RESUMO
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
Assuntos
Infecções por Flavivirus/genética , Flavivirus/fisiologia , Proteínas de Membrana/metabolismo , Animais , Povo Asiático/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Sistemas CRISPR-Cas , Linhagem Celular , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/fisiologia , Replicação Viral , Vírus da Febre Amarela/fisiologia , Zika virus/fisiologiaRESUMO
During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.
Assuntos
Autofagossomos/enzimologia , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Proteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Animais Geneticamente Modificados , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia , Células COS , Sistemas CRISPR-Cas , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Chlorocebus aethiops , Genótipo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas Musculares/metabolismo , Fenótipo , Fosfatos de Fosfatidilinositol/metabolismo , Proteolipídeos/metabolismo , Interferência de RNA , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , TransfecçãoRESUMO
The endoplasmic reticulum (ER) is a central hub for the biogenesis of various organelles and lipid-containing structures. Recent studies suggest that vacuole membrane protein 1 (VMP1) and transmembrane protein 41B (TMEM41B), multispanning ER membrane proteins, regulate the formation of many of these ER-derived structures, including autophagosomes, lipid droplets, lipoproteins, and double-membrane structures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. VMP1 and TMEM41B possess a DedA domain that is widely distributed not only in eukaryotes but also in prokaryotes and predicted to adopt a characteristic structure containing two reentrant loops. Furthermore, recent studies show that both proteins have lipid scrambling activity. Based on these findings, the potential roles of VMP1 and TMEM41B in the dynamic remodeling of ER membranes and the biogenesis of ER-derived structures are discussed.
Assuntos
Autofagia , Proteínas de Membrana/genética , Retículo Endoplasmático/genética , HumanosRESUMO
BACKGROUND: Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson's disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. OBJECTIVE: This study aimed to characterize how altered VMP1 expression affects NLRP3 inflammasome activation and mitochondrial function. METHODS: VMP1 expression was depleted in a monocytic cell line using CRISPR-Cas9. The effect of VMP1 on NLRP3 inflammasome activation was examined by stimulating cells with LPS and ATP or α-synuclein fibrils. Inflammasome activation was determined by caspase-1 activation using both a FLICA assay and a biosensor as well as by the release of proinflammatory molecules measured by ELISA. RNA-sequencing was utilized to define global gene expression changes resulting from VMP1 deletion. SERCA activity and mitochondrial function were investigated using various fluorescence microscopy-based approaches including a novel method that assesses the function of individual mitochondria in a cell. RESULTS: Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 inflammasome activation and release of proinflammatory molecules. Examination of the VMP1-dependent changes in these cells revealed that VMP1 deficiency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. CONCLUSIONS: Collectively, these studies reveal VMP1 as a negative regulator of inflammatory responses, and we postulate that decreased expression of VMP1 can aggravate the inflammatory sequelae associated with neurodegenerative diseases like PD.
Assuntos
Inflamassomos , Doenças Mitocondriais , Humanos , Inflamassomos/metabolismo , Proteínas de Membrana/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Vacúolos/metabolismoRESUMO
The asymmetric distribution of lipids, maintained by flippases/floppases and scramblases, plays a pivotal role in various physiologic processes. Scramblases are proteins that move phospholipids between the leaflets of the lipid bilayer of the cellular membrane in an energy-independent manner. Recent studies have indicated that viral infection is closely related to cellular lipid distribution. The level and distribution of phosphatidylserine (PtdSer) in cells have been demonstrated to be critical regulators of viral infections. Previous studies have supported that the infection of human immunodeficiency virus (HIV), Zika virus, Ebola virus (EBOV), influenza virus, and dengue fever virus require the externalization of phospholipids mediated by scramblases, which are also involved in the pathogenicity of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we review the relationship of scramblases with viruses and the potential viral effector proteins that might utilize host scramblases.
Assuntos
COVID-19 , Viroses , Infecção por Zika virus , Zika virus , Humanos , SARS-CoV-2 , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismoRESUMO
The autophagy protein ATG2, proposed to transfer bulk lipid from the endoplasmic reticulum (ER) during autophagosome biogenesis, interacts with ER residents TMEM41B and VMP1 and with ATG9, in Golgi-derived vesicles that initiate autophagosome formation. In vitro assays reveal TMEM41B, VMP1, and ATG9 as scramblases. We propose a model wherein membrane expansion results from the partnership of a lipid transfer protein, moving lipids between the cytosolic leaflets of apposed organelles, and scramblases that reequilibrate the leaflets of donor and acceptor organelle membranes as lipids are depleted or augmented. TMEM41B and VMP1 are implicated broadly in lipid homeostasis and membrane dynamics processes in which their scrambling activities likely are key.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Autofagossomos/metabolismo , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Modelos Biológicos , Modelos Teóricos , Biogênese de Organelas , Proteínas de Transferência de Fosfolipídeos/fisiologiaRESUMO
Autophagy, a catabolic process orchestrating the degradation of proteins and organelles within lysosomes, is pivotal for maintaining cellular homeostasis. However, its dual role in cancer involves preventing malignant transformation while fostering progression and therapy resistance. Vacuole Membrane Protein 1 (VMP1) is an essential autophagic protein whose expression, per se, triggers autophagy, being present in the whole autophagic flux. In pancreatic cancer, VMP1-whose expression is linked to the Kirsten Rat Sarcoma Virus (KRAS) oncogene-significantly contributes to disease promotion, progression, and chemotherapy resistance. This investigation extends to breast cancer, colon cancer, hepatocellular carcinoma, and more, highlighting VMP1's nuanced nature, contingent on specific tissue contexts. The examination of VMP1's interactions with micro-ribonucleic acids (miRNAs), including miR-21, miR-210, and miR-124, enhances our understanding of its regulatory network in cancer. Additionally, this article discusses VMP1 gene fusions, especially with ribosomal protein S6 kinase B1 (RPS6KB1), shedding light on potential implications for tumor malignancy. By deciphering the molecular mechanisms linking VMP1 to cancer progression, this exploration paves the way for innovative therapeutic strategies to disrupt these pathways and potentially improve treatment outcomes.
Assuntos
Carcinoma Hepatocelular , Neoplasias do Colo , Neoplasias Hepáticas , Proteínas de Membrana , MicroRNAs , Humanos , Autofagia/genética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , MicroRNAs/genéticaRESUMO
TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown. Here, we conduct a remote homology search and identify a new clade consisting mainly of bacterial proteins of unknown function that are members of the Pfam family PF06695. Phylogenetic analysis reveals that the TMEM41, VMP1, DedA and PF06695 families form a superfamily with a common origin, which we term the DedA superfamily. Coevolution-based structural prediction suggests that the DedA domain contains two reentrant loops facing each other in the membrane. This topology is biochemically verified by the substituted cysteine accessibility method. The predicted structure is topologically similar to that of the substrate-binding region of Na+-coupled glutamate transporter solute carrier 1 (SLC1) proteins. A potential ion-coupled transport function of the DedA superfamily proteins is discussed. This article has an associated First Person interview with the joint first authors of the paper.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Proteínas de Bactérias , Retículo Endoplasmático/genética , Humanos , Membranas Intracelulares , Proteínas de Membrana/genética , FilogeniaRESUMO
Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin-RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance.
