RESUMO
Hepatocellular carcinoma (HCC) remains the most prevalent form of primary liver cancer, characterized by late detection and suboptimal response to current therapies. The tumour microenvironment, especially the role of M2 macrophages, is pivotal in the progression and prognosis of HCC. We applied the machine learning algorithm-CIBERSORT, to quantify cellular compositions within the HCC TME, focusing on M2 macrophages. Gene expression profiles were analysed to identify key molecules, with ATP6V1E1 as a primary focus. We employed Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis to investigate the molecular pathways and prognostic significance of ATP6V1E1. A prognostic model was developed using multivariate Cox regression analysis based on ATP6V1E1-related molecules, and functional impacts were assessed through cell proliferation assays. M2 macrophages were the dominant cell type in the HCC TME, significantly correlating with adverse survival outcomes. ATP6V1E1 was robustly associated with advanced disease stages and poor prognostic features such as vascular invasion and elevated alpha-fetoprotein levels. GSEA linked high ATP6V1E1 expression to critical oncogenic pathways, including immunosuppression and angiogenesis, and reduced activity in metabolic processes like bile acid and fatty acid metabolism. The prognostic model stratified HCC patients into distinct risk categories, showing high predictive accuracy (1-year AUC = 0.775, 3-year AUC = 0.709 and 5-year AUC = 0.791). In vitro assays demonstrated that ATP6V1E1 knockdown markedly inhibited the proliferation of HCC cells. The study underscores the significance of M2 macrophages and ATP6V1E1 in HCC, highlighting their potential as therapeutic and prognostic targets.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Aprendizado de Máquina , Macrófagos , Microambiente Tumoral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Humanos , Macrófagos/metabolismo , Microambiente Tumoral/genética , Prognóstico , Regulação Neoplásica da Expressão Gênica , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Masculino , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Estimativa de Kaplan-Meier , Algoritmos , Pessoa de Meia-IdadeRESUMO
Vacuolar-type H+-ATPases (V-ATPases) play a crucial role in the life cycle of agricultural pests and represent a promising target for the development of novel insecticides. In this study, S18, a derivative of vanillin acquired from Specs database using a structure-based virtual screening methodology, was first identified as a V-ATPase inhibitor. It binds to subunit A of the enzyme with a Kd of 1 nM and exhibits insecticidal activity against M. separata. Subsequently, using S18 as the lead compound, a new series of vanillin derivatives were rationally designed and efficiently synthesized. and their biological activities were assessed. Among them, compound 3b-03 showed the strongest insecticidal activity against M. separata by effectively targeting the V-ATPase subunit A with Kd of 0.803 µM. Isothermal titration calorimetric measurements and docking results provided insights into its interaction with subunit A of V-ATPase, which could facilitate future research aimed at the development of novel chemical insecticides.
Assuntos
Benzaldeídos , Inseticidas , Simulação de Acoplamento Molecular , ATPases Vacuolares Próton-Translocadoras , Inseticidas/química , Inseticidas/farmacologia , Inseticidas/síntese química , Animais , Benzaldeídos/química , Benzaldeídos/farmacologia , Relação Estrutura-Atividade , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Estrutura Molecular , HalogenaçãoRESUMO
Head and neck squamous cell carcinoma (HNSCC) present a significant challenge due to its heterogeneity and limited treatment options, often resulting in severe side effects and poor survival rates with conventional chemoradiotherapy. Here, we investigated the anticancer activity of halogenated benzoate derivatives of cleistanthin A, ECDD-S16 and ECDD-S18, in HNSCC cells. Our findings revealed that ECDD-S18 exhibited remarkable cytotoxicity, surpassing that of cisplatin with minimal impact on normal and cisplatin-sensitive cells. Notably, ECDD-S18 induced apoptosis in a dose-dependent manner and effectively targeted vacuolar ATPase (V-ATPase), impairing lysosomal acidification. Intriguingly, ECDD-S18 inhibited autophagic flux, as evidenced by increased autophagosome but decreased autolysosome formation. Furthermore, proteomic analysis demonstrated downregulation of cathepsin D (CTSD), the lysosomal protease in ECDD-S18-treated HNSCC cells, concurrent with suppressed cell migration. ECDD-S18 also decreased expression of mesenchymal markers, suggesting inhibition of epithelial-mesenchymal transition (EMT). Importantly, cotreatment with ECDD-S18 and cisplatin enhanced the reduction in cell viability. Collectively, our results indicated that the anticancer activity of ECDD-S18 partly stems from its ability to disrupt lysosomal acidification and inhibit autophagy via targeted inhibition of V-ATPase. These findings underscore the therapeutic promise of ECDD-S18 in HNSCC treatment, either alone or in combination with existing drugs, while mitigating toxicity to normal cells.
Assuntos
Autofagia , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , ATPases Vacuolares Próton-Translocadoras , Humanos , Autofagia/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Cisplatino/farmacologia , Movimento Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Catepsina D/metabolismoRESUMO
The vacuolar-type H+-translocating ATPase (V-ATPase) is the major proton pump for intraorganellar acidification. Therefore, the integrity of the V-ATPase is closely associated with cellular homeostasis, and mutations in genes encoding V-ATPase components and assembly factors have been reported in certain types of diseases. For instance, the recurrent mutations of ATP6AP1, a gene encoding a V-ATPase accessory protein, have been associated with cancers and immunodeficiency. With the aim of studying V-ATPase-related mutations using the yeast model system, we report that Big1 is another homologue of ATP6AP1 in yeast cells, and we characterize the role of Big1 in maintaining a fully functional V-ATPase. In addition to its role in acidifying the vacuole or lysosome, our data support the concept that the V-ATPase may function as part of a signaling pathway to regulate macroautophagy/autophagy through a mechanism that is independent from Tor/MTOR.
Assuntos
Autofagia , Lisossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Vacúolos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Lisossomos/metabolismo , Vacúolos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Mutação/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The natural lignan diphyllin has shown promising antitumor activity, although its clinical advancement has been impeded by challenges such as low solubility, poor metabolic stability, and limited potency. In response, we developed and synthesized two sets of diphyllin 4-C derivatives, comprising six ester derivatives and eight 1, 2, 3-triazole derivatives. Notably, among these derivatives, 1, 2, 3-triazole derivatives 7c and 7e demonstrated the most potent cytotoxic effects, with IC50 values ranging from 0.003 to 0.01 µM. Treatment with 0.2 µM of 7c and 7e resulted in a reduction of V-ATPase activity in HGC-27 cells to 23% and 29%, respectively.
Assuntos
Antineoplásicos , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Lignanas/farmacologia , Lignanas/química , Lignanas/síntese química , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Azidas/síntese química , Azidas/química , Azidas/farmacologiaRESUMO
Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H+ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction.
Assuntos
Encéfalo , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Encéfalo/patologia , Encéfalo/metabolismo , Animais , Lisossomos/metabolismo , Lisossomos/enzimologia , Encefalopatias/genética , Encefalopatias/metabolismo , Encefalopatias/enzimologia , Encefalopatias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismoRESUMO
Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.
Assuntos
Autofagossomos , Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte , Lisossomos , Lisossomos/metabolismo , Humanos , Autofagossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Sistemas CRISPR-Cas , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Hidrolases/metabolismo , Hidrolases/genética , Células HeLa , Células HEK293RESUMO
The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.
Assuntos
Neoplasias Pulmonares , Anticorpos de Domínio Único , ATPases Vacuolares Próton-Translocadoras , Animais , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/imunologia , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Feminino , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Linhagem Celular Tumoral , Neoplasias da Mama/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Camundongos Endogâmicos BALB C , Humanos , Invasividade NeoplásicaRESUMO
Anaplastic thyroid cancer (ATC) is among the most aggressive and metastatic malignancies, often resulting in fatal outcomes due to the lack of effective treatments. Prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has shown potential as an antineoplastic agent against various human tumors. However, its effects on ATC and the underlying mechanism remain unclear. Here, we demonstrate that PA exhibits significant anti-ATC activity both in vitro and in vivo by inducing GSDME-dependent pyroptosis in ATC cells. Mechanistically, PA promotes lysosomal membrane permeabilization (LMP), leading to the release of cathepsins that activate caspase 8/3 to cleave GSDME. Remarkably, PA significantly upregulates three key functional subunits of V-ATPase-ATP6V1A, ATP6V1B2, and ATP6V0C-resulting in lysosomal over-acidification. This over-acidification exacerbates LMP and subsequent lysosomal damage. Neutralization of lysosomal lumen acidification or inhibition/knockdown of these V-ATPase subunits attenuates PA-induced lysosomal damage, pyroptosis and growth inhibition of ATC cells, highlighting the critical role for lysosomal acidification and LMP in PA's anticancer effects. In summary, our findings uncover a novel link between PA and lysosomal damage-dependent pyroptosis in cancer cells. PA may act as a V-ATPase agonist targeting lysosomal acidification, presenting a new potential therapeutic option for ATC treatment.
Assuntos
Lisossomos , Piroptose , Carcinoma Anaplásico da Tireoide , ATPases Vacuolares Próton-Translocadoras , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Humanos , Piroptose/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sapogeninas/farmacologia , Camundongos , Camundongos Nus , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , GasderminasRESUMO
Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.
Assuntos
Proteínas Relacionadas à Autofagia , ATPases Vacuolares Próton-Translocadoras , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Animais , Camundongos , Ligação Proteica , Neurônios/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia , Células HEK293 , Células-Tronco Pluripotentes Induzidas/metabolismo , Influenza Humana/virologia , Influenza Humana/metabolismo , Influenza Humana/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos KnockoutRESUMO
ATP6AP2 knockout in the renal nephron impairs receptor-mediated endocytosis, increasing urinary albumin and glucose excretion and impairing weight gain. Nonesterified fatty acids (NEFA) in urine are bound to albumin and reabsorbed in the proximal tubule through receptor-mediated endocytosis by the megalin-cubilin complex. We hypothesized that ATP6AP2 knockout increases urinary NEFA excretion through a reduction in megalin. Ten-week-old male C57BL/6 mice with nephron specific inducible ATP6AP2 knockout and noninduced controls were fed either normal diet (ND 12% fat) or high fat diet (HFD 45% fat) for 6 months. ATP6AP2 knockout significantly increased urine albumin:creatinine ratio in both ND and HFD fed mice while normalized urine NEFA concentration increased 489% and 259% in ND and HFD knockout mice compared to respective controls. Knockout decreased renal cortical megalin mRNA by 47% on ND and 49% on HFD while megalin protein expression decreased by 36% and 44% respectively. At the same time, markers of mTOR activity were increased while autophagy was impaired. Our results indicate that nephron specific ATP6AP2 knockout increases urinary NEFA excretion in the setting of impaired receptor-mediated endocytosis. Further investigation should determine whether ATP6AP2 contributes to obesity related ectopic lipid deposition in the proximal tubule.
Assuntos
Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Néfrons , Animais , Masculino , Camundongos , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Córtex Renal/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Néfrons/metabolismo , Receptor de Pró-Renina , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ATPases Vacuolares Próton-Translocadoras , Caenorhabditis elegans/genética , Animais , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Divisão Celular Assimétrica , Apoptose , Epigênese Genética , Nucleossomos/metabolismoRESUMO
Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-ß1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-ß1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.
Assuntos
Linfócitos T CD8-Positivos , Colesterol , Neoplasias Colorretais , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Humanos , Animais , Colesterol/metabolismo , Camundongos , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta1/metabolismo , Memória Imunológica , ATPases Vacuolares Próton-Translocadoras/metabolismo , Microambiente Tumoral/imunologia , Receptores X do Fígado/metabolismo , Hidroxicolesteróis/metabolismo , Hidroxicolesteróis/farmacologia , Pirrolidinas/farmacologia , Proteína Smad3/metabolismo , Camundongos Endogâmicos C57BL , Carbamatos/farmacologiaRESUMO
Diabetes-associated periodontitis (DP) presents severe inflammation and resistance to periodontal conventional treatment, presenting a significant challenge in clinical management. In this study, we investigated the underlying mechanism driving the hyperinflammatory response in gingival epithelial cells (GECs) of DP patients. Our findings indicate that lysosomal dysfunction under high glucose conditions leads to the blockage of autophagy flux, exacerbating inflammatory response in GECs. Single-cell RNA sequencing and immunohistochemistry analyses of clinical gingival epithelia revealed dysregulation in the lysosome pathway characterized by reduced levels of lysosome-associated membrane glycoprotein 2 (LAMP2) and V-type proton ATPase 16 kDa proteolipid subunit c (ATP6V0C) in subjects with DP. In vitro stimulation of human gingival epithelial cells (HGECs) with a hyperglycemic microenvironment showed elevated release of proinflammatory cytokines, compromised lysosomal acidity and blocked autophagy. Moreover, HGECs with deficiency in ATP6V0C demonstrated impaired autophagy and heightened inflammatory response, mirroring the effects of high glucose stimulation. Proteomic analysis of acetylation modifications identified altered acetylation levels in 28 autophagy-lysosome pathway-related proteins and 37 sites in HGECs subjected to high glucose stimulation or siATP6V0C. Overall, our finding highlights the pivotal role of lysosome impairment in autophagy obstruction in DP and suggests a potential impact of altered acetylation of relevant proteins on the interplay between lysosome dysfunction and autophagy blockage. These insights may pave the way for the development of effective therapeutic strategies against DP.
Assuntos
Autofagia , Células Epiteliais , Gengiva , Lisossomos , Periodontite , Humanos , Lisossomos/metabolismo , Acetilação , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Gengiva/metabolismo , Gengiva/patologia , Periodontite/metabolismo , Periodontite/patologia , Periodontite/complicações , Masculino , Feminino , ATPases Vacuolares Próton-Translocadoras/metabolismo , Pessoa de Meia-Idade , Glucose/farmacologia , AdultoRESUMO
In this issue of Structure, Oot and Wilkens1 present new mechanistic insights to finally merge the function of V-ATPase and TLDc domain proteins. They show that TLDc proteins directly affect V-ATPase activity and assembly, expanding our understanding of how V-ATPase and TLDc proteins exert a plethora of biological functions.
Assuntos
ATPases Vacuolares Próton-Translocadoras , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Humanos , Domínios ProteicosRESUMO
The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.
Assuntos
Camundongos Knockout , ATPases Vacuolares Próton-Translocadoras , Animais , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Camundongos , Rim/metabolismo , Genes Essenciais/genéticaRESUMO
Accumulation of α-synuclein (α-Syn) has been implicated in proteasome and autophagy dysfunction in Parkinson's disease (PD). High frequency electrical stimulation (HFS) mimicking clinical parameters used for deep brain stimulation (DBS) in vitro or DBS in vivo in preclinical models of PD have been found to reduce levels of α-Syn and, in certain cases, provide possible neuroprotection. However, the mechanisms by which this reduction in α-Syn improves cellular dysfunction associated with α-Syn accumulation remains elusive. Using HFS parameters that recapitulate DBS in vitro, we found that HFS led to a reduction of mutant α-Syn and thereby limited proteasome and autophagy impairments due to α-Syn. Additionally, we observed that HFS modulates via the ATP6V0C subunit of V-ATPase and mitigates α-Syn mediated autophagic dysfunction. This study highlights a role for autophagy in reduction of α-Syn due to HFS which may prove to be a viable approach to decrease pathological protein accumulation in neurodegeneration.
Assuntos
Autofagia , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Animais , Estimulação Elétrica/métodos , Estimulação Encefálica Profunda/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , CamundongosRESUMO
Emerging evidence indicates that fatty acid (FA) metabolic pathways regulate host immunity to vertebrate viruses. However, information on FA signaling in plant virus infection remains elusive. In this study, we demonstrate the importance of fatty acid desaturase (FAD), an enzyme that catalyzes the rate-limiting step in the conversion of saturated FAs into unsaturated FAs, during infection by a plant RNA virus. We previously found that the rare Kua-ubiquitin-conjugating enzyme (Kua-UEV1) fusion protein FAD4 from Nicotiana benthamiana (NbFAD4) was downregulated upon turnip mosaic virus (TuMV) infection. We now demonstrate that NbFAD4 is unstable and is degraded as TuMV infection progresses. NbFAD4 is required for TuMV replication, as it interacts with TuMV replication protein 6K2 and colocalizes with viral replication complexes. Moreover, NbFAD4 overexpression dampened the accumulation of immunity-related phytohormones and FA metabolites, and its catalytic activity appears to be crucial for TuMV infection. Finally, a yeast 2-hybrid library screen identified the vacuolar H+-ATPase component ATP6V0C as involved in NbFAD4 degradation and further suppression of TuMV infection. This study reveals the intricate role of FAD4 in plant virus infection, and sheds light on a new mechanism by which a V-ATPase is involved in plant antiviral defense.
Assuntos
Ácidos Graxos Dessaturases , Nicotiana , Proteínas de Plantas , Potyvirus , ATPases Vacuolares Próton-Translocadoras , Replicação Viral , Nicotiana/virologia , Nicotiana/genética , Nicotiana/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Dessaturases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Potyvirus/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Ácidos Graxos/metabolismo , Doenças das Plantas/virologia , Interações Hospedeiro-Patógeno , Reguladores de Crescimento de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , ProteóliseRESUMO
Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H+-ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H+-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.
Assuntos
Células Epiteliais Alveolares , ATPases Vacuolares Próton-Translocadoras , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Camundongos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Organelas/metabolismo , Camundongos KnockoutRESUMO
During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.