Wulffmedeiros5796

Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug.Subclonal reconstruction from bulk tumor DNA sequencing has become a pillar of cancer evolution studies, providing insight into the clonality and relative ordering of mutations and mutational processes. We provide an outline of the complex computational approaches used for subclonal reconstruction from single and multiple tumor samples. We identify the underlying assumptions and uncertainties in each step and suggest best practices for analysis and quality assessment. This guide provides a pragmatic resource for the growing user community of subclonal reconstruction methods.Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a β-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.Complement hyperactivation, angiopathic thrombosis and protein-losing enteropathy (CHAPLE disease) is a lethal disease caused by genetic loss of the complement regulatory protein CD55, leading to overactivation of complement and innate immunity together with immunodeficiency due to immunoglobulin wasting in the intestine. We report in vivo human data accumulated using the complement C5 inhibitor eculizumab for the medical treatment of patients with CHAPLE disease. selleck compound We observed cessation of gastrointestinal pathology together with restoration of normal immunity and metabolism. We found that patients rapidly renormalized immunoglobulin concentrations and other serum proteins as revealed by aptamer profiling, re-established a healthy gut microbiome, discontinued immunoglobulin replacement and other treatments and exhibited catch-up growth. Thus, we show that blockade of C5 by eculizumab effectively re-establishes regulation of the innate immune complement system to substantially reduce the pathophysiological manifestations of CD55 deficiency in humans.Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.In chronic hepatitis C virus (HCV) infection, exhausted HCV-specific CD8+ T cells comprise memory-like and terminally exhausted subsets. However, little is known about the molecular profile and fate of these two subsets after the elimination of chronic antigen stimulation by direct-acting antiviral (DAA) therapy. link2 Here, we report a progenitor-progeny relationship between memory-like and terminally exhausted HCV-specific CD8+ T cells via an intermediate subset. link3 Single-cell transcriptomics implicated that memory-like cells are maintained and terminally exhausted cells are lost after DAA-mediated cure, resulting in a memory polarization of the overall HCV-specific CD8+ T cell response. However, an exhausted core signature of memory-like CD8+ T cells was still detectable, including, to a smaller extent, in HCV-specific CD8+ T cells targeting variant epitopes. These results identify a molecular signature of T cell exhaustion that is maintained as a chronic scar in HCV-specific CD8+ T cells even after the cessation of chronic antigen stimulation.Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression ( less then 0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/β-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.Hydrogen storage materials are the key to hydrogen energy utilization. However, current materials can hardly meet the storage capacity and/or operability requirements of practical applications. Here we report an advancement in hydrogen storage performance and related mechanism based on a hydrofluoric acid incompletely etched MXene, namely, a multilayered Ti2CTx (T is a functional group) stack that shows an unprecedented hydrogen uptake of 8.8 wt% at room temperature and 60 bar H2. Even under completely ambient conditions (25 °C, 1 bar air), Ti2CTx is still able to retain ~4 wt% hydrogen. The hydrogen storage is stable and reversible in the material, and the hydrogen release is controllable by pressure and temperature below 95 °C. The storage mechanism is deduced to be a nanopump-effect-assisted weak chemisorption in the sub-nanoscale interlayer space of the material. Such a storage approach provides a promising strategy for designing practical hydrogen storage materials.Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. In the present study, we determined cryo-electron microscopy (cryo-EM) structures of several variants of Sec61-Sec62-Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and luminal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.