RESUMEN
Mitochondria and endoplasmic reticulum contacts (MERCs) control multiple cellular processes, including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double-negative (DN) stage, we studied their role in early mouse thymocyte development. We found that T cell-specific knockout of Hspa9, which encodes GRP75, a protein that mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. The IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study identifies the essential role of GRP75-dependent MERCs in early thymocyte development and the governing facts of cell survival and differentiation in the DN stage.
RESUMEN
Follicular helper T (Tfh) cells are important for generating humoral immune responses by helping B cells form germinal centers (GCs) and the production of high-affinity antibodies. However, aberrant Tfh cell expansion also contributes to the generation of self-reactive autoantibodies and promotes autoantibody-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Protein phosphatase 2A catalytic subunit alpha isoform (PP2A Cα) expression levels are elevated in peripheral T cells of SLE patients and positively correlate with autoantibody titers and disease activity. Here, we demonstrate a critical role of PP2A in Tfh differentiation by using T cell restricted PP2A Cα deficient mice. We observed impaired Tfh differentiation and GC response in two different classical Tfh induction models. Mechanistic studies revealed that downregulation of protein translation of the Tfh lineage transcription factor BCL6 in PP2A deficient T cells. Importantly, we found that PP2A deficiency by either gene knockout or chemical inhibition alleviated lupus severity in mice. Lastly, we confirmed a positive correlation between PP2A Cα and BCL6 protein levels in human CD4+ T cells from patients with SLE. In summary, our study revealed a critical role of PP2A in regulating Tfh cells and suggests it is a potential therapeutic target for lupus.
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Lupus Eritematoso Sistémico , Linfocitos T Colaboradores-Inductores , Humanos , Ratones , Animales , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Autoanticuerpos , Linfocitos B , Diferenciación CelularRESUMEN
T-cell receptors (TCRs) and B-cell receptors (BCRs) are critical in recognizing antigens and activating the adaptive immune response. Stochastic V(D)J recombination generates massive TCR/BCR repertoire diversity. Single-cell immune profiling with transcriptome analysis allows the high-throughput study of individual TCR/BCR clonotypes and functions under both normal and pathological settings. However, a comprehensive database linking these data is not yet readily available. Here, we present the human Antigen Receptor database (huARdb), a large-scale human single-cell immune profiling database that contains 444 794 high confidence T or B cells (hcT/B cells) with full-length TCR/BCR sequence and transcriptomes from 215 datasets. All datasets were processed in a uniform workflow, including sequence alignment, cell subtype prediction, unsupervised cell clustering, and clonotype definition. We also developed a multi-functional and user-friendly web interface that provides interactive visualization modules for biologists to analyze the transcriptome and TCR/BCR features at the single-cell level. HuARdb is freely available at https://huarc.net/database with functions for data querying, browsing, downloading, and depositing. In conclusion, huARdb is a comprehensive and multi-perspective atlas for human antigen receptors.
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Bases de Datos Genéticas , Receptores de Antígenos de Linfocitos B/clasificación , Receptores de Antígenos de Linfocitos T/clasificación , Programas Informáticos , Linfocitos B , Humanos , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de la Célula Individual , Transcriptoma/genética , Recombinación V(D)J/genéticaRESUMEN
This study aims to investigate the influence of excessive oxidative stress on cardiac injury during acute myocardial ischemia (AMI), with a focus on apoptosis, autophagy, and inflammatory cell infiltration, and to detect the role of hydrogen sulfide (H2S) in this process. We found that SOD1 knockout (KO) mice showed excessive oxidative stress and exacerbated myocardium injury after AMI. Increased apoptosis and inflammation response in the ischemic myocardium contribute to this deterioration, whereas enhanced autophagy plays a protective role. Myocardial inflammation after AMI was much more severe in SOD1 KO mice than in wild-type mice. Pretreatment with the H2S donor NaHS reduced autophagy and apoptosis levels in the ischemic myocardium and alleviated the regional inflammation response in the cardiac tissues of SOD1 KO mice. Moreover, autophagy and apoptosis levels were significantly enhanced in SOD1 knockdown primary neonatal rat cardiomyocytes (NRCMs) under glucose deprivation. Pretreatment with NaHS can partially inhibit this elevation. Taken together, we found that excessive oxidative stress can aggravate cardiac injury during AMI. Exogenous H2S can alleviate cardiac injury during AMI by reducing apoptosis and inflammation response in heart tissues under oxidative stress.