Assuntos
Proteínas de Membrana , Neoplasias , Processamento de Proteína Pós-Traducional , Humanos , Autofagia/genética , Macroautofagia , Proteínas de Membrana/metabolismo , Ubiquitina , UbiquitinaçãoRESUMO
Caudal fin regeneration is regulated by a variety of mechanisms, but the role of long non-coding RNA (lncRNA) has rarely been studied. The present study aimed to describe the landscape of lncRNAs during caudal fin regeneration using whole transcriptome sequencing, and then to conduct a functional study on the target lncRNAs using real-time fluorescent quantitative PCR (RT-qPCR), in situ hybridization, and the CRISPR/Cas9 method for lncRNA gene knockout. The results of the transcriptome sequencing showed that a total of 381 lncRNAs were differentially expressed, among which ENSDART00000154324 (lincRNA-154324) was found to be highly related to caudal fin regeneration, and thus it was chosen as the target lncRNA for the subsequent functional study. The results regarding the temporal and spatial expression of lincRNA-154324 and the gene knockout results from CRISPR/Cas9 indicated that lincRNA-154324 is involved in the caudal fin regeneration of zebrafish. Importantly, we serendipitously discovered that the cis correlation coefficient between lincRNA-154324 and its neighboring gene vacuole membrane protein 1 (vmp1) is extremely high, and they are essential for the process of caudal fin regeneration. Moreover, studies have found that vmp1 plays an important role in protein secretion, organelle formation, multicellular development, and autophagy. Collectively, our result may provide a framework for the identification and analysis of lncRNAs involved in the regeneration of the zebrafish caudal fin.
Assuntos
RNA Longo não Codificante , Peixe-Zebra , Animais , Hibridização In Situ , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cicatrização , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Malignant glioma, especially glioblastoma, is a highly aggressive disease with a dismal prognosis. Vacuole membrane protein 1 (VMP1) is a critical autophagy-associated protein with roles in oncogenesis and tumor progression. However, the contribution of VMP1 to glioma development as well as its prognostic value has not been established. METHODS: The expression of VMP1 and clinicopathologic data for 1996 glioma samples were collected from authoritative public databases to explore its prognostic value. Lentiviral CRISPR-Cas9 gene editing system was performed to deplete VMP1 expression. Apoptosis assays, cell cycle assays, colony formation assays, and EdU incorporation analysis were conducted to validate the biological function of VMP1. Transmission electron microscopy was used to determine the role of VMP1 in regulating autophagy. RESULTS: VMP1 overexpression was associated with advanced disease and had a poor prognosis in patients with glioma. The depletion of VMP1 by CRISPR-Cas9 gene editing significantly inhibited cell proliferation, increased cell death, and induced cell cycle arrest. Mechanistically, VMP1 knockout blocked autophagic flux and thus sensitized glioma cells to radiotherapy and chemotherapy. Moreover, a nomogram model showed that VMP1 expression has high prognostic value for determining survival in glioma. CONCLUSIONS: Our results provide insights into the pathological and biological functions of VMP1, including its roles in promoting tumor growth and progression, and support its value as a new diagnostic and prognostic biomarker for glioma.
Assuntos
Autofagia , Glioma/metabolismo , Glioma/patologia , Proteínas de Membrana/metabolismo , Apoptose , Autofagia/genética , Biomarcadores , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Edição de Genes/métodos , Humanos , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Estadiamento de Neoplasias , PrognósticoRESUMO
The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.
Assuntos
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Vacúolos/metabolismo , Animais , Autofagia/fisiologia , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico/fisiologiaRESUMO
Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.
Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , HumanosRESUMO
Generation and turnover of phosphatidylinositol 3-phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy-related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P-enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.
Assuntos
Autofagia , Dictyostelium/metabolismo , Proteínas de Membrana/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Protozoários/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Transdução de SinaisRESUMO
Bois noir is an economically important grapevine yellows that is induced by 'Candidatus Phytoplasma solani' and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the 'Ca. P. solani' genetic variability associated to the nettle-H. obsoletus and bindweed-H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of 'Ca. P. solani' isolates was carried out using polymerase chain reaction/restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed-H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some 'Ca. P. solani' profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.
Assuntos
Proteínas de Bactérias/genética , Convolvulus/microbiologia , Variação Genética , Hemípteros/microbiologia , Phytoplasma/fisiologia , Urtica dioica/microbiologia , Animais , Itália , Dados de Sequência Molecular , Filogenia , Phytoplasma/genética , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Vitis/microbiologiaRESUMO
Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.
Assuntos
Apoptose , Autofagia , Neoplasias Colorretais/patologia , Proteínas de Membrana/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína Beclina-1 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/ultraestrutura , Meios de Cultura Livres de Soro , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estaurosporina/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Vacuole membrane protein 1 (VMP1) is an integral membrane protein that plays a pivotal role in cellular processes, particularly in the regulation of autophagy. Autophagy, a self-degradative mechanism, is essential for maintaining cellular homeostasis by degradation and recycling damaged organelles and proteins. VMP1 involved in the autophagic processes include the formation of autophagosomes and the subsequent fusion with lysosomes. Moreover, VMP1 modulates endoplasmic reticulum (ER) calcium levels, which is significant for various cellular functions, including protein folding and cellular signaling. Recent studies have also linked VMP1 to the cellular response against viral infections and lipid droplet (LD). Dysregulation of VMP1 has been observed in several pathological conditions, including neurodegenerative diseases such as Parkinson's disease (PD), pancreatitis, hepatitis, and tumorogenesis, underscoring its potential as a therapeutic target. This review aims to provide an overview of VMP1's multifaceted roles and its implications in disease pathology.
RESUMO
Upon entering host cells, ß-coronaviruses specifically induce generation of replication organelles (ROs) from the endoplasmic reticulum (ER) through their nonstructural protein 3 (nsp3) and nsp4 for viral genome transcription and replication. The most predominant ROs are double-membrane vesicles (DMVs). The ER-resident proteins VMP1 and TMEM41B, which form a complex to regulate autophagosome and lipid droplet (LD) formation, were recently shown to be essential for ß-coronavirus infection. Here we report that VMP1 and TMEM41B contribute to DMV generation but function at different steps. TMEM41B facilitates nsp3-nsp4 interaction and ER zippering, while VMP1 is required for subsequent closing of the paired ER into DMVs. Additionally, inhibition of phosphatidylserine (PS) formation by siPTDSS1 partially reverses the DMV and LD defects in VMP1 KO cells, suggesting that appropriate PS levels also contribute to DMV formation. This work provides clues to the mechanism of how host proteins collaborate with viral proteins for endomembrane reshaping to promote viral infection.
Assuntos
Infecções por Coronavirus , Coronavirus , Autofagia , Retículo Endoplasmático , Proteínas de Membrana , Espécies Reativas de Oxigênio , Replicação ViralRESUMO
Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson's disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 inflammasome activation and release of proinflammatory molecules. Examination of the VMP1 dependent changes in these cells revealed that VMP1 deficiency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. Autophagic defects were also observed in VMP1 depleted macrophages. Collectively, these studies reveal VMP1 as a negative regulator of inflammatory responses, and we postulate that decreased expression of VMP1 can aggravate the inflammatory sequelae associated with neurodegenerative diseases like PD.
RESUMO
Background: There is growing evidence that immune cells are strongly associated with the prognosis and treatment of clear cell renal cell carcinoma (ccRCC). Our aim is to construct an immune subtype-related model to predict the prognosis of ccRCC patients and to provide guidance for finding appropriate treatment strategies. Methods: Based on single-cell analysis of the GSE152938 dataset from the GEO database, we defined the immune subtype-related genes in ccRCC. Immediately afterwards, we used Cox regression and Lasso regression to build a prognostic model based on TCGA database. Then, we carried out a series of evaluation analyses around the model. Finally, we proved the role of VMP1 in ccRCC by cellular assays. Result: Initially, based on TCGA ccRCC patient data and GEO ccRCC single-cell data, we successfully constructed a prognostic model consisting of five genes. Survival analysis showed that the higher the risk score, the worse the prognosis. We also found that the model had high predictive accuracy