RESUMO
Premature leaf senescence significantly reduces rice yields. Despite identifying numerous factors influencing these processes, the intricate genetic regulatory networks governing leaf senescence demand further exploration. We report the characterization of a stably inherited, ethyl methanesulfonate(EMS)-induced rice mutant with wilted leaf tips from seedling till harvesting, designated lts2. This mutant exhibits dwarfism and early senescence at the leaf tips and margins from the seedling stage when compared to the wild type. Furthermore, lts2 displays a substantial decline in both photosynthetic activity and chlorophyll content. Transmission electron microscopy revealed the presence of numerous osmiophilic granules in chloroplast cells near the senescent leaf tips, indicative of advanced cellular senescence. There was also a significant accumulation of H2O2, alongside the up-regulation of senescence-associated genes within the leaf tissues. Genetic mapping situated lts2 between SSR markers Q1 and L12, covering a physical distance of approximately 212 kb in chr.1. No similar genes controlling a premature senescence leaf phenotype have been identified in the region, and subsequent DNA and bulk segregant analysis (BSA) sequencing analyses only identified a single nucleotide substitution (C-T) in the exon of LOC_Os01g35860. These findings position the lts2 mutant as a valuable genetic model for elucidating chlorophyll metabolism and for further functional analysis of the gene in rice.
Assuntos
Clorofila , Mutação , Oryza , Folhas de Planta , Oryza/genética , Oryza/metabolismo , Oryza/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/metabolismo , Clorofila/metabolismo , Senescência Vegetal/genética , Mapeamento Cromossômico , Fenótipo , Regulação da Expressão Gênica de Plantas , Fotossíntese/genética , Genes de Plantas , Peróxido de Hidrogênio/metabolismoRESUMO
The "Indica to Japonica" initiative in China focuses on adapting Japonica rice varieties from the northeast to the unique photoperiod and temperature conditions of lower latitudes. While breeders can select varieties for their adaptability, the sensitivity to light and temperature often complicates and prolongs the process. Addressing the challenge of cultivating high-yield, superior-quality Japonica rice over expanded latitudinal ranges swiftly, in the face of these sensitivities, is critical. Our approach harnesses the CRISPR-Cas9 technology to edit the EHD1 gene in the premium northeastern Japonica cultivars Jiyuanxiang 1 and Yinongxiang 12, which are distinguished by their exceptional grain quality-increased head rice rates, gel consistency, and reduced chalkiness and amylose content. Field trials showed that these new ehd1 mutants not only surpass the wild types in yield when grown at low latitudes but also retain the desirable traits of their progenitors. Additionally, we found that disabling Ehd1 boosts the activity of Hd3a and RFT1, postponing flowering by approximately one month in the ehd1 mutants. This research presents a viable strategy for the accelerated breeding of elite northeastern Japonica rice by integrating genomic insights with gene-editing techniques suitable for low-latitude cultivation.
RESUMO
B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.
Assuntos
Antígenos CD28 , Linfócitos T CD8-Positivos , Camundongos , Animais , Antígenos CD28/metabolismo , Antígenos CD/metabolismo , Ligantes , Membranas Sinápticas/metabolismo , Antígeno B7-2 , Glicoproteínas de Membrana/metabolismo , Antígeno B7-1/metabolismo , Moléculas de Adesão Celular , Ativação LinfocitáriaRESUMO
CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.
Assuntos
Antígenos CD28 , Imunoconjugados , Humanos , Antígeno CTLA-4/metabolismo , Antígenos CD28/metabolismo , Antígenos CD/metabolismo , Ligantes , Antígenos de Diferenciação , Abatacepte/farmacologia , Antígeno B7-2 , Glicoproteínas de Membrana/metabolismo , Antígeno B7-1/metabolismo , Moléculas de Adesão CelularRESUMO
Chromatin regulators (CRs) regulate epigenetic patterns on a partial or global scale, playing a critical role in affecting multi-target gene expression. As chromatin immunoprecipitation sequencing (ChIP-seq) data associated with CRs are rapidly accumulating, a comprehensive resource of CRs needs to be built urgently for collecting, integrating, and processing these data, which can provide abundant annotated information on CR upstream and downstream regulatory analyses as well as CR-related analysis functions. This study established an integrative CR resource, named CRdb (http://cr.liclab.net/crdb/), with the aim of curating a large number of available resources for CRs and providing extensive annotations and analyses of CRs to help biological researchers clarify the regulation mechanism and function of CRs. The CRdb database comprised a total of 647 CRs and 2,591 ChIP-seq samples from more than 300 human tissues and cell types. These samples have been manually curated from NCBI GEO/SRA and ENCODE. Importantly, CRdb provided the abundant and detailed genetic annotations in CR-binding regions based on ChIP-seq. Furthermore, CRdb supported various functional annotations and upstream regulatory information on CRs. In particular, it embedded four types of CR regulatory analyses: CR gene set enrichment, CR-binding genomic region annotation, CR-TF co-occupancy analysis, and CR regulatory axis analysis. CRdb is a useful and powerful resource that can help in exploring the potential functions of CRs and their regulatory mechanism in diseases and biological processes.
Assuntos
Cromatina , Bases de Dados Genéticas , Genômica , Humanos , Cromatina/genética , Bases de Dados Factuais , Genoma , Anotação de Sequência MolecularRESUMO
Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD28/metabolismo , Humanos , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Because of labor shortages or resource scarcity, direct seeding is the preferred method for rice (Oryza sativa. L) cultivation, and it necessitates direct seeding at the current density. In this study, two density of direct seeding with high and normal density were selected to identify the genes involved in shade-avoidance syndrome. Phenotypic and gene expression analysis showed that densely direct seeding (DDS) causes a set of acclimation responses that either induce shade avoidance or toleration. When compared to normal direct seeding (NDS), plants cultivated by DDS exhibit constitutive shade-avoidance syndrome (SAS), in which the accompanying solar radiation drops rapidly from the middle leaf to the base leaf during flowering. Simulation of shade causes rapid reduction in phytochrome gene expression, changes in the expression of multiple miR156 or miR172 genes and photoperiod-related genes, all of which leads to early flowering and alterations in the plant architecture. Furthermore, DDS causes senescence by downregulating the expression of chloroplast synthesis-related genes throughout almost the entire stage. Our findings revealed that DDS is linked to SAS, which can be employed to breed density-tolerant rice varieties more easily and widely.
RESUMO
Transcription co-factors (TcoFs) play crucial roles in gene expression regulation by communicating regulatory cues from enhancers to promoters. With the rapid accumulation of TcoF associated chromatin immunoprecipitation sequencing (ChIP-seq) data, the comprehensive collection and integrative analyses of these data are urgently required. Here, we developed the TcoFBase database (http://tcof.liclab.net/TcoFbase), which aimed to document a large number of available resources for mammalian TcoFs and provided annotations and enrichment analyses of TcoFs. TcoFBase curated 2322 TcoFs and 6759 TcoFs associated ChIP-seq data from over 500 tissues/cell types in human and mouse. Importantly, TcoFBase provided detailed and abundant (epi) genetic annotations of ChIP-seq based TcoF binding regions. Furthermore, TcoFBase supported regulatory annotation information and various functional annotations for TcoFs. Meanwhile, TcoFBase embedded five types of TcoF regulatory analyses for users, including TcoF gene set enrichment, TcoF binding genomic region annotation, TcoF regulatory network analysis, TcoF-TF co-occupancy analysis and TcoF regulatory axis analysis. TcoFBase was designed to be a useful resource that will help reveal the potential biological effects of TcoFs and elucidate TcoF-related regulatory mechanisms.
Assuntos
Bases de Dados Genéticas , Redes Reguladoras de Genes , Software , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Cromatina/química , Cromatina/metabolismo , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Internet , Camundongos , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismoRESUMO
A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.
Assuntos
Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Domínios de Homologia de src , Comunicação Celular , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Células Jurkat , Receptor de Morte Celular Programada 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Imunológicos/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.
Assuntos
Apoptose , Caspase 8/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Caspase 8/genética , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Transcription-induced chimeric RNAs are an emerging area of research into molecular signatures for disease biomarker and therapeutic target development. Despite their importance, little is known for chimeric RNAs-relevant roles and the underlying mechanisms for cancer pathogenesis and progression. Here we describe a unique ASTN2-PAPPAantisense chimeric RNA (A-PaschiRNA) that could be the first reported chimeric RNA derived from the splicing of exons and intron antisense of two neighboring genes, respectively. Aberrant A-PaschiRNA level in ESCC tissues was associated with tumor progression and patients' outcome. In vitro and in vivo studies demonstrated that A-PaschiRNA aggravated ESCC metastasis and enhanced stemness through modulating OCT4. Mechanistic studies demonstrated that ERK5-mediated non-canonical PAF1 activity was required for A-PaschiRNA-induced cancer malignancy. The study defined an undocumented function of chimeric RNAs in aggravating cancer stemness and metastasis.
Assuntos
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Glicoproteínas/genética , Proteínas do Tecido Nervoso/genética , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/genética , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , RNA/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.
Assuntos
Proliferação de Células/genética , Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Animais , Complexo CD3/genética , Complexo CD3/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Citocinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Receptor de Morte Celular Programada 1/genética , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Imunológicos/genética , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/imunologia , Espectrometria de Massas em TandemRESUMO
Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.
Assuntos
Antígeno B7-1/metabolismo , Antígeno B7-H1/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunoterapia/métodos , Ipilimumab/farmacologia , Células Jurkat , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/terapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Evasion of apoptosis is a major contributing factor to the development of chemo- and radiotherapy resistance. Therefore, activation of non-apoptotic programmed cell death (PCD) could be an effective alternative against apoptosis-resistant cancers. In this study, we demonstrated in vitro and in vivo that metformin can induce pyroptosis, a non-apoptotic PCD, in esophageal squamous cell carcinoma (ESCC), a commonly known chemo-refractory cancer, especially at its advanced stages. Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogene and upregulated PELP1 in advanced stages of ESCC is highly associated with cancer progression and patient outcomes. Intriguingly, metformin treatment leads to gasdermin D (GSDMD)-mediated pyroptosis, which is abrogated by forced expression of PELP1. Mechanistically, metformin induces pyroptosis of ESCC by targeting miR-497/PELP1 axis. Our findings suggest that metformin and any other pyroptosis-inducing reagents could serve as alternative treatments for chemo- and radiotherapy refractory ESCC or other cancers sharing the same pyroptosis mechanisms.
Assuntos
Proteínas Correpressoras/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Metformina/farmacologia , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Correpressoras/biossíntese , Proteínas Correpressoras/genética , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Piroptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Piroptose , Aminoácidos/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Forma Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Necrose , Proteínas de Ligação a Fosfato , Multimerização Proteica , Transporte Proteico , Relação Estrutura-AtividadeAssuntos
Processamento Eletrônico de Dados/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Algoritmos , Linhagem Celular , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Processamento Eletrônico de Dados/normas , Humanos , Espectrometria de Massas/normas , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteômica/normas , SoftwareRESUMO
Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells.
Assuntos
Necrose , Fosfopeptídeos/análise , Fosfoproteínas/análise , Proteoma/análise , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Espectrometria de Massas , Camundongos , Fosfopeptídeos/imunologia , Fosfoproteínas/imunologia , Fosforilação , Proteoma/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.
Assuntos
Proteínas de Bactérias/química , Proteínas Quinases/química , Cristalografia por Raios X , Histidina Quinase , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Streptococcus mutans/metabolismoRESUMO
The γ-aminobutyric acid A receptor-associated protein (Gabarap) functions in γ-aminobutyric acid A receptor trafficking and postsynaptic localization in neurons, but its physiological roles in other systems have not been studied. In this study, we report that Gabarap-deficient mice are more susceptible to mortality in two sepsis models. An underlying mechanism of this higher mortality rate in Gabarap(-/-) septic mice is the higher level of proinflammatory cytokine expression in Gabarap(-/-) mice versus wild-type mice. In vitro studies show that Nlrp3 inflammasome activation is enhanced by Gabarap deficiency, as evidenced by more casapse-1 activation, more IL-1ß, and more IL-18 secretion in LPS- and ATP-treated Gabarap(-/-) macrophages. The Gabarap deficiency led to inefficient clearance of damaged mitochondria in LPS plus ATP-treated macrophages, resulting in more mitochondrial ROS and the release of mitochondrial DNA into cytosol. Both ROS and mitochondrial DNA are known to promote inflammasome activation. These results demonstrate that Gabarap functions in the immune system. It is involved in mitochondrial quality control in macrophages, and thus it influences Nlrp3 inflammasome-dependent inflammatory responses.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/genética , Deleção de Genes , Inflamassomos , Inflamação/genética , Inflamação/metabolismo , Proteínas de Membrana/genética , Animais , Proteínas Reguladoras de Apoptose , Transporte Biológico , Caspase 1/metabolismo , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Genes Letais , Predisposição Genética para Doença , Inflamação/imunologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitofagia/genética , Mitofagia/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio/metabolismo , Sepse/genética , Sepse/mortalidadeRESUMO
Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3(+/+)) and RIP3 knockout (RIP3(-/-)) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3(+/+) and RIP3(-/-) mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3(+/+) and RIP3(-/-) mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3(+/+) macrophages, 121 were detected exclusively from RIP3(+/+) MEFs, 286 phosphopeptides were induced more in RIP3(+/+) macrophages than in RIP3(-/-) macrophages and 26 phosphopeptides had higher induction in RIP3(+/+) MEFs than in RIP3(-/-) cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists.