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Complications affecting the lung are hallmarks of severe coronavirus disease 2019 (COVID-19). While there is evidence for autoimmunity in severe COVID-19, the exact mechanisms remain unknown. Here, we established a prospective observational cohort to study lung specific autoantibodies (auto-Abs). Incubation of plasma from severe COVID-19 patients with healthy human lung tissue revealed the presence of IgA antibodies binding to surfactant-producing pneumocytes. Enzyme-linked immunosorbent assays (ELISA) and protein pull-downs using porcine surfactant confirmed the presence of auto-Abs binding to surfactant proteins in severe COVID-19 patients. Mass spectrometry and ELISAs with recombinant proteins identified IgA auto-Abs that target human surfactant proteins B and C. In line with these findings, lungs of deceased COVID-19 patients showed reduced pulmonary surfactant. Our data suggest that IgA-driven autoimmunity against surfactant may result in disease progression of COVID-19.

It is not well understood why diabetic individuals are more prone to develop severe COVID-19. To this, we here established a human kidney organoid model promoting early hallmarks of diabetic kidney disease development. Upon SARS-CoV-2 infection, diabetic-like kidney organoids exhibited higher viral loads compared with their control counterparts. Genetic deletion of the angiotensin-converting enzyme 2 (ACE2) in kidney organoids under control or diabetic-like conditions prevented viral detection. Moreover, cells isolated from kidney biopsies from diabetic patients exhibited altered mitochondrial respiration and enhanced glycolysis, resulting in higher SARS-CoV-2 infections compared with non-diabetic cells. Conversely, the exposure of patient cells to dichloroacetate (DCA), an inhibitor of aerobic glycolysis, resulted in reduced SARS-CoV-2 infections. Our results provide insights into the identification of diabetic-induced metabolic programming in the kidney as a critical event increasing SARS-CoV-2 infection susceptibility, opening the door to the identification of new interventions in COVID-19 pathogenesis targeting energy metabolism.

Simple Summary Diffusely infiltrating gliomas are frequent brain tumors with variable prognosis. In addition to the blood pigment’s role of oxygen transportation, the metabolic pathway synthesizing heme has been shown to play a role in the biochemistry of various tumors. In this study we thus investigated the impact of heme biosynthesis factors mRNA expression on the survival in glioma patients and observed a progressive decrease in survival time with increasing mRNA expression signature. This association was present for overall as well as progression-free survival and remained statistically significant after correction for established prognostic factors such as patient age and tumor grade. Abstract Diffusely infiltrating gliomas are characterized by a variable clinical course, and thus novel prognostic biomarkers are needed. The heme biosynthesis cycle constitutes a fundamental metabolic pathway and might play a crucial role in glioma biology. The aim of this study was thus to investigate the role of the heme biosynthesis mRNA expression signature on prognosis in a large glioma patient cohort. Glioma patients with available sequencing data on heme biosynthesis expression were retrieved from The Cancer Genome Atlas (TCGA). In each patient, the heme biosynthesis mRNA expression signature was calculated and categorized into low, medium, and high expression subgroups. Differences in progression-free and overall survival between these subgroups were investigated including a multivariate analysis correcting for WHO grade, tumor subtype, and patient age and sex. In a total of 693 patients, progression-free and overall survival showed a strictly monotonical decrease with increasing mRNA expression signature subgroups. In detail, median overall survival was 134.2 months in the low, 79.9 months in the intermediate, and 16.5 months in the high mRNA expression signature subgroups, respectively. The impact of mRNA expression signature on progression-free and overall survival was independent of the other analyzed prognostic factors. Our data indicate that the heme biosynthesis mRNA expression signature might serve as an additional novel prognostic marker in patients with diffusely infiltrating gliomas to optimize postoperative management.

Summary The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIP, HOIL-1L, and SHARPIN, promotes tumor necrosis factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT201A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production, and liver damage markers. Cells derived from Hoil-1lT201A;R208A/T201A;R208A mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that HOIL-1L NZF cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo., Graphical abstract, Highlights • An RBR-type E3 ligase HOIL-1L decodes linear ubiquitin chains via the NZF domain • HOIL-1L NZF is essential for proper responses to LPS and TNF-induced shock in mice • Intact HOIL-1L NZF is required for activating the TNF-induced NF-kB pathway • HOIL-1L NZF cooperates with SHARPIN to control inflammation in mice, Biological sciences; Molecular biology; Immune response

The dual anticytokine and antiviral actions of baricitinib reduce SARS-CoV-2 infectivity in organoids and morbidity in people., Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 and improves the pathologies of cardiovascular disease and acute lung injury. To address whether the carboxypeptidase enzymatic activity of ACE2 is protective against COVID-19, we investigated the effects of B38-CAP, an ACE2-like enzyme, on SARS-CoV-2-induced lung injury. Expression of ACE2 protein was significantly downregulated in the lungs of SARS-CoV-2-infected hamsters. Recombinant S1 domain or receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein also directly downregulated ACE2 expression and elevated Ang II levels and considerably worsened acid-induced lung injury in hamsters. Treatment with B38-CAP downregulated Spike RBD-induced high Ang II levels, severe inflammation and pulmonary edema through its ACE2-like enzymatic activity. Consistently, elevated cytokine mRNA levels and impaired lung functions were improved by B38-CAP treatment. Moreover, in SARS-CoV-2-infected humanized ACE2 transgenic mice, B38-CAP significantly improved the pathologies of lung injury, alleviated the cytokine storms and downregulated viral RNA levels. These results provide the first experimental in vivo evidence that increasing ACE2-like enzymatic activity is a potential and powerful therapeutic strategy for lung pathologies in COVID-19.

