream effectors JNK and p38 were increased in the livers of Card6-HKO mice but repressed in those of Card6-HTG mice. Furthermore, ASK1 knockdown normalized the effect of CARD6 deficiency on the activation of NF-κB, JNK and p38, while ASK1 overexpression abrogated the suppressive effect of CARD6. CARD6 was also shown to interact with ASK1. Mutant CARD6 that lacked the ability to interact with ASK1 could not inhibit ASK1 and failed to protect against hepatic I/R injury. Conclusions CARD6 is a novel protective factor against hepatic I/R injury that suppresses inflammation and liver cell death by inhibiting the ASK1 signaling pathway. Lay summary The protein CARD6 plays an important role during the process of liver blood flow restriction (ischemia) and restoration (reperfusion). By suppressing the activity of ASK1, CARD6 can protect against hepatocyte injury. Targeting CARD6 is a potential strategy for prevention and treatment of ischemia/reperfusion injury.

Durable influenza protection Vaccines are indispensable for the control and prevention of influenza, but there are several challenges to efficacy. Some individuals respond poorly to vaccination, and virus variation makes targeting optimal antigens difficult. Broadly neutralizing antibodies are one solution, but they have their own pitfalls, including limited cross-reactivity to both influenza A and B strains and the need for repeated injections. Now, Laursen et al. have developed multidomain antibodies with breadth and potency. Administered intranasally to mice with an adeno-associated virus vector, the antibodies provided durable and continuous protection from a panoply of influenza strains. Science , this issue p. 598

Hepatic ischemia-reperfusion (IR) injury is a common clinical issue lacking effective therapy and validated pharmacological targets. Here, using integrative 'omics' analysis, we identified an arachidonate 12-lipoxygenase (ALOX12)-12-hydroxyeicosatetraenoic acid (12-HETE)-G-protein-coupled receptor 31 (GPR31) signaling axis as a key determinant of the hepatic IR process. We found that ALOX12 was markedly upregulated in hepatocytes during ischemia to promote 12-HETE accumulation and that 12-HETE then directly binds to GPR31, triggering an inflammatory response that exacerbates liver damage. Notably, blocking 12-HETE production inhibits IR-induced liver dysfunction, inflammation and cell death in mice and pigs. Furthermore, we established a nonhuman primate hepatic IR model that closely recapitulates clinical liver dysfunction following liver resection. Most strikingly, blocking 12-HETE accumulation effectively attenuated all pathologies of hepatic IR in this model. Collectively, this study has revealed previously uncharacterized metabolic reprogramming involving an ALOX12-12-HETE-GPR31 axis that functionally determines hepatic IR procession. We have also provided proof of concept that blocking 12-HETE production is a promising strategy for preventing and treating IR-induced liver damage.

Several reports have described the presence of antibodies against Alzheimer’s disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1705-y) contains supplementary material, which is available to authorized users.

Background: Cardiac hypertrophy and its resultant heart failure are among the most common causes of mortality worldwide. Abnormal protein degradation, especially the impaired lysosomal degradation of large organelles and membrane proteins, is involved in the progression of cardiac hypertrophy. However, the underlying mechanisms have not been fully elucidated. Methods: We investigated cardiac transmembrane BAX inhibitor motif containing 1 (TMBIM1) mRNA and protein expression levels in samples from patients with heart failure and mice with aortic banding (AB)–induced cardiac hypertrophy. We generated cardiac-specific Tmbim1 knockout mice and cardiac-specific Tmbim1 -overexpressing transgenic mice and then challenged them with AB surgery. We used microarray, confocal image, and coimmunoprecipitation analyses to identify the downstream targets of TMBIM1 in cardiac hypertrophy. Tmbim1 / Tlr4 double-knockout mice were generated to investigate whether the effects of TMBIM1 on cardiac hypertrophy were Toll-like receptor 4 (TLR4) dependent. Finally, lentivirus-mediated TMBIM1 overexpression in a monkey AB model was performed to evaluate the therapeutic potential of TMBIM1. Results: TMBIM1 expression was significantly downregulated on hypertrophic stimuli in both human and mice heart samples. Silencing cardiac Tmbim1 aggravated AB-induced cardiac hypertrophy. This effect was blunted by Tmbim1 overexpression. Transcriptome profiling revealed that the TLR4 signaling pathway was disrupted dramatically by manipulation of Tmbim1 . The effects of TMBIM1 on cardiac hypertrophy were shown to be dependent on TLR4 in double-knockout mice. Fluorescent staining indicated that TMBIM1 promoted the lysosome-mediated degradation of activated TLR4. Coimmunoprecipitation assays confirmed that TMBIM1 directly interacted with tumor susceptibility gene 101 via a PTAP motif and accelerated the formation of multivesicular bodies that delivered TLR4 to the lysosomes. Finally, lentivirus-mediated TMBIM1 overexpression reversed AB-induced cardiac hypertrophy in monkeys. Conclusions: TMBIM1 protects against pathological cardiac hypertrophy through promoting the lysosomal degradation of activated TLR4. Our findings reveal the central role of TMBIM1 as a multivesicular body regulator in the progression of pathological cardiac hypertrophy, as well as the role of vesicle trafficking in signaling regulation during cardiac hypertrophy. Moreover, targeting TMBIM1 could be a novel therapeutic strategy for treating cardiac hypertrophy and heart failure.

Background & Aims Hepatic ischaemia–reperfusion (I/R) injury is characterised by severe inflammation and extensive cell death. Multiple signalling pathways, including NF-κB and mitogen-activated protein kinase (MAPK)/c-Jun NH2-terminal kinase (JNK), have important roles in this process. Identifying the unknown critical regulators of these signalling pathways could provide potential targets for therapeutic application. Dual-specificity phosphatase 14 (DUSP14) acts as a negative regulator of NF-κB signalling. However, its function in hepatic I/R injury is unknown. Methods Hepatocyte-specific Dusp14 knockout (HKO) and transgenic (TG) mice were subjected to hepatic I/R surgery to examine Dusp14 function in vivo . Primary hepatocytes isolated from Dusp14 -HKO and Dusp14 -TG mice were cultured and subjected to hypoxia/reoxygenation insult in vitro . Inflammatory cytokine production was measured using quantitative reverse transcription PCR and ELISA. Liver damage was analysed using histopathology. Co-immunoprecipitation and pull-down assays followed by Western blot were performed to detect Dusp14 and transforming growth factor (Tgf)-β-activated kinase 1 (Tak1) interactions. Results Dusp14 was significantly downregulated in liver tissues from liver transplantation patients and mice subjected to hepatic I/R surgery. Dusp14 -HKO and Dusp14 -TG mouse models demonstrated that Dusp14 reduced cell death, ameliorated inflammation, and promoted hepatocyte proliferation and/or regeneration. Dusp14 also suppressed NF-κB and MAPK signalling via a physical interaction with Tak1, leading to its subsequent inhibition. Tak1 inhibition by 5Z-7-ox abolished Dusp14 function in vivo , indicating that TAK1 is required for Dusp14 function in hepatic I/R injury. Finally, mutant Dusp14 lost the ability to bind Tak1 and failed to protect against hepatic I/R injury. Conclusions Dusp14 is a protective factor in hepatic I/R injury, and the Dusp14-Tak1-Jnk1/2 regulatory axis is important for the pathogenesis of hepatic I/R injury. Modulation of this axis could be a novel therapeutic strategy to prevent or interfere with this pathological process. Lay summary Reductions in the level of the protein Dusp14 are closely associated with liver damage caused by inadequate blood supply followed by restoration of blood flow to the liver. Dusp14 protects against liver damage by suppressing the activity of Tak1. Targeting Dusp14 could be a strategy for prevention and treatment of this disease.

Background Vinexin β is a novel adaptor protein that regulates cellular adhesion, cytoskeletal reorganization, signal transduction, and transcription; however, the exact role that vinexin β plays in atherosclerosis remains unknown. Methods and Results Immunoblot analysis showed that vinexin β expression is upregulated in the atherosclerotic lesions of both patients with coronary heart disease and hyperlipemic apolipoprotein E–deficient mice and is primarily localized in macrophages indicated by immunofluorescence staining. The high‐fat diet–induced double‐knockout mice exhibited lower aortic plaque burdens than apolipoprotein E −/− littermates and decreased macrophage content. Vinexin β deficiency improved plaque stability by attenuating lipid accumulation and increasing smooth muscle cell content and collagen. Moreover, the bone marrow transplant experiment demonstrated that vinexin β deficiency exerts atheroprotective effects in hematopoietic cells. Consistent with these changes, the mRNA expression of proinflammatory cytokines were downregulated in vinexin β −/− apolipoprotein E −/− mice, whereas the anti‐inflammatory M2 macrophage markers were upregulated. The immunohistochemical staining and in vitro experiments showed that deficiency of vinexin β inhibited the accumulation of monocytes and the migration of macrophages induced by tumor necrosis factor α–stimulated human umbilical vein endothelial cells as well as macrophage proliferation. Finally, the inhibitory effects exerted by vinexin β deficiency on foam cell formation, nuclear factor κB activation, and inflammatory cytokine expression were largely reversed by constitutive Akt activation, whereas the increased expression of the nuclear factor κB subset promoted by adenoviral vinexin β was dramatically suppressed by inhibition of AKT . Conclusions Vinexin β deficiency attenuates atherogenesis primarily by suppressing vascular inflammation and inactivating Akt–nuclear factor κB signaling. Our data suggest that vinexin β could be a therapeutic target for the treatment of atherosclerosis.

Background Atherosclerosis is a chronic disease that is closely related to inflammation and macrophage apoptosis, which leads to secondary necrosis and proinflammatory responses in advanced lesions. Caspase‐activated DN ase ( CAD ) is a double‐strand specific endonuclease that leads to the subsequent degradation of chromosome DNA during apoptosis. However, whether CAD is involved in the progression of atherosclerosis remains elusive. Methods and Results CAD −/− ApoE −/− and ApoE −/− littermates were fed a high‐fat diet for 28 weeks to develop atherosclerosis. Human specimens were collected from coronary heart disease ( CHD ) patients who were not suitable for transplantation. CAD expression was increased in the atheromatous lesions of CHD patients and high‐fat diet‐treated ApoE‐deficient mice. Further investigation demonstrated that CAD deficiency inhibited high‐fat diet‐induced atherosclerosis, as evidenced by decreased atherosclerotic plaques, inhibited inflammatory response, and macrophage apoptosis, as well as enhanced stability of plaques in CAD −/− ApoE −/− mice compared to the ApoE −/− controls. Bone marrow transplantation verified the effect of CAD on atherosclerosis from macrophages. Mechanically, the decrease in the phosphorylated levels of mitogen‐activated protein kinase (MAPK) kinase/extracellular signal‐regulated kinase 1 and 2 ( MEK ‐ ERK 1/2) that resulted from CAD knockout and the activation of nuclear factor kappa B signaling mediated by CAD stimulation that was suppressed by inhibiting ERK 1/2 phosphorylation revealed the potential association between the role of CAD in atherosclerosis and the MAPK signaling pathway. Conclusions In conclusion, CAD deficiency protects against atherosclerosis through inhibiting inflammation and macrophage apoptosis, which is partially through inactivation of the MEK ‐ ERK 1/2 signaling pathway. This finding provides a promising therapeutic target for treating atherosclerosis.

Oncostatin M (OSM) is a secreted cytokine mainly involved in chronic inflammatory and cardiovascular diseases through binding to OSM receptor β (OSMR-β). Recent studies demonstrated that the presence of OSM contributed to the destabilization of atherosclerotic plaque. To investigate whether OSMR-β deficiency affects atherosclerosis, male OSMR-β−/−ApoE−/− mice were generated and utilized. Here we observed that OSMR-β expression was remarkably upregulated in both human and mouse atherosclerotic lesions, which were mainly located in macrophages. We found that OSMR-β deficiency significantly ameliorated atherosclerotic burden in aorta and aortic root relative to ApoE-deficient littermates and enhanced the stability of atherosclerotic plaques by increasing collagen and smooth muscle cell content, while decreasing macrophage infiltration and lipid accumulation. Moreover, bone marrow transplantation of OSMR-β−/− hematopoietic cells to atherosclerosis-prone mice displayed a consistent phenotype. Additionally, we observed a relatively reduced level of JAK2 and signal transducer and activator of transcription (STAT)3 in vivo and under Ox-LDL stimulation in vitro. Our findings suggest that OSMR-β deficiency in macrophages improved high-fat diet-induced atherogenesis and plaque vulnerability. Mech­anistically, the protective effect of OSMR-β deficiency on atherosclerosis may be partially attributed to the inhibition of the JAK2/STAT3 activation in macrophages, whereas OSM stimulation can activate the signaling pathway.

Although pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide, our understanding of the molecular mechanisms underlying this disease is still poor. Here, we demonstrate that suppressor of IKKɛ (SIKE), a negative regulator of the interferon pathway, attenuates pathological cardiac hypertrophy in rodents and non-human primates in a TANK-binding kinase 1 (TBK1)/AKT-dependent manner. Sike-deficient mice develop cardiac hypertrophy and heart failure, whereas Sike-overexpressing transgenic (Sike-TG) mice are protected from hypertrophic stimuli. Mechanistically, SIKE directly interacts with TBK1 to inhibit the TBK1-AKT signalling pathway, thereby achieving its anti-hypertrophic action. The suppression of cardiac remodelling by SIKE is further validated in rats and monkeys. Collectively, these findings identify SIKE as a negative regulator of cardiac remodelling in multiple animal species due to its inhibitory regulation of the TBK1/AKT axis, suggesting that SIKE may represent a therapeutic target for the treatment of cardiac hypertrophy and heart failure., Identifying pathways that cause pathological cardiac hypertrophy holds great therapeutic potential. Here the authors discover one such pathway and show that SIKE, an inhibitor of interferon signalling, prevents pathological but not physiological cardiac hypertrophy by interacting with TBK1 and modulating the TBK1/AKT signalling in rodents and monkeys.

Background Angiotensin‐converting enzyme 3 ( ACE 3) is a recently defined homolog of ACE . However, the pathophysiological function of ACE 3 is largely unknown. Here, we aim to explore the role of ACE 3 in pathological cardiac hypertrophy. Methods and Results Neonatal rat cardiomyocytes ( NRCM s) with gain and loss of function of ACE 3 and mice with global knockout or cardiac‐specific overexpression of ACE 3 were used in this study. In cultured cardiomyocytes, ACE 3 conferred protection against angiotensin II (Ang II )‐induced hypertrophic growth. Cardiac hypertrophy in mice was induced by aortic banding ( AB ) and the extent of hypertrophy was analyzed through echocardiographic, pathological, and molecular analyses. Our data demonstrated that ACE 3‐deficient mice exhibited more pronounced cardiac hypertrophy and fibrosis and a strong decrease in cardiac contractile function, conversely, cardiac‐specific ACE 3‐overexpressing mice displayed an attenuated hypertrophic phenotype, compared with control mice, respectively. Analyses of the underlying molecular mechanism revealed that ACE 3‐mediated protection against cardiac hypertrophy by suppressing the activation of mitogen‐activated protein kinase kinase ( MEK )‐regulated extracellular signal‐regulated protein kinase ( ERK 1/2) signaling, which was further evidenced by the observation that inhibition of the MEK ‐ ERK 1/2 signaling by U0126 rescued the exacerbated hypertrophic phenotype in ACE 3‐deficient mice. Conclusions Our comprehensive analyses suggest that ACE 3 inhibits pressure overload‐induced cardiac hypertrophy by blocking the MEK ‐ ERK 1/2 signaling pathway.

Aggregation of the hyperphosphorylated protein tau into neurofibrillary tangles and neuropil threads is a hallmark of Alzheimer disease (AD). Identification and characterization of the epitopes recognized by anti-tau antibodies might shed light on the molecular mechanisms of AD pathogenesis. Here we report on the biochemical and structural characterization of a tau-specific monoclonal antibody CBTAU-24.1, which was isolated from the human memory B cell repertoire. Immunohistochemical staining with CBTAU-24.1 specifically detects pathological tau structures in AD brain samples. The crystal structure of CBTAU-24.1 Fab with a phosphorylated tau peptide revealed recognition of a unique epitope (Ser235-Leu243) in the tau proline-rich domain. Interestingly, the antibody can bind tau regardless of phosphorylation state of its epitope region and also recognizes both monomeric and paired helical filament tau irrespective of phosphorylation status. This human anti-tau antibody and its unique epitope may aid in development of diagnostics and/or therapeutic AD strategies.

Nat. Med. 23, 439–449 (2017); published online 20 February 2017; corrected after print 26 July 2017 In the version of this article initially published, the authors inadvertently left out information in the Online Methods section regarding a second injection of AAV8-CFLAR(S1) 7 weeks after the first injection in the monkey experiments to ensure stable expression of CFLAR(S1) in the livers of the monkeys that received the injections.