RESUMEN
The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)-induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER-resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid-sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto-ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA-responsive ubiquitin ligase which, together with ADIPOR2, defines an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.
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Hidrolasas , Fluidez de la Membrana , Animales , Ácidos Grasos/metabolismo , Hidrolasas/metabolismo , Mamíferos , Proteínas de la Membrana/metabolismo , Fosfolípidos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.
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Enfermedades Desmielinizantes , Sulfoglicoesfingolípidos , Humanos , Glicoesfingolípidos/metabolismo , Proteínas Portadoras/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Vaina de Mielina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Enfermedades Desmielinizantes/patologíaRESUMEN
The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
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Mieloma Múltiple , Semaforinas , Membrana Celular/metabolismo , Humanos , Factores Inmunológicos , Inmunoterapia , Proteínas de la Membrana , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Proteómica , Semaforinas/genética , Semaforinas/metabolismoRESUMEN
Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
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Vesículas Extracelulares/metabolismo , Mitocondrias/metabolismo , Células-Madre Neurales/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/ultraestructuraRESUMEN
The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.
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Proteínas Bacterianas/metabolismo , Células Dendríticas/inmunología , Sinapsis Inmunológicas/inmunología , Receptores Acoplados a Proteínas G/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/metabolismo , Salmonella entericaRESUMEN
Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.
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Antígeno B7-H1/biosíntesis , Antígeno B7-H1/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Animales , Antígeno B7-H1/inmunología , Sistemas CRISPR-Cas , Línea Celular , Membrana Celular/metabolismo , Endosomas/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Lisosomas/metabolismo , Ratones , Proteolisis , Proteoma/metabolismo , Especificidad por Sustrato , Linfocitos T/inmunología , Linfocitos T/metabolismo , Escape del Tumor/inmunologíaRESUMEN
Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.
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Regulación Viral de la Expresión Génica , Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , ARN Viral/metabolismo , Ribonucleasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Fibroblastos/metabolismo , Fibroblastos/virología , Herpes Simple/genética , Herpes Simple/patología , Herpes Simple/virología , Humanos , Biosíntesis de Proteínas , Proteoma , ARN Viral/genética , Ribonucleasas/genética , Transcriptoma , Proteínas Virales/genéticaRESUMEN
Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins.
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Hemo-Oxigenasa 1/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Retículo Endoplásmico/enzimología , Técnicas de Inactivación de Genes , Células HeLa , Hemo-Oxigenasa 1/genética , Humanos , Espectrometría de Masas , Proteínas de la Membrana/genética , Proteolisis , Receptores de Superficie Celular/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Tetherin (BST2/CD317) is a viral restriction factor that anchors enveloped viruses to host cells and limits viral spread. The HIV-1 Vpu accessory protein counteracts tetherin by decreasing its cell surface expression and targeting it for ubiquitin-dependent endolysosomal degradation. Although the Vpu-mediated downregulation of tetherin has been extensively studied, the molecular details are not completely elucidated. We therefore used a forward genetic screen in human haploid KBM7 cells to identify novel genes required for tetherin trafficking. Our screen identified WDR81 as a novel gene required for tetherin trafficking and degradation in both the presence and absence of Vpu. WDR81 is a BEACH-domain containing protein that is also required for the degradation of EGF-stimulated epidermal growth factor receptor (EGFR) and functions in a complex with the WDR91 protein. In the absence of WDR81 the endolysosomal compartment appears swollen, with enlarged early and late endosomes and reduced delivery of endocytosed dextran to cathepsin-active lysosomes. Our data suggest a role for the WDR81-WDR91 complex in the fusion of endolysosomal compartments and the absence of WDR81 leads to impaired receptor trafficking and degradation.
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Antígenos CD/metabolismo , Proteínas Portadoras/metabolismo , Lisosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas Ligadas a GPI/metabolismo , VIH-1/metabolismo , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Transporte de Proteínas , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.
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Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Empalme del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transcripción Genética , Animales , Arginina/genética , Arginina/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Células HeLa , Humanos , Metilación , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteínas Asociadas a Matriz Nuclear/química , Factores de Transcripción de Octámeros/química , Factor de Empalme Asociado a PTB , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/genética , Proteínas de Unión al ARN/química , Proteínas Represoras/genética , Ribonucleoproteínas/genéticaRESUMEN
We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS-based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.
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Proteoma/análisis , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Escherichia coli/química , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/metabolismo , Respuesta al Choque TérmicoRESUMEN
Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.
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The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.
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COVID-19 , SARS-CoV-2 , Anticuerpos , Anticuerpos Antivirales , Humanos , Células Asesinas Naturales , ProteómicaRESUMEN
EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.
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Enfermedad Granulomatosa Crónica , NADPH Oxidasas , Humanos , Animales , Ratones , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fagocitos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The regulation of synaptic connectivity in the brain is vital to proper functioning and development of the CNS. Formation of neural networks in the CNS has been shown to be heavily influenced by astrocytes, which secrete factors, including thrombospondin (TSP) family proteins, that promote synaptogenesis. However, whether this process is different between males and females has not been thoroughly investigated. In this study, we found that cortical neurons purified from newborn male rats showed a significantly more robust synaptogenic response compared with female-derived cells when exposed to factors secreted from astrocytes. This difference was driven largely by the neuronal response to TSP2, which increased synapses in male neurons while showing no effect on female neurons. Blockade of endogenous 17ß-estradiol (E2) production with letrozole normalized the TSP response between male and female cells, indicating a level of regulation by estrogen signaling. Our results suggest that male and female neurons show a divergent response to TSP synaptogenic signaling, contributing to sex differences in astrocyte-mediated synaptic connectivity.