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced Kras

Introduction: In contrast to other adult tissues myocardium cannot be sufficiently regenerated following significant myocardial infarction (MI). Efficient cardiac regeneration was demonstrated in neonatal mouse myocardial injury models. Similar observations were reported in other neonatal mammals including newborn human babies suffering from MI. The mechanisms of neonatal mammalian cardiac regeneration remain unclear. Here we show the crucial role of IGF1R which was unraveled in a time-course transcriptome analysis of neonatal mouse hearts. Methods: IGF1R was specifically knocked-down (KD) in cardiomyocytes of wildtype postnatal day one (P1) mice using adeno-associated virus (rAAV9) delivered shRNAmirs. KD of Renilla served as control (REN-CTRL). 5x10 13 viral genomes per kg bodyweight were injected intrathoracally. Three batches of rAAV9 with different shRNAmirs were used for the IGF1R-KD groups. Viral transduction was confirmed by bioluminescence imaging on luciferase containing rAAV9 copies and by immunofluorescence staining of rAAV9 delivered GFP reporter. KD efficiency was confirmed in vitro using a retrovirus system and in vivo. On P2 mice underwent either left anterior descending artery (LAD) ligation for induction of MI or SHAM surgery. Cardiac function was subsequently assessed by echocardiography 1 day post injury (dpi) and 21 dpi. Thereafter, hearts were harvested and left ventricular fibrosis was analyzed histologically. Results: LAD ligation resulted in significant MI in both, IGF1R-KD and REN-CTRL, LAD groups as proven by a markedly reduced ejection fraction (EF) 1 dpi. Importantly, 21 dpi IGF1R-KD and REN-CTRL SHAM groups displayed normal cardiac function proving no effect on neonatal cardiac growth and development of only KD of IGF1R without LAD ligation. In contrast, LAD ligation IGF1R-KD mice presented significantly reduced EF 21dpi compared to the other 3 groups. Histological analysis revealed significant fibrosis in the IGF1R-KD LAD hearts compared to the other 3 groups. Conclusions: Whereas IGF1R-KD or control rAAV9 does not alter physiological cardiac development, KD of IGF1R markedly impairs neonatal cardiac regeneration in neonatal mice after MI suggesting a crucial role in neonatal cardiac regeneration.

Background Cardiac remodeling and subsequent heart failure remain critical issues after myocardial infarction (MI). Complete cardiac regeneration was shown recently in a neonatal mouse model of MI. This cardiac regenerative potential is limited to the first few postnatal days and its decline parallels with the maturation of the adaptive immune system. Purpose Herein, we hypothesized that the T-cell maturation status critically impacts the myocardial healing outcomes in neonates and contributes to the shift from regenerative to scarring phenotype observed shortly after birth. Methods The post-MI immune responses were characterized in postnatal day one (P1, regenerative) compared to seven-day old (P7, scarring) mice subjected to permanent left anterior descending artery (LAD) ligation. The myocardial leukocyte infiltrate was phenotyped by flow cytometry at 36 hours and five days after LAD ligation. Next, we studied neonatal post-MI repair in lymphocyte-deficient Rag2 knock-out (KO) mice subjected to LAD ligation. Moreover, we adoptively transferred syngeneic splenic Thy 1.1+ T-cells obtained from adult donors into P1 versus P7 recipients and then assessed their impact on post-MI healing. Results LAD ligation induced a robust early inflammatory response (36h post-MI) in both age groups. The in situ inflammation was, nevertheless, rapidly resolved in P1-, but not in P7-infarcted animals. The distinct age groups showed a similar profile of cardiac myeloid cell infiltration but showed remarkable differences in the lymphoid compartment. P1-infarcted mice showed an early recruitment of γδT-cells, whereas P7-infarcted mice exhibited a prominent infiltration of αβT-cells. Of note, neonatal cardiac regeneration was not altered in neonatal lymphocyte-deficient (Rag2 KO) animals. However, the adoptive transfer of adult T-cells had several consequences in neonatal and one week old mice subjected to ischemic injury. P1-infarcted mice transferred with adult T-cells showed an adult-like healing phenotype, marked by an irreversible cardiac functional impairment (assessed by echocardiography) and increased fibrosis. This is in sharp contrast to the regenerative phenotype typically observed in untreated age-matched controls. Furthermore, P7-infarcted mice transferred with adult T-cell showed significantly decreased survival rate after LAD ligation. Conclusion Neonatal hearts demonstrate rapid clearance of the ischemia-induced leukocyte infiltration, further reflecting the known fact of fast cardiac regeneration in newborn rodents. Of note, the adoptive transfer of adult T-cells into neonate recipients partially blocked cardiac regeneration and promoted an irreversible functional impairment. These data indicate that the cardiac repair process, and its related “regeneration vs. scarring” dichotomy, is critically impacted by the T-cell development status. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Innsbruck Medical University, Medizinischer Forschungsfonds Tirol

c‐Jun N‐terminal kinases (JNKs) are constitutively activated in mammalian brains and are indispensable for their development and neural functions. MKK7 is an upstream activator of all JNKs. However, whether the common JNK signaling pathway regulates the brain's control of social behavior remains unclear. Here, we show that female mice in which Mkk7 is deleted specifically in mature neurons (Mkk7flox/floxSyn‐Cre mice) give birth to a normal number of pups but fail to raise them due to a defect in pup retrieval. To explore the mechanism underlying this abnormality, we performed comprehensive behavioral tests. Mkk7flox/floxSyn‐Cre mice showed normal locomotor functions and cognitive ability but exhibited depression‐like behavior. cDNA microarray analysis of mutant brain revealed an altered gene expression pattern. Quantitative RT‐PCR analysis demonstrated that mRNA expression levels of genes related to neural signaling pathways and a calcium channel were significantly different from controls. In addition, loss of neural MKK7 had unexpected regulatory effects on gene expression patterns in oligodendrocytes. These findings indicate that MKK7 has an important role in regulating the gene expression patterns responsible for promoting normal social behavior and staving off depression., c‐Jun N‐terminal kinase (JNK) signaling has well established, important roles in mammalian brain development and neural functions. However, whether the mitogen‐activated protein kinase kinase 7 (MKK7), a specific upstream activator of JNK, regulates social behavior remains unclear. In this study, we demonstrate that the MKK7‐JNK pathway in mature neurons regulates mouse mental status and plays a critical role in parental behavior.

BackgroundWhile the pathogenesis of coronavirus disease 2019 (COVID-19) is becoming increasingly clear, there is little data on IgA response, the first line of bronchial immune defense.ObjectiveTo determine, whether COVID-19 is associated with a vigorous total IgA response and whether IgA autoantibodies are associated with complications of severe illness. Since thrombotic events are frequent in severe COVID-19 and resemble hypercoagulation of antiphospholipid syndrome (APS), our approach focused on antiphospholipid antibodies (aPL).Materials and methodsIn this retrospective cohort study we compared clinical data and aPL from 64 patients with COVID-19 from three independent centers (two in Switzerland, one in Liechtenstein). Samples were collected from April 9, 2020 to May 1, 2020. Total IgA and aPL were measured with FDA-approved commercially available clinical diagnostic kits.ResultsClinical records of the 64 patients with COVID-19 were reviewed and divided into a cohort with mild illness (mCOVID, n=26 [41%]), a discovery cohort with severe illness (sdCOVD, n=14 [22%]) and a confirmation cohort with severe illness (scCOVID, n=24 [38%]). Severe illness was significantly associated with increased total IgA (sdCOVID, P=0.01; scCOVID, PPP=0.003; scCOVID, PPConclusionsHigher total IgA and IgA-aPL were consistently associated with severe illness. These novel data strongly suggest that a vigorous antiviral IgA-response triggered in the bronchial mucosa induces systemic autoimmunity.

BACKGROUND AND AIMS Mitogen-activated protein kinase kinase (MKK) 7 and MKK4 are upstream activators of c-Jun NH2 -terminal kinases (JNKs) and have been shown to be required for the early development of the liver. Although it has been suggested that MKK7 might be involved in the regulation of hepatocyte proliferation, the functional role of MKK7 in the liver has remained unclear. APPROACH AND RESULTS Here, we examined phenotypic alterations in liver-specific or hepatocyte/hematopoietic cell-specific MKK7 knockout (KO) mice, which were generated by crossing MKK7LoxP/LoxP with albumin-cyclization recombination (Alb-Cre) or myxovirus resistance protein 1-Cre mice, respectively. The livers of Alb-Cre-/+ MKK7LoxP/LoxP mice developed without discernible tissue disorganization. MKK7 KO mice responded normally to liver injuries incurred by partial hepatectomy or injection of CCl4 . However, tissue repair following CCl4 -induced injury was delayed in MKK7 KO mice compared with that of control mice. Furthermore, after repeated injections of CCl4 for 8 weeks, the liver in MKK7 KO mice showed intense fibrosis with increased protractive hepatocyte proliferation, suggesting that MKK7 deficiency might affect regenerative responses of hepatocytes in the altered tissue microenvironment. MKK7 KO hepatocytes demonstrated normal proliferative activity when cultured in monolayers. However, MKK7 KO significantly suppressed branching morphogenesis of hepatocyte aggregates within a collagen gel matrix. Microarray analyses revealed that suppression of branching morphogenesis in MKK7 KO hepatocytes was associated with a reduction in mRNA expression of transgelin, glioma pathogenesis related 2, and plasminogen activator urokinase-type (Plau); and forced expression of these genes in MKK7 KO hepatocytes partially recovered the attenuated morphogenesis. Furthermore, hepatocyte-specific overexpression of Plau rescued the impaired tissue repair of MKK7 KO mice following CCl4 -induced injury. CONCLUSIONS MKK7 is dispensable for the regenerative proliferation of hepatocytes but plays important roles in repair processes following parenchymal destruction, possibly through modulation of hepatocyte-extracellular matrix interactions.

RANKL and RANK are expressed in different cell types and tissues throughout the body. They were originally described for their essential roles in bone remodeling and the immune system but have subsequently been shown to provide essential signals from regulating mammary gland homeostasis during pregnancy to modulating tumorigenesis. The success of RANKL/RANK research serves as a paragon for translational research from the laboratory to the bedside. The case in point has been the development of Denosumab, a RANKL-blocking monoclonal antibody which has already helped millions of patients suffering from post-menopausal osteoporosis and skeletal related events in cancer. Here we will provide an overview of the pathway from its origins to its clinical relevance in disease, with a special focus on emerging evidence demonstrating the therapeutic value of targeting the RANKL/RANK/OPG axis not only in breast cancer but also as an addition to the cancer immunotherapy arsenal.

The role of angiotensin converting enzyme 2 has expanded from regulating the renin angiotensin system to regulating intestinal amino acid homeostasis and the gut microbiome. Recently, angiotensin converting enzyme 2 was identified as a primary receptor for severe acute respiratory syndrome coronaviruses 1 and 2 being expressed in multiple tissues including the luminal surface of the gut. In this brief perspective, we examine the role of angiotensin converting enzyme 2 as the receptor for severe acute respiratory syndrome coronavirus 2 and the impact of coronavirus disease 19 infection on the gut microbiome and on the gut epithelium.

How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1α through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.

Bone tissues have trabecular bone with a high bone turnover and cortical bone with a low turnover. The mechanisms by which the turnover rate of these bone tissues is determined remain unclear. Osteocytes secrete sclerostin, a Wnt/β-catenin signaling antagonist, and inhibit bone formation. We found that sclerostin expression in cortical bone is more marked than in trabecular bone in Sost reporter mice. Leukemia inhibitory factor (LIF) secreted from osteoclasts reportedly suppressed sclerostin expression and promoted bone formation. Here, we report that osteoclasts downregulate sclerostin expression in trabecular bone and promote bone turnover. Treatment of C57BL/6 mice with an anti-RANKL antibody eliminated the number of osteoclasts and LIF-positive cells in trabecular bone. The number of sclerostin-positive cells was increased in trabecular bone, while the number of β-catenin-positive cells and bone formation were decreased in trabecular bone. Besides, Tnfsf11 heterozygous (Rankl+/−) mice exhibited a decreased number of LIF-positive cells and increased number of sclerostin-positive cells in trabecular bone. Rankl+/− mice exhibited a decreased number of β-catenin-positive cells and reduced bone formation in trabecular bone. Furthermore, in cultured osteoclasts, RANKL stimulation increased Lif mRNA expression, suggesting that RANKL signal increased LIF expression. In conclusion, osteoclasts downregulate sclerostin expression and promote trabecular bone turnover.

Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) and Golgi apparatus of antibody-producing cells, reduced antibody secretion, and aberrant IgG N-glycosylation. The data uncover a key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans., Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

A novel infectious disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was detected in Wuhan, China, in December 2019. The disease (COVID-19) spread rapidly, reaching epidemic proportions in China, and has been found in 27 other countries. As of February 27, 2020, over 82,000 cases of COVID-19 were reported, with > 2800 deaths. No specific therapeutics are available, and current management includes travel restrictions, patient isolation, and supportive medical care. There are a number of pharmaceuticals already being tested [1, 2], but a better understanding of the underlying pathobiology is required. In this context, this article will briefly review the rationale for angiotensin-converting enzyme 2 (ACE2) receptor as a specific target.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1–7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure., The enzyme ACE2 is involved in cardiac pathology and can counteract heart failure and other cardio-pulmonary diseases. Here the authors show that bacteria produce an ACE2-like enzyme that is effective in suppressing cardiac hypertrophy and fibrosis in mice.

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2.

Summary: Mitochondrial oxidative phosphorylation (OXPHOS) and substrate utilization critically regulate the function of hypothalamic proopiomelanocortin (POMC)-expressing neurons. Here, we demonstrate that inactivation of apoptosis-inducing factor (AIF) in POMC neurons mildly impairs mitochondrial respiration and decreases firing of POMC neurons in lean mice. In contrast, under diet-induced obese conditions, POMC-Cre-specific inactivation of AIF prevents obesity-induced silencing of POMC neurons, translating into improved glucose metabolism, improved leptin, and insulin sensitivity, as well as increased energy expenditure in AIFΔPOMC mice. On a cellular level, AIF deficiency improves mitochondrial morphology, facilitates the utilization of fatty acids for mitochondrial respiration, and increases reactive oxygen species (ROS) formation in POMC neurons from obese mice, ultimately leading to restored POMC firing upon HFD feeding. Collectively, partial impairment of mitochondrial function shifts substrate utilization of POMC neurons from glucose to fatty acid metabolism and restores their firing properties, resulting in improved systemic glucose and energy metabolism in obesity. : Timper et al. show that mild impairment of mitochondrial OXPHOS upon deletion of AIF or MPC1 in hypothalamic POMC-expressing neurons increases their fatty acid utilization and ROS formation, prevents obesity-induced silencing of these neurons, and improves systemic glucose metabolism in obesity. Keywords: hypothalamic POMC-neurons, apoptosis-inducing factor, AIF, mitochondrial, pyruvate carrier-1, MPC-1, mitochondrial oxidative phosphorylation, fatty acid, oxidation, reactive oxygen species, ROS, obesity

Physical exercise induces cell proliferation in the adult hippocampus in rodents. Serotonin (5-HT) and angiotensin (Ang) II are important mediators of the pro-mitotic effect of physical activity. Here, we examine precursor cells in the adult brain of mice lacking angiotensin-converting enzyme (ACE) 2, and explore the effect of an acute running stimulus on neurogenesis. ACE2 metabolizes Ang II to Ang-(1–7) and is essential for the intestinal uptake of tryptophan (Trp), the 5-HT precursor. In ACE2-deficient mice, we observed a decrease in brain 5-HT levels and no increase in the number of BrdU-positive cells following exercise. Targeting the Ang II/AT1 axis by blocking the receptor, or experimentally increasing Trp/5-HT levels in the brain of ACE2-deficient mice, did not rescue the running-induced effect. Furthermore, mice lacking the Ang-(1–7) receptor, Mas, presented a normal neurogenic response to exercise. Our results identify ACE2 as a novel factor required for exercise-dependent modulation of adult neurogenesis and essential for 5-HT metabolism.

The tumor necrosis factor (TNF) receptor RANK (TNFRSF11A) and its ligand RANKL (TNFSF11) regulate osteoclast development and bone metabolism. They also control stem cell expansion and proliferation of mammary epithelial cells via the sex hormone progesterone. As such, RANKL and RANK have been implicated in the onset of hormone-induced breast cancer. Recently, RANK/RANKL were identified as crucial regulators for BRCA1 mutation-driven breast cancer. Current prevention strategies for BRCA1 mutation carriers are associated with wide-ranging risks; therefore, the search for alternative, non-invasive strategies is of paramount importance. We summarize here the functions of the RANKL/RANK pathway in mammalian physiology and focus on its recently uncovered role in breast cancer. We propose that anti-RANKL therapy should be pursued as a preventative strategy for breast cancer.

// Moritz Horn 1 , Virginia Kroef 1 , Kira Allmeroth 1 , Nicole Schuller 5 , Stephan Miethe 1 , Martin Peifer 3, 4 , Josef M. Penninger 5 , Ulrich Elling 5 and Martin S. Denzel 1, 2 1 Max Planck Institute for Biology of Aging, Cologne D-50931, Germany 2 CECAD-Cluster of Excellence University of Cologne, Cologne D-50931, Germany 3 Center for Molecular Medicine Cologne, University of Cologne, Cologne D-50931, Germany 4 Department of Translational Genomics, Center of Integrated Oncology Cologne–Bonn, Medical Faculty University of Cologne, Cologne D-50931, Germany 5 Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna Biocenter, Vienna A-1030, Austria Correspondence to: Martin S. Denzel, email: Martin.Denzel@age.mpg.de Keywords: haploid stem cells; forward genetic screens; target identification; interaction site mapping; chemoresistance prediction Received: November 07, 2017 Accepted: January 16, 2018 Published: January 23, 2018 ABSTRACT Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis. Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca 2+ pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes. Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) frequently entails complications that bear similarities to autoimmune diseases. To date, there are little data on possible immunoglobulin (Ig) A-mediated autoimmune responses. Here, we aim to determine whether COVID-19 is associated with a vigorous total IgA response and whether IgA antibodies are associated with complications of severe illness. Since thrombotic events are frequent in severe COVID-19 and resemble hypercoagulation of antiphospholipid syndrome, our approach focused on antiphospholipid antibodies (aPL). METHODS: In this retrospective cohort study, clinical data and aPL from 64 patients with COVID-19 were compared from 3 independent tertiary hospitals (1 in Liechtenstein, 2 in Switzerland). Samples were collected from 9 April to 1 May 2020. RESULTS: Clinical records of 64 patients with COVID-19 were reviewed and divided into a cohort with mild illness (mCOVID; 41%), a discovery cohort with severe illness (sdCOVID; 22%) and a confirmation cohort with severe illness (scCOVID; 38%). Total IgA, IgG, and aPL were measured with clinical diagnostic kits. Severe illness was significantly associated with increased total IgA (sdCOVID, P = .01; scCOVID, P < .001), but not total IgG. Among aPL, both cohorts with severe illness significantly correlated with elevated anticardiolipin IgA (sdCOVID and scCOVID, P < .001), anticardiolipin IgM (sdCOVID, P = .003; scCOVID, P< .001), and anti-beta 2 glycoprotein-1 IgA (sdCOVID and scCOVID, P< .001). Systemic lupus erythematosus was excluded from all patients as a potential confounder. CONCLUSIONS: Higher total IgA and IgA-aPL were consistently associated with severe illness. These novel data strongly suggest that a vigorous antiviral IgA response, possibly triggered in the bronchial mucosa, induces systemic autoimmunity.

The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K–protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K–PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K–PKB–GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.

The apelin pathway has emerged as a critical regulator of cardiovascular homeostasis and disease. However, the exact role of pyr1-apelin-13 in angiotensin (Ang) II–mediated heart disease remains unclear. We used apelin-deficient (APLN −/y ) and apolipoprotein E knockout mice to evaluate the regulatory roles of pyr1-apelin-13. The 1-year aged APLN −/y mice developed myocardial hypertrophy and dysfunction with reduced angiotensin-converting enzyme 2 levels. Ang II infusion (1.5 mg kg −1 d −1 ) for 4 weeks potentiated oxidative stress, pathological hypertrophy, and myocardial fibrosis in young APLN −/y hearts resulting in exacerbation of cardiac dysfunction. Importantly, daily administration of 100 μg/kg pyr1-apelin-13 resulted in upregulated angiotensin-converting enzyme 2 levels, decreased superoxide production and expression of hypertrophy- and fibrosis-related genes leading to attenuated myocardial hypertrophy, fibrosis, and dysfunction in the Ang II–infused apolipoprotein E knockout mice. In addition, pyr1-apelin-13 treatment largely attenuated Ang II–induced apoptosis and ultrastructural injury in the apolipoprotein E knockout mice by activating Akt and endothelial nitric oxide synthase phosphorylation signaling. In cultured neonatal rat cardiomyocytes and cardiofibroblasts, exposure of Ang II decreased angiotensin-converting enzyme 2 protein and increased superoxide generation, cellular proliferation, and migration, which were rescued by pyr1-apelin-13, and Akt and endothelial nitric oxide synthase agonist stimulation. The increased superoxide generation and apoptosis in cultured cardiofibroblasts in response to Ang II were strikingly prevented by pyr1-apelin-13 which was partially reversed by cotreatment with the Akt inhibitor MK2206. In conclusion, pyr1-apelin-13 peptide pathway is a negative regulator of aging-mediated and Ang II–mediated adverse myocardial remodeling and dysfunction and represents a potential candidate to prevent and treat heart disease.

Ricin is one of the most feared bioweapons in the world due to its extreme toxicity and easy access. Since no antidote exists, it is of paramount importance to identify the pathways underlying ricin toxicity. Here, we demonstrate that the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase Fut9 are key regulators of ricin toxicity. Genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin via deregulated intracellular trafficking. Importantly, cells from a patient with SLC35C1 deficiency are also resistant to ricin. Mechanistically, we confirm that reduced fucosylation leads to increased sialylation of Lewis X structures and thus masking of ricin-binding sites. Inactivation of the sialyltransferase responsible for modifications of Lewis X (St3Gal4) increases the sensitivity of cells to ricin, whereas its overexpression renders cells more resistant to the toxin. Thus, we have provided unprecedented insights into an evolutionary conserved modular sugar code that can be manipulated to control ricin toxicity.

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Objective A RANKL-binding peptide, WP9QY (W9), is known to inhibit mouse osteoclastogenesis by stimulating the production of autocrine factors such as bone morphogenetic proteins (BMPs) to induce osteoblast differentiation. In the present study, we investigated whether osteoblastic differentiation is mediated by RANKL signaling. Methods The effect of W9 on the differentiation of osteoclasts and osteoblasts was examined in mouse bone-marrow cultures, and in a mouse co-culture system consisting of primary osteoblasts derived from RANKL-deficient or wild-type (WT) newborn mouse calvariae, with WT-derived bone marrow mononuclear cells. Results The addition of the W9 peptide to the WT mouse bone-marrow culture simultaneously inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation, and stimulated alkaline phosphatase (ALP)-positive osteoblastic calcified nodule formation. RANKL-deficient osteoblasts exhibited weak ALP activity compared to WT osteoblasts. W9 treatment strongly inhibited TRAP-positive osteoclast formation, and stimulated ALP-positive osteoblast differentiation in co-cultures of WT-derived osteoblasts and bone-marrow cells, in the presence of bone-resorbing factors. In contrast, W9 exerted only a weak effect on ALP-positive osteoblast differentiation in co-cultures with RANKL-deficient osteoblasts, even in the presence of the W9 peptide, parathyroid hormone, and/or BMP-2. Conclusions The W9 peptide inhibited RANKL-mediated osteoclast formation in osteoblasts. It also directly stimulated osteoblast differentiation, both via RANKL signaling-mediated autocrine factors, and alternative mechanisms.

Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell-dependent antigen sampling and B cell-dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.

Aims Elabela/Toddler/Apela (ELA) has been identified as a novel endogenous peptide ligand for APJ/Apelin receptor/Aplnr. ELA plays a crucial role in early cardiac development of zebrafish as well as in maintenance of self-renewal of human embryonic stem cells. Apelin was the first identified APJ ligand, and exerts positive inotropic heart effects and regulates the renin-angiotensin system. The aim of this study was to investigate the biological effects of ELA in the cardiovascular system. Methods and results Continuous infusion of ELA peptide significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and impaired contractility in mice. ELA treatment reduced mRNA expression levels of genes associated with heart failure and fibrosis. The cardioprotective effects of ELA were diminished in APJ knockout mice, indicating that APJ is the key receptor for ELA in the adult heart. Mechanistically, ELA downregulated angiotensin-converting enzyme (ACE) expression in the stressed hearts, whereas it showed little effects on angiotensin-converting enzyme 2 (ACE2) expression, which are distinct from the effects of Apelin. FoxM1 transcription factor, which induces ACE expression in the stressed hearts, was downregulated by ELA but not by Apelin. ELA antagonized angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Conclusion The ELA-APJ axis protects from pressure overload-induced heart failure possibly via suppression of ACE expression and pathogenic angiotensin II signalling. The different effects of ELA and Apelin on the expression of ACE and ACE2 implicate fine-tuned mechanisms for a ligand-induced APJ activation and downstream signalling.

CLP1 plays an essential role in the protein complex involved in mRNA 3'-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5' end of RNAs. From the evidence that phosphorylation of the 5' end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1K/K) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5' end of RNAs in the siRNA pathway, with no apparent involvement of NOL9.

The polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, regulates muscle wasting and atrophy. Previously, we elucidated the PABPN1-dependent proteome and found that levels of structural proteins, sarcomeric and cytoskeletal, were highly altered. We identified MURC, a plasma membrane-associated protein, to be affected by the cytoskeletal stability and suggest that MURC is a novel marker for impaired regeneration in muscles. We also studied the spatial organization of muscle structural proteins in 2D and 3D cell models with reduced PABPN1 levels (named here as shPAB). We show that dysregulation of cytoskeletal proteins in the shPab proteome is associated with a cytoskeleton lacking a polarized organization in muscle cells. We show that consequently, the cell mechanical features as well as myogenic differentiation are significantly reduced. We then show that restoring cytoskeletal stability, by actin overexpression in shPAB was beneficial for cell fusion and for the expression of sarcomeric proteins in shPAB models. We suggest that poor cytoskeleton mechanical features are caused by altered expression levels and contribute to aging-associated muscle wasting and atrophy.

Neuron-immune interaction in the dorsal root ganglia (DRG) plays a pivotal role in the neuropathic pain development after nerve injury. Sigma-1 receptor (Sig-1R) is expressed by DRG neurons but its role in neuropathic pain is not fully understood. We investigated the effect of peripheral Sig-1R on neuroinflammation in the DRG after spared (sciatic) nerve injury (SNI) in mice. Nerve injury induced a decrease in NeuN staining along with the nuclear eccentricity and ATF3 expression in the injured DRG. Sig-1R was present in all DRG neurons examined, and after SNI this receptor translocated to the periphery of the soma and the vicinity of the nucleus, especially in injured ATF3 + neurons. In WT mice, injured DRG produced the chemokine CCL2, and this was followed by massive infiltration of macrophages/monocytes, which clustered mainly around sensory neurons with translocated Sig-1R, accompanied by robust IL-6 increase and mechanical allodynia. In contrast, Sig-1R knockout (Sig-1R-KO) mice showed reduced levels of CCL2, decreased macrophage/monocyte infiltration into DRG, and less IL-6 and neuropathic mechanical allodynia after SNI. Our findings point to an important role of peripheral Sig-1R in sensory neuron-macrophage/monocyte communication in the DRG after peripheral nerve injury; thus, these receptors may contribute to the neuropathic pain phenotype, Neurofarmacología del dolor de la Universidad de Granada (CTS-109), FPU grants from the Spanish Ministry of Education, Culture and Sports., Spanish Ministry of Economy and Competitiveness (MINECO, grant SAF2016-80540-R), the Junta de Andalucía (grant CTS 109), Esteve Pharmaceuticals, European Regional Development Fund (ERDF)

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000– 5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

The tRNA ligase complex (tRNA-LC) mediates splicing of pre-tRNAs, and the Xbp1-mRNA during the unfolded protein response (UPR). The presence of a cysteine residue and two metal ions in the active site of RTCB, the catalytic subunit of the tRNA-LC, suggests that the tRNA-LC could be susceptible to oxidative inactivation in aerobic conditions. Here, we report that the tRNA-LC is sensitive to oxidation and reveal a co-evolutionary association with PYROXD1, a conserved and essential, yet uncharacterizedoxidoreductase. We show that PYROXD1 preserves the activity of the tRNA-LC in mammalian cells by converting RTCB-bound NAD(P)H into NAD(P)+, thereby sustaining pre-tRNA splicing and UPR. NAD(P)+, typically an oxidative co-enzyme, acts here as an antioxidant by abrogating the local, copper-mediated generation of reactive oxygen species that inactivate the tRNA-LC. We also show that the ability of PYROXD1 to protect the tRNA-LC against oxidation is impaired in genetic variants that cause myopathies in humans. Thus, we have established that the tRNA-LC is a redox-regulated metalloenzyme, and PYROXD1 an oxidoreductase that preserves tRNA-LC activity in aerobic environments through a novel, protecting mechanism. Whether metalloenzymes other than tRNA-LC require such mechanism to maintain their activity remains to be elucidated.

Communications biology 3(1), 342 (2020). doi:10.1038/s42003-020-1072-4, Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules., Published by Springer Nature, London

Apoptosis inducing factor (AIF) has been shown to be a major contributor to neuron loss in the immature brain after hypoxia-ischemia (HI). Indeed, mice bearing a hypomorphic mutation causing reduced AIF expression are protected against neonatal HI. To further investigate the possible molecular mechanisms of this neuroprotection, we generated an AIF knock-in mouse by introduction of a latent transgene coding for flagged AIF protein into the Rosa26 locus, followed by its conditional activation by a ubiquitously expressed Cre recombinase. Such AIF transgenic mice overexpress the pro-apoptotic splice variant of AIF (AIF1) at both the mRNA (5.9 times higher) and protein level (2.4 times higher), but not the brain-specific AIF splice-isoform (AIF2). Excessive AIF did not have any apparent effects on the phenotype or physiological functions of the mice. However, brain injury (both gray and white matter) after neonatal HI was exacerbated in mice overexpressing AIF, coupled to enhanced translocation of mitochondrial AIF to the nucleus as well as enhanced caspase-3 activation in some brain regions, as indicated by immunohistochemistry. Altogether, these findings corroborate earlier studies demonstrating that AIF plays a causal role in neonatal HI brain injury.

Human life expectancy has significantly increased due to nutritional access, improved hygiene, or medical advances such as vaccinations. Consequently, morbidities shifted from infectious diseases a...

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions1,2. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes1-3. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.

Immunometabolism explores how the intracellular metabolic pathways in immune cells can regulate their function under different micro-environmental and (patho-)-physiological conditions (Pearce, 2010; Buck et al., 2015; O'Neill and Pearce, 2016). In the last decade great advances have been made in studying and manipulating metabolic programs in immune cells. Immunometabolism has primarily focused on glycolysis, the TCA cycle and oxidative phosphorylation (OXPHOS) as well as free fatty acid synthesis and oxidation. These pathways are important for providing the energy needs of cell growth, membrane rigidity, cytokine production and proliferation. In this review, we will however, highlight the specific role of iron metabolism at the cellular and organismal level, as well as how the bioavailability of this metal orchestrates complex metabolic programs in immune cell homeostasis and inflammation. We will also discuss how dysregulation of iron metabolism contributes to alterations in the immune system and how these novel insights into iron regulation can be targeted to metabolically manipulate immune cell function under pathophysiological conditions, providing new therapeutic opportunities for autoimmunity and cancer.

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR

We have created synthetic peptide libraries to benchmark crosslinking mass spectrometry search engines for different types of crosslinker. The unique benefit of using a library is knowing which identified crosslinks are true and which are false. Here we have used mass spectrometry data generated from measurement of the peptide libraries to evaluate the most frequently applied search algorithms in crosslinking mass-spectrometry. When filtered to an estimated false discovery rate of 5%, false crosslink identification ranged from 5.2% to 11.3% for search engines with inbuilt validation strategies for error estimation. When different external validation strategies were applied to one single search output, false crosslink identification ranged from 2.4% to a surprising 32%, despite being filtered to an estimated 5% false discovery rate. Remarkably, the use of MS-cleavable crosslinkers did not reduce the false discovery rate compared to non-cleavable crosslinkers, results from which have far-reaching implications in structural biology. We anticipate that the datasets acquired during this research will further drive optimisation and development of search engines and novel data-interpretation technologies, thereby advancing our understanding of vital biological interactions.

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. Osteoclasts secrete small extracellular vesicles that stimulate osteoblasts, promoting bone formation via receptor activator of nuclear factor-kappa B ligand (RANKL), thereby linking bone formation and resorption.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site‐specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)‐induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF‐κB reporter relative to wild‐type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated Hoip(K778R/K778R) knock‐in mice, which show no overt developmental phenotypes; however, in response to TNF, Hoip(K778R/K778R) mouse embryonic fibroblasts display mildly suppressed NF‐κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF‐induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in Hoip(K778R/K778R) mice, which is rescued by knockout of TNFR1. We propose that site‐specific ubiquitination of HOIP regulates a LUBAC‐dependent switch between survival and apoptosis in TNF signaling.