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Sulfuro de Hidrógeno/uso terapéutico , Isquemia Miocárdica/tratamiento farmacológico , Enfermedad Aguda , Animales , Sulfuro de Hidrógeno/farmacología , Masculino , Ratones , Ratones Noqueados , Estrés Oxidativo , Ratas , Ratas Sprague-DawleyRESUMEN
Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems. To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. The growth of the mutant virus can then be regulated by the degradation and splicing of the protein. We found that an ultrafast gp41-1 split intein was able to rescue or degrade the protein of interest (POI) by removing or adding a strong degron through protein splicing. As a result, the function of the POI was turned on or off during the process. Using HCMV essential gene IE1/IE2, we confirmed that imPS worked remarkably well in conditionally regulating protein stability during viral infection. This conditional approach is likely to be applicable for dissecting the gene functions of HCMV or other viruses.
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Citomegalovirus/genética , Genes Esenciales , Genes Virales , Biología Molecular/métodos , Virología/métodos , Citomegalovirus/crecimiento & desarrollo , Células HEK293 , Humanos , Inteínas/genética , Viabilidad Microbiana , Estabilidad ProteicaRESUMEN
Aims. To examine the expression patterns of hydrogen sulphide- (H2S-) producing enzymes in ischaemic heart tissue and plasma levels of H2S after 2 weeks of NaHS treatment after myocardial infarction (MI) and to clarify the role of endogenous H2S in the MI process. Results. After MI surgery, 2 weeks of treatment with the H2S donor NaHS alleviated ischaemic injury. Meanwhile, in ischemia myocardium, three H2S-producing enzymes, cystathionine γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) significantly increased. Plasma H2S levels were also elevated. In vitro, NaHS treatment protected cardiomyocytes from hypoxic injury and raised CBS levels in a concentration-dependent manner. Different from in vivo results, however, CSE or 3-MST expression did not change. NaHS treatment increased the activity of CSE/CBS but not of 3-MST. When CSE was either knocked down (in vitro) or knocked out (in vivo), H2S levels significantly decreased, which subsequently exacerbated the ischaemic injury. Meanwhile, the expressions of CBS and 3-MST increased due to compensation. Conclusions. Exogenous H2S treatment changed the expressions of three H2S-producing enzymes and H2S levels after MI, suggesting a new and indirect regulatory mechanism for H2S production and its contribution to cardiac protection. Endogenous H2S plays an important role in protecting ischaemic tissue after MI.
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Sulfuro de Hidrógeno/metabolismo , Infarto del Miocardio/enzimología , Sulfuros/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Citoprotección/efectos de los fármacos , Sulfuro de Hidrógeno/sangre , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Isquemia Miocárdica/patología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas Sprague-Dawley , Sulfurtransferasas/metabolismoRESUMEN
UNLABELLED: The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multifunctional HCMV protein pUL38 previously has been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2) (J. N. Moorman et al., Cell Host Microbe 3:253-262, 2008, http://dx.doi.org/10.1016/j.chom.2008.03.002). pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL3825-331) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL3825-331 lost the ability to interact with TSC2 but retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL3825-331 replicated with â¼10-fold less efficiency than the wild-type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOI-dependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23 and 24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL3825-331. We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. Therefore, this study identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners. IMPORTANCE: HCMV, like other viruses, depends exclusively on its host cell to propagate. Therefore, it has developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. mTORC1 is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both TSC2-dependent and TSC2-independent manners. The pUL38-independent mode of mTORC1 activation also has been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV.