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Because of their unique capacity to cross-present Ags to CD8+ T cells, mouse lymphoid tissue-resident CD8+ dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8+ DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8+ DC lines and primary CD8+ DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8+ DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8+ DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8- DCs. [ABSTRACT FROM AUTHOR]

Protein secretion allows communication of distant cells in an organism and controls a broad range of physiological functions. We describe a quantitative, high-resolution mass spectrometric workflow to detect and quantify proteins that are released from immune cells upon receptor ligation. We quantified the time-resolved release of 775 proteins, including 52 annotated cytokines from only 150,000 primary Toll-like receptor 4-activated macrophages per condition. Achieving low picogram sensitivity, we detected secreted proteins whose abundance increased by a factor of more than 10,000 upon stimulation. Secretóme to transcriptome comparisons revealed the transcriptionally decoupled release of lysosomal proteins. From genetic models, we defined secretory profiles that depended on distinct intracellular signaling adaptors and showed that secretion of many proinflammatory proteins is safeguarded by redundant mechanisms, whereas signaling adaptor synergy promoted the release of anti-inflammatory proteins. [ABSTRACT FROM AUTHOR]

Caspase-1 serves an essential function in the initiation of inflammation by proteolytically maturing the cytokines interleukin 1β and interleukin 18. Several Nod-like receptors activate caspase-1 in response to microbial and 'danger' signals by assembling cytosolic protein complexes called 'inflammasomes'. We show here that superoxide dismutase 1 (SOD1) regulates caspase-1 activation. In SOD1-deficient macrophages, higher superoxide production decreased the cellular redox potential and specifically inhibited caspase-1 by reversible oxidation and glutathionylation of the redox-sensitive cysteine residues Cys397 and Cys362. Conversely, hypoxic conditions abrogated caspase-1 inhibition. In vivo, SOD1-deficient mice produced less caspase-1-dependent cytokines and were less susceptible to lipopolysaccharide-induced septic shock. Our findings identify a physiological post-translational mechanism in the control of caspase-1-mediated inflammatory processes. [ABSTRACT FROM AUTHOR]

The natural host for Ebola virus, presumed to be an animal, has not yet been identified despite an extensive search following several major outbreaks in Africa. A straightforward approach used to determine animal contact with Ebola virus is by assessing the presence of specific antibodies in serum. This approach however has been made very difficult by the absence of specific reagents required for the detection of antibodies from the majority of wild animal species. In this study, we isolated a human monoclonal antibody Fab fragment, KZ51, that reacts with an immunodominant epitope on Ebola virus nucleoprotein (NP) that is conserved on all four Ebola virus subtypes. The antibody KZ51 represents a major specificity as sera from all convalescent patients tested (10/10) and sera from guinea pigs infected with each of the four Ebola virus subtypes competed strongly with KZ51 for binding to radiation-inactivated Ebola virus. These features allowed us to develop a novel assay for the detection of seroconversion irrespective of Ebola virus subtype or animal species. In this assay, the binding of KZ51 Fab-phage particles is used as an indicator assay and the presence of specific antibodies against Ebola virus in sera is indicated by binding competition. A prominent feature of the assay is that the Fab-phage particles may be prestained with a dye so that detection of binding can be directly determined by visual inspection. The assay is designed to be both simple and economical to enable its use in the field. [Copyright &y& Elsevier]

Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)–based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease. Image 1 • MS-based proteomics enables the unsupervised detection of intercellular signaling proteins and their post-translational modifications. • Specialized biochemical strategies provide comprehensive views on secreted proteins, cell-surface receptors and their interactions. • Proximity labeling methods enable the characterization of dynamic signaling complexes at cellular interfaces. • Advancements in metabolic protein labeling strategies facilitate cell type–specific signaling analyses in heterocellular environments. [ABSTRACT FROM AUTHOR]

The article offers information on the identification of gelatinase substrates at the blood-brain barrier that play a role in astroglial barrier function. Using a sensitive label-free mass spectrometry-based secretome approach, the study identifies potential substrates for MMP-9 and MMP-2 at the cell surface of primary astrocytes, including known substrates and previously unknown MMP-dependent events involved in cell-cell and cell-matrix interactions.

Inflammasomes are multimeric protein complexes that are activated through a NOD-like receptor and regulate the proteolytic activation of caspase-1 and cytokines, like IL-1β. The NLRP3 inflammasome is implicated in many human pathologies including infections, autoinflammatory syndromes, chronic inflammation and metabolic diseases; however, the molecular mechanisms of activation are not fully understood. In this study we show that NLRP3 inflammasome activation requires intracellular copper. A clinically approved copper chelator, tetrathiomolybdate, inhibited the canonical NLRP3 but not the AIM2, NLRC4, and NLRP1 inflammasomes or NF-kB-dependent priming. We demonstrate that NLRP3 inflammasome activation is blocked by removing copper from the active site of superoxide dismutase 1, recapitulating impaired inflammasome function in superoxide dismutase 1-deficient mice. This regulation is specific to macrophages, but not monocytes, both in mice and humans. In vivo, depletion of bioavailable copper resulted in attenuated caspase-1-dependent inflammation and reduced susceptibility to LPS-induced endotoxic shock. Our results indicate that targeting the intracellular copper homeostasis has potential for the treatment of NLRP3-dependent diseases. [ABSTRACT FROM AUTHOR]

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-β1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets. [ABSTRACT FROM AUTHOR]

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane-specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CARriers of the TGN to the cell Surface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)-G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic. [ABSTRACT FROM AUTHOR]

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post‐transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP‐APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS‐based quantification. We report the time‐resolved interplay of more than 50 previously undescribed modification and hundreds of protein–protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site‐specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll‐like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost‐effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells. SYNOPSIS: A robust proteomics strategy is used to elucidate the time‐resolved interplay of protein‐protein interactions (PPIs) and post‐translational modifications (PTMs) in human immune cells with a focus on TRAF2, MAPK14, and MAP3K7. An experimental strategy to reveal dynamic signaling checkpoints in primary cells is developed.Crosstalk between protein modifications and interactions in signaling complexes is identified.TRAF2 ISGylation, phosphorylation, and its ARHGEF18 interaction in monocytes are discovered.Differential drug modes of action for MAPK14 inhibitors are dissected. [ABSTRACT FROM AUTHOR]

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterolbinding pocket reduced CD81-cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations ofCD81with andwithout cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol- unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells. [ABSTRACT FROM AUTHOR]

Cytokines are signaling molecules secreted and sensed by immune and other cell types, enabling dynamic intercellular communication. Although a vast amount of data on these interactions exists, this information is not compiled, integrated or easily searchable. Here we report immuneXpresso, a text-mining engine that structures and standardizes knowledge of immune intercellular communication. We applied immuneXpresso to PubMed to identify relationships between 340 cell types and 140 cytokines across thousands of diseases. The method is able to distinguish between incoming and outgoing interactions, and it includes the effect of the interaction and the cellular function involved. These factors are assigned a confidence score and linked to the disease. By leveraging the breadth of this network, we predicted and experimentally verified previously unappreciated cell–cytokine interactions. We also built a global immune-centric view of diseases and used it to predict cytokine–disease associations. This standardized knowledgebase (http://www.immunexpresso.org) opens up new directions for interpretation of immune data and model-driven systems immunology. [ABSTRACT FROM AUTHOR]

Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis ( ALS) and frontotemporal lobar degeneration ( FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased β2-transferrin levels in patient CSF. Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling. [ABSTRACT FROM AUTHOR]

Glucagon is believed to be a pancreas-specific hormone, and hyperglucagonemia has been shown to contribute significantly to the hyperglycemic state of patients with diabetes. This hyperglucagonemia has been thought to arise from α-cell insensitivity to suppressive effects of glucose and insulin combined with reduced insulin secretion. We hypothesized that postabsorptive hyperglucagonemia represents a gut-dependent phenomenon and subjected 10 totally pancreatectomized patients and 10 healthy control subjects to a 75-g oral glucose tolerance test and a corresponding isoglycemic intravenous glucose infusion. We applied novel analytical methods of plasma glucagon (sandwich ELISA and mass spectrometry-based proteomics) and show that 29-amino acid glucagon circulates in patients without a pancreas and that glucose stimulation of the gastrointestinal tract elicits significant hyperglucagonemia in these patients. These findings emphasize the existence of extrapancreatic glucagon (perhaps originating from the gut) in man and suggest that it may play a role in diabetes secondary to total pancreatectomy. [ABSTRACT FROM AUTHOR]

Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial p-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. [ABSTRACT FROM AUTHOR]

Preclinical studies show that blocking Interleukin–1 (IL–1) retards the progression of Amyotrophic Lateral Sclerosis (ALS). We assessed the safety of Anakinra (ANA), an IL–1 receptor antagonist, in ALS patients. In a single arm pilot study we treated 17 ALS patients with ANA (100 mg) daily for one year. We selected patients with dominant or exclusive lower motor neuron degeneration (LMND) presentation, as peripheral nerves may be more accessible to the drug. Our primary endpoint was safety and tolerability. Secondary endpoints included measuring disease progression with the revised ALS functional rating scale (ALSFRSr). We also quantified serum inflammatory markers. For comparison, we generated a historical cohort of 47 patients that fit the criteria for enrolment, disease characteristics and rate of progression of the study group. Only mild adverse events occurred in ALS patients treated with ANA. Notably, we observed lower levels of cytokines and the inflammatory marker fibrinogen during the first 24 weeks of treatment. Despite of this, we could not detect a significant reduction in disease progression during the same period in patients treated with ANA compared to controls as measured by the ALSFRSr. In the second part of the treatment period we observed an increase in serum inflammatory markers. Sixteen out of the 17 patients (94%) developed antibodies against ANA. This study showed that blocking IL–1 is safe in patients with ALS. Further trials should test whether targeting IL–1 more efficiently can help treating this devastating disease. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Hexanucleotide repeat expansion in C9orf72 is the most common pathogenic mutation in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the lack of an ATG start codon, the repeat expansion is translated in all reading frames into dipeptide repeat (DPR) proteins, which form insoluble, ubiquitinated, p62-positive aggregates that are most abundant in the cerebral cortex and cerebellum. To specifically analyze DPR toxicity and aggregation, we expressed DPR proteins from synthetic genes containing a start codon but lacking extensive GGGGCC repeats. Poly-Gly-Ala (GA) formed p62-positive cytoplasmic aggregates, inhibited dendritic arborization and induced apoptosis in primary neurons. Quantitative mass spectrometry analysis to identify poly-GA co-aggregating proteins revealed a significant enrichment of proteins of the ubiquitin-proteasome system. Among the other interacting proteins, we identified the transport factor Unc119, which has been previously linked to neuromuscular and axonal function, as a poly-GA co-aggregating protein. Strikingly, the levels of soluble Unc119 are strongly reduced upon poly-GA expression in neurons, suggesting a loss of function mechanism. Similar to poly-GA expression, Unc119 knockdown inhibits dendritic branching and causes neurotoxicity. Unc119 overexpression partially rescues poly-GA toxicity suggesting that poly-GA expression causes Unc119 loss of function. In C9orf72 patients, Unc119 is detectable in 9.5 % of GA inclusions in the frontal cortex, but only in 1.6 % of GA inclusions in the cerebellum, an area largely spared of neurodegeneration. A fraction of neurons with Unc119 inclusions shows loss of cytosolic staining. Poly-GA-induced Unc119 loss of function may thereby contribute to selective vulnerability of neurons with DPR protein inclusions in the pathogenesis of C9orf72 FTLD/ALS. [ABSTRACT FROM AUTHOR]

Vaccination is the most effective method to prevent infectious diseases. However, approaches to identify novel vaccine candidates are commonly laborious and protracted. While surface proteins are suitable vaccine candidates and can elicit antibacterial antibody responses, systematic approaches to define surfomes from gram-negatives have rarely been successful. Here we developed a combined discovery-driven mass spectrometry and computational strategy to identify bacterial vaccine candidates and validate their immunogenicity using a highly prevalent gram-negative pathogen, Helicobacter pylori, as a model organism. We efficiently isolated surface antigens by enzymatic cleavage, with a design of experiment based strategy to experimentally dissect cell surface-exposed from cytosolic proteins. From a total of 1,153 quantified bacterial proteins, we thereby identified 72 surface exposed antigens and further prioritized candidates by computational homology inference within and across species. We next tested candidate-specific immune responses. All candidates were recognized in sera from infected patients, and readily induced antibody responses after vaccination of mice. The candidate jhp_0775 induced specific B and T cell responses and significantly reduced colonization levels in mouse therapeutic vaccination studies. In infected humans, we further show that jhp_0775 is immunogenic and activates IFNγ secretion from peripheral CD4+ and CD8+ T cells. Our strategy provides a generic preclinical screening, selection and validation process for novel vaccine candidates against gram-negative bacteria, which could be employed to other gram-negative pathogens. [ABSTRACT FROM AUTHOR]

Summary Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs. [ABSTRACT FROM AUTHOR]

NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. Because of its involvement in many diseases such as atherosclerosis and Alzheimer’s Disease, a better understanding of NLRP3 regulation may help to shape therapeutic approaches. The aim of this study was to identify post-translational modifications on NLRP3 and their impact on inflammasome activation. We applied mass spectrometry of NLRP3 to identify post-translational modifications. We identified ubiquitinated and phosphorylated residues. To analyze the function of the modifications, we mutated the residues in NLRP3 expression plasmids and used them for reconstitution of NLRP3-deficient macrophages or overexpression studies in HEK293T cells. Ubiquitinylated residues were found in the NACHT and LRR domains of NLRP3 and mutation of these residues resulted in hyperactive NLRP3. In addition, mutation of one of the phosphorylated residues to a phosphomimetic residue showed reduced IL-1beta release and caspase-1 cleavage upon NLRP3 inflammasome activation, whereas mutation to alanine had no effect. Further studies in HEK293T cells found that the recruitment of the downstream adaptor ASC as well as the self-interaction of NLRP3 were reduced with the phosphomimetic residue. Thus, NLRP3 is likely regulated by post-translational modification and needs to be deubiquitinylated and possibly dephosphorylated before activation. Knowledge about the modifications and the pathways involved may help shape novel strategies for treating diseases that involve NLRP3. [ABSTRACT FROM AUTHOR]

Summary Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses. [ABSTRACT FROM AUTHOR]

A correction to the article "Evidence of Extrapancreatic Glucagon Secretion in Man" is presented.

Sensing of nucleic acids (NAs) by Toll-like receptors (TLRs) is crucial in the host defence against viruses and bacteria. Yet, these TLRs can contribute to autoimmunity. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the ER into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 show defective signalling of endosomal TLRs and are at increased risk to develop herpes simplex virus encephalitis. To date, molecular insights into UNC93B1-mediated regulation of human NA-sensing TLRs are lacking. In this study, we aimed to investigate the role of trafficking motifs and post-translational modifications of UNC93B1 for human endosomal TLR activity. To this end, we utilised mass spectrometry in order to identify post-translational modification sites and prepared mutant forms of UNC93B1 by site-directed mutagenesis. EBV immortalized B cells from UNC93B1-deficient patients as well as UNC93B1 KO THP1s generated by CRISPR-Cas-based genome editing were reconstituted with wild type or mutant forms of UNC93B1 and assessed for endosomal TLR activity. The localization of UNC93B1 was analysed by confocal microscopy. The destruction of either trafficking motifs or post-translational modification sites resulted in receptor-, ligand-, and cell-type specific defects of human endosomal TLRs. Furthermore, mass spectrometry revealed that wild type versus mutant forms of UNC93B1 differed in terms of interaction partners and cellular compartment signatures. Our study reveals another level of complexity of UNC93B1-mediated TLR regulation and thereby contributes to the understanding of nucleic-acid sensing TLR biology. [ABSTRACT FROM AUTHOR]