摘要：nflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

摘要：Background:Non-alcoholic Fatty Liver Disease (NAFLD), now better known as Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and its progression to Nonalcoholic Steatohepatitis (NASH), more recently referred to as Metabolic (Dysfunction)-Associated Steatohepatitis (MASH) are the most common causes of liver failure and chronic liver damage. The new names emphasize the metabolic involvement both in relation to liver function and pathological features with extrahepatic manifestations. This study aims to explore the role of the immunometabolic enzyme ATP citrate lyase (ACLY), with a critical function in lipogenesis, carbohydrate metabolism, gene expression and inflammation.Methods:ACLY function was investigated in TNFα-triggered human hepatocytes and in PBMC-derived macrophages from MASH patients. Evaluation of expression levels was carried out by western blotting and/or RT-qPCR. In the presence or absence of ACLY inhibitors, ROS, lipid peroxidation and GSSG oxidative stress biomarkers were quantified. Chromatin immunoprecipitation (ChIP), transient transfections, immunocytochemistry, histone acetylation quantitation were used to investigate ACLY function in gene expression reprogramming. IL-6 and IL-1β were quantified by Lumit immunoassays.Results:Mechanistically, ACLY inhibition reverted lipid accumulation and oxidative damage while reduced secretion of inflammatory cytokines in TNFα-triggered human hepatocytes. These effects impacted not only on lipid metabolism but also on other crucial features of liver function such as redox status and production of inflammatory mediators. Moreover, ACLY mRNA levels together with those of malic enzyme 1 (ME1) increased in human PBMC-derived macrophages from MASH patients when compared to age-matched healthy controls. Remarkably, a combination of hydroxycitrate (HCA), the natural ACLY inhibitor, with red wine powder (RWP) significantly lowered ACLY and ME1 mRNA amount as well as IL-6 and IL-1β production in macrophages from subjects with MASH.Conclusion:Collectively, our findings for the first time highlight a broad spectrum of ACLY functions in liver as well as in the pathogenesis of MASH and its diagnostic and therapeutic potential value.

摘要：Diethylene glycol is a toxic industrial solvent resulting in a well-defined toxidrome. Diglycolic acid (DGA) has been identified as the metabolite responsible for the nephrotoxicity and hepatotoxicity. These studies assess the mechanism of DGA-induced neurotoxicity, specifically addressing the known ability of DGA to chelate calcium (Ca2+) in solution and inhibit mitochondrial complex II. SH-SY5Y cells were seeded into 96-well plates to assess intracellular Ca2+chelation, complex II activity, mitochondrial membrane potential (ΔΨm), ATP production, and release of inflammatory cytokines TNF-α and IL-1β with 2-, 4-, 6-, 24-, and 48-h DGA exposure. Peak Ca2+chelation occurred at 4 h in cells treated with 6.25-50 mM DGA; however, effects were transient. Complex II activity was significantly decreased at all DGA concentrations tested, with 12.5 mM DGA causing 80% inhibition and 25 and 50 mM DGA causing 97 and 100% inhibition, respectively. Subsequently, 12.5-50 mM DGA concentrations significantly decreased ΔΨm at all time points. 50 mM DGA significantly increased release of TNF-α and IL-1β after 24 and 48 h with significantly decreased ATP production observed at the same time points and concentration. These studies demonstrate that the DGA-induced mechanism of SH-SY5Y cell death involves complex II inhibition leading to mitochondrial depolarization, and subsequent ATP depletion with accompanying inflammatory cytokine release. These results indicate a direct mechanism of DGA-induced neurotoxicity in vitro, similarly observed in other DEG-affected target organs.

摘要：Lipid droplets are cytosolic fat storage organelles present in most eukaryotic cells. Long regarded merely as inert fat reservoirs, they are now emerging as major regulators of cellular metabolism. They act as hubs that coordinate the pathways of lipid uptake, distribution, storage, and use in the cell. Recent studies have revealed that they are also essential components of the cellular stress response. One of the hallmark characteristics of lipid droplets is their capacity to buffer excess lipids and to finely tune their subsequent release based on specific cellular requirements. This simple feature of lipid droplet biology, buffering and delayed release of lipids, forms the basis for their pleiotropic roles in the cellular stress response. In stressed cells, lipid droplets maintain energy and redox homeostasis and protect against lipotoxicity by sequestering toxic lipids into their neutral lipid core. Their mobility and dynamic interactions with mitochondria enable an efficient delivery of fatty acids for optimal energy production. Lipid droplets are also involved in the maintenance of membrane and organelle homeostasis by regulating membrane composition, preventing lipid peroxidation and removing damaged proteins and lipids. Finally, they also engage in a symbiotic relationship with autophagy and act as reservoirs of bioactive lipids that regulate inflammation and immunity. Thus, lipid droplets are central managers of lipid metabolism that function as safeguards against various types of cellular stress

摘要：Glucose-dependent insulinotropic polypeptide (GIP) communicates nutrient intake from the gut to islets, enabling optimal levels of insulin secretion via the GIP receptor (GIPR) on β cells. The GIPR is also expressed in α cells, and GIP stimulates glucagon secretion; however, the role of this action in the postprandial state is unknown. Here, we demonstrate that GIP potentiates amino acid-stimulated glucagon secretion, documenting a similar nutrient-dependent action to that described in β cells. Moreover, we demonstrate that GIP activity in α cells contributes to insulin secretion by invoking paracrine α to β cell communication. Last, specific loss of GIPR activity in α cells prevents glucagon secretion in response to a meal stimulus, limiting insulin secretion and driving glucose intolerance. Together, these data uncover an important axis by which GIPR activity in α cells is necessary to coordinate the optimal level of both glucagon and insulin secretion to maintain postprandial homeostasis.

摘要：Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.

摘要：Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.

摘要：Long half-life of therapeutic Abs and Fc fusion proteins is crucial to their efficacy and is, in part, regulated by their interaction with neonatal Fc receptor (FcRn). However, the current methods (e.g., surface plasmon resonance and biolayer interferometry) for measurement of interaction between IgG and FcRn (IgG/FcRn) require either FcRn or IgG to be immobilized on the surface, which is known to introduce experimental artifacts and have led to conflicting data. To study IgG/FcRn interactions in solution, without a need for surface immobilization, we developed a novel (to our knowledge), solution-based homogeneous binding immunoassay based on NanoBiT luminescent protein complementation technology. We optimized the assay (NanoBiT FcRn assay) for human FcRn, mouse FcRn, rat FcRn, and cynomolgus FcRn and used them to determine the binding affinities of a panel of eight Abs. Assays could successfully capture the modulation in IgG/FcRn binding based on changes in Fc fragment of the Abs. We also looked at the individual contribution of Fc and F(ab)2on the IgG/FcRn interaction and found that Fc is the main driver for the interaction at pH 6. Our work highlights the importance of using orthogonal methods to validate affinity data generated using biosensor platforms. Moreover, the simple add-and-read format of the NanoBiT FcRn assay is amenable for high-throughput screening during early Ab discovery phase.

摘要：Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

摘要：Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation. This immunoassay takes less than two hours to complete in a homogeneous "Add and Read" format and was successfully used to monitor multiple signaling pathways' activation through specific nodes of phosphorylation (e.g pIκBα, pAKT, and pSTAT3). We also tested deactivation of these pathways with small and large molecule inhibitors and obtained the expected pharmacology. This approach does not require cell engineering. Therefore, the phosphorylation of an endogenous substrate is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific inhibitors.