RESUMO
Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidilserinas/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/metabolismo , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cultura Primária de Células , Transporte Proteico/fisiologia , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.
Assuntos
Ritmo Circadiano/imunologia , Inflamação/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Animais , Degranulação Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação/imunologia , Camundongos , Neutrófilos/imunologia , Pneumonia/complicações , Pneumonia/imunologia , Proteoma/imunologia , Proteoma/metabolismo , Síndrome do Desconforto Respiratório/imunologiaRESUMO
Succinate is a signaling metabolite sensed extracellularly by succinate receptor 1 (SUNCR1). The accumulation of succinate in macrophages is known to activate a pro-inflammatory program; however, the contribution of SUCNR1 to macrophage phenotype and function has remained unclear. Here we found that activation of SUCNR1 had a critical role in the anti-inflammatory responses in macrophages. Myeloid-specific deficiency in SUCNR1 promoted a local pro-inflammatory phenotype, disrupted glucose homeostasis in mice fed a normal chow diet, exacerbated the metabolic consequences of diet-induced obesity and impaired adipose-tissue browning in response to cold exposure. Activation of SUCNR1 promoted an anti-inflammatory phenotype in macrophages and boosted the response of these cells to type 2 cytokines, including interleukin-4. Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects, who had lower expression of SUCNR1 in adipose-tissue-resident macrophages. Our findings highlight the importance of succinate-SUCNR1 signaling in determining macrophage polarization and assign a role to succinate in limiting inflammation.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Receptores Acoplados a Proteínas G/imunologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Ácido Succínico/imunologia , Ácido Succínico/metabolismo , Ácido Succínico/farmacologia , Células THP-1RESUMO
Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.
Assuntos
COVID-19 , Neoplasias Pulmonares , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Proteínas de Membrana Lisossomal , Autofagia , ProteínasRESUMO
Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.
Assuntos
Caveolina 1/metabolismo , Metástase Neoplásica/patologia , Neoplasias/patologia , Animais , Movimento Celular , Fibroblastos/patologia , Humanos , Melanoma/patologia , Camundongos , Camundongos KnockoutRESUMO
Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expressions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses.
Assuntos
Calcineurina , Dióxido de Nitrogênio , Camundongos , Animais , Calcineurina/metabolismo , Ácido Oleico , Processamento de Proteína Pós-Traducional , Ácidos Graxos/metabolismoRESUMO
This study investigated the influence of photoperiod (day length) on the efficacy of grape seed proanthocyanidin extract (GSPE) in mitigating metabolic disorders in obese rats fed a cafeteria diet. Rats were exposed to standard (L12), long (L18), or short (L6) photoperiods and treated with GSPE or vehicle. In the standard photoperiod, GSPE reduced body weight gain (50.5%), total cholesterol (37%), and triglycerides (34.8%), while increasing the expression of hepatic metabolic genes. In the long photoperiod, GSPE tended to decrease body weight gain, increased testosterone levels (68.3%), decreased liver weight (12.4%), and decreased reverse serum amino acids. In the short photoperiod, GSPE reduced glycemia (~10%) and lowered triglyceride levels (38.5%), with effects modified by diet. The standard photoperiod showed the greatest efficacy against metabolic syndrome-associated diseases. The study showed how day length affects GSPE's benefits and underscores considering biological rhythms in metabolic disease therapies.
Assuntos
Extrato de Sementes de Uva , Fígado , Fotoperíodo , Proantocianidinas , Animais , Proantocianidinas/farmacologia , Extrato de Sementes de Uva/farmacologia , Ratos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Masculino , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Ratos Wistar , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Obesidade/etiologiaRESUMO
BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.
Assuntos
Fragilidade , Cardiopatias , Hipertensão Pulmonar , Adulto , Animais , DNA Mitocondrial/genética , Fragilidade/patologia , Cardiopatias/patologia , Heteroplasmia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Camundongos , Mitocôndrias/genéticaRESUMO
Disruptions of the light/dark cycle and unhealthy diets can promote misalignment of biological rhythms and metabolic alterations, ultimately leading to an oxidative stress condition. Grape seed proanthocyanidin extract (GSPE), which possesses antioxidant properties, has demonstrated its beneficial effects in metabolic-associated diseases and its potential role in modulating circadian disruptions. Therefore, this study aimed to assess the impact of GSPE administration on the liver oxidant system of healthy and diet-induced obese rats undergoing a sudden photoperiod shift. To this end, forty-eight photoperiod-sensitive Fischer 344/IcoCrl rats were fed either a standard (STD) or a cafeteria diet (CAF) for 6 weeks. A week before euthanizing, rats were abruptly transferred from a standard photoperiod of 12 h of light/day (L12) to either a short (6 h light/day, L6) or a long photoperiod (18 h light/day, L18) while receiving a daily oral dose of vehicle (VH) or GSPE (25 mg/kg). Alterations in body weight gain, serum and liver biochemical parameters, antioxidant gene and protein expression, and antioxidant metabolites were observed. Interestingly, GSPE partially ameliorated these effects by reducing the oxidative stress status in L6 through an increase in GPx1 expression and in hepatic antioxidant metabolites and in L18 by increasing the NRF2/KEAP1/ARE pathway, thereby showing potential in the treatment of circadian-related disorders by increasing the hepatic antioxidant response in a photoperiod-dependent manner.
Assuntos
Extrato de Sementes de Uva , Proantocianidinas , Ratos , Animais , Antioxidantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fotoperíodo , Ratos Wistar , Fator 2 Relacionado a NF-E2/metabolismo , Extrato de Sementes de Uva/farmacologia , Extrato de Sementes de Uva/uso terapêutico , Proantocianidinas/metabolismo , Obesidade/tratamento farmacológico , Obesidade/etiologia , Obesidade/metabolismo , Fígado/metabolismoRESUMO
The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2 + IV2 , I + IV, and I + III2 + IV2 . To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1-/- fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Fatores de Processamento de Serina-Arginina/metabolismo , Peixe-Zebra , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/genética , Feminino , Camundongos , Membranas Mitocondriais/metabolismo , Fosforilação Oxidativa , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Respiratory chain complexes can super-assemble into quaternary structures called supercomplexes that optimize cellular metabolism. The interaction between complexes III (CIII) and IV (CIV) is modulated by supercomplex assembly factor 1 (SCAF1, also known as COX7A2L). The discovery of SCAF1 represented strong genetic evidence that supercomplexes exist in vivo. SCAF1 is present as a long isoform (113 amino acids) or a short isoform (111 amino acids) in different mouse strains. Only the long isoform can induce the super-assembly of CIII and CIV, but it is not clear whether SCAF1 is required for the formation of the respirasome (a supercomplex of CI, CIII2 and CIV). Here we show, by combining deep proteomics and immunodetection analysis, that SCAF1 is always required for the interaction between CIII and CIV and that the respirasome is absent from most tissues of animals containing the short isoform of SCAF1, with the exception of heart and skeletal muscle. We used directed mutagenesis to characterize SCAF1 regions that interact with CIII and CIV and discovered that this interaction requires the correct orientation of a histidine residue at position 73 that is altered in the short isoform of SCAF1, explaining its inability to interact with CIV. Furthermore, we find that the CIV subunit COX7A2 is replaced by SCAF1 in supercomplexes containing CIII and CIV and by COX7A1 in CIV dimers, and that dimers seem to be more stable when they include COX6A2 rather than the COX6A1 isoform.
Assuntos
Membranas Mitocondriais/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/químicaRESUMO
Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.
Assuntos
Envelhecimento/genética , Núcleo Celular/genética , DNA Mitocondrial/genética , Variação Genética/genética , Metabolismo/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Envelhecimento/fisiologia , Animais , Feminino , Genoma Mitocondrial/genética , Haplótipos , Insulina/metabolismo , Longevidade/genética , Masculino , Metabolismo/fisiologia , Metabolômica , Camundongos , Camundongos Congênicos , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Obesidade/genética , Obesidade/metabolismo , Fenótipo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Encurtamento do Telômero , Transcriptoma , Resposta a Proteínas não DobradasRESUMO
The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+ channels can influence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independent of K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently of K+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here, we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.
Assuntos
Proliferação de Células , Ativação do Canal Iônico , Canal de Potássio Kv1.3/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Canais KATP/metabolismo , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/genética , Potenciais da Membrana , Mutação , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of ß-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated ß-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of ß-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.
Assuntos
Actinas/metabolismo , Sinapses Imunológicas/enzimologia , Isoenzimas/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Profilinas/metabolismo , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Células Cultivadas , Cisteína/metabolismo , Ativação Enzimática , Complexo de Golgi/enzimologia , Complexo de Golgi/imunologia , Complexo de Golgi/metabolismo , Humanos , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Isoenzimas/química , Isoenzimas/genética , Proteínas Luminescentes/antagonistas & inibidores , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Profilinas/genética , Proteína Quinase C/química , Proteína Quinase C/genética , Proteína Quinase C-theta , Transporte Proteico , Pseudópodes , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Cadherin-based intercellular adhesions are essential players in epithelial homeostasis, but their dynamic regulation during tissue morphogenesis and remodeling remain largely undefined. Here, we characterize an unexpected role for the membrane-anchored metalloproteinase MT2-MMP in regulating epithelial cell quiescence. Following co-immunoprecipitation and mass spectrometry, the MT2-MMP cytosolic tail was found to interact with the zonula occludens protein-1 (ZO-1) at the apical junctions of polarized epithelial cells. Functionally, MT2-MMP localizes in the apical domain of epithelial cells where it cleaves E-cadherin and promotes epithelial cell accumulation, a phenotype observed in 2D polarized cells as well as 3D cysts. MT2-MMP-mediated cleavage subsequently disrupts apical E-cadherin-mediated cell quiescence resulting in relaxed apical cortical tension favoring cell extrusion and re-sorting of Src kinase activity to junctional complexes, thereby promoting proliferation. Physiologically, MT2-MMP loss of function alters E-cadherin distribution, leading to impaired 3D organoid formation by mouse colonic epithelial cells ex vivo and reduction of cell proliferation within intestinal crypts in vivo Taken together, these studies identify an MT2-MMP-E-cadherin axis that functions as a novel regulator of epithelial cell homeostasis in vivo.
Assuntos
Caderinas/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Metaloproteinase 15 da Matriz/metabolismo , Junções Aderentes/metabolismo , Caderinas/genética , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Junções Intercelulares/metabolismo , Junções Íntimas/metabolismoRESUMO
It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to ßHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by ßIII-tubulin (ßIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with ßIII-tubulin and ßIII tubulin-mediated drug resistance. This supports the idea that overexpression of ßIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.
Assuntos
Microtúbulos/química , Compostos Policíclicos/química , Tubulina (Proteína)/química , Resistencia a Medicamentos Antineoplásicos , Ácido Edético/química , Células HeLa , Humanos , Espectrometria de Massas , Taxoides/químicaRESUMO
HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics, including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4(+)T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8(+)T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6(-/-)CD8(+)T cells to Rag1(-/-)mice demonstrated specific impairment in CD8(+)T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin-1-dynactin-mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFN)γ production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Citotoxicidade Imunológica/imunologia , Histona Desacetilases/metabolismo , Linfócitos T Citotóxicos/imunologia , Vacínia/imunologia , Animais , Transporte Biológico/fisiologia , Células Cultivadas , Citotoxicidade Imunológica/genética , Complexo Dinactina/antagonistas & inibidores , Desacetilase 6 de Histona , Histona Desacetilases/genética , Interferon gama/metabolismo , Cinesinas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to ß-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of ß-tubulin.
Assuntos
Sítios de Ligação/fisiologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Colchicina/metabolismo , Macrolídeos/metabolismo , Nucleotídeos/metabolismo , Taxoides/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Produtos Biológicos/metabolismo , Bovinos , Humanos , Ligantes , Microtúbulos/metabolismoRESUMO
Dectin-1 (Clec7a) is a paradigmatic C-type lectin receptor that binds Syk through a hemITAM motif and couples sensing of pathogens such as fungi to induction of innate responses. Dectin-1 engagement triggers a plethora of activating events, but little is known about the modulation of such pathways. Trying to define a more precise picture of early Dectin-1 signaling, we explored the interactome of the intracellular tail of the receptor in mouse dendritic cells. We found unexpected binding of SHIP-1 phosphatase to the phosphorylated hemITAM. SHIP-1 colocalized with Dectin-1 during phagocytosis of zymosan in a hemITAM-dependent fashion. Moreover, endogenous SHIP-1 relocated to live or heat-killed Candida albicans-containing phagosomes in a Dectin-1-dependent manner in GM-CSF-derived bone marrow cells (GM-BM). However, SHIP-1 absence in GM-BM did not affect activation of MAPK or production of cytokines and readouts dependent on NF-κB and NFAT. Notably, ROS production was enhanced in SHIP-1-deficient GM-BM treated with heat-killed C. albicans, live C. albicans, or the specific Dectin-1 agonists curdlan or whole glucan particles. This increased oxidative burst was dependent on Dectin-1, Syk, PI3K, phosphoinositide-dependent protein kinase 1, and NADPH oxidase. GM-BM from CD11c∆SHIP-1 mice also showed increased killing activity against live C. albicans that was dependent on Dectin-1, Syk, and NADPH oxidase. These results illustrate the complexity of myeloid C-type lectin receptor signaling, and how an activating hemITAM can also couple to intracellular inositol phosphatases to modulate selected functional responses and tightly regulate processes such as ROS production that could be deleterious to the host.