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Astrocitos , Factores Sexuales , Trombospondinas , Animales , Femenino , Masculino , Neurogénesis , Neuronas , Ratas , SinapsisRESUMEN
The rise in rates of opioid abuse in recent years in the United States has led to a dramatic increase in the incidence of neonatal abstinence syndrome (NAS). Despite improved understanding of NAS and its acute symptoms, there remains a paucity of information regarding the long-term effects of prenatal exposure to drugs of abuse on neurological development. The primary goal of this study was to investigate the effects of prenatal drug exposure on synaptic connectivity within brain regions associated with the mesolimbic dopamine pathway, the primary reward pathway associated with drug abuse and addiction, in a mouse model. Our secondary goal was to examine the role of the Ca+2 channel subunit α2δ-1, known to be involved in key developmental synaptogenic pathways, in mediating these effects. Pregnant mouse dams were treated orally with either the opioid drug buprenorphine (commonly used in medication-assisted treatment for substance use patients), gabapentin (neuropathic pain drug that binds to α2δ-1 and has been increasingly co-abused with opioids), a combination of both drugs, or vehicle daily from gestational day 6 until postnatal day 11. Confocal fluorescence immunohistochemistry (IHC) imaging of the brains of the resulting wild-type (WT) pups at postnatal day 21 revealed a number of significant alterations in excitatory and inhibitory synaptic populations within the anterior cingulate cortex (ACC), nucleus accumbens (NAC), and medial prefrontal cortex (PFC), particularly in the buprenorphine or combinatorial buprenorphine/gabapentin groups. Furthermore, we observed several drug- and region-specific differences in synaptic connectivity between WT and α2δ-1 haploinsufficient mice, indicating that critical α2δ-1-associated synaptogenic pathways are disrupted with early life drug exposure.
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The cellular response to interferon (IFN) is essential for antiviral immunity, IFN-based therapy and IFN-related disease. The plasma membrane (PM) provides a critical interface between the cell and its environment, and is the initial portal of entry for viruses. Nonetheless, the effect of IFN on PM proteins is surprisingly poorly understood, and has not been systematically investigated in primary immune cells. Here, we use multiplexed proteomics to quantify IFNα2a-stimulated PM protein changes in primary human CD14+ monocytes and CD4+ T cells from five donors, quantifying 606 and 482 PM proteins respectively. Comparison of cell surface proteomes revealed a remarkable invariance between donors in the overall composition of the cell surface from each cell type, but a marked donor-to-donor variability in the effects of IFNα2a. Furthermore, whereas only 2.7% of quantified proteins were consistently upregulated by IFNα2a at the surface of CD4+ T cells, 6.8% of proteins were consistently upregulated in primary monocytes, suggesting that the magnitude of the IFNα2a response varies according to cell type. Among these differentially regulated proteins, we found the viral target Endothelin-converting enzyme 1 (ECE1) to be an IFNα2a-stimulated protein exclusively upregulated at the surface of CD4+ T cells. We therefore provide a comprehensive map of the cell surface of IFNα2a-stimulated primary human immune cells, including previously uncharacterized interferon stimulated genes (ISGs) and candidate antiviral factors.
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Linfocitos T CD4-Positivos/inmunología , Enzimas Convertidoras de Endotelina/inmunología , Interferón-alfa/farmacología , Monocitos/inmunología , Linfocitos T CD4-Positivos/citología , Humanos , Monocitos/citología , ProteómicaRESUMEN
Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Integración Viral , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.
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Cromatografía de Fase Inversa/métodos , Espectrometría de Masas/métodos , Fragmentos de Péptidos/análisis , Mapeo Peptídico/métodos , Proteómica/métodos , Acetonitrilos , Cationes , Núcleo Celular/química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Marcaje Isotópico , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteoma/química , Tripsina/metabolismoRESUMEN
BACKGROUND: Over half of new cancer cases occur in patients aged > or = 65 years. Many older patients can benefit from intensive cancer therapies, yet evidence suggests that this population is undertreated. METHODS: To assess preferences and influential factors in geriatric cancer management, practicing U.S. medical oncologists completed a survey containing four detailed vignettes exploring colon, breast, lung, and prostate cancer treatment. Participants were randomly assigned one of two surveys with vignettes that were identical except for patient age (<65 years or >70 years). RESULTS: Physicians in each survey group (n = 200) were demographically similar. Intensive therapy was significantly less likely to be recommended for an older than for a younger, but otherwise identical, patient in two of the scenarios. For a woman with metastatic colon cancer (Eastern Cooperative Oncology Group [ECOG] score, 1) for whom chemotherapy was recommended, nearly all oncologists chose an intensive regimen if the patient's age was 63; but if her age was 85, one fourth of the oncologists chose a less intensive treatment. Likewise, for stage IIA breast cancer (ECOG score, 0), 93% recommended intensive adjuvant treatment for a previously healthy patient aged 63; but only 66% said they would do so if the patient's age was 75. Oncologists commonly identified patient age as an influence on treatment choice, but were even more likely to cite performance status as a determining factor. CONCLUSIONS: Advanced age can deter oncologists from choosing intensive cancer therapy, even if patients are highly functional and lack comorbidities. Education on tailoring cancer treatment and a greater use of comprehensive geriatric assessment may reduce cancer undertreatment in the geriatric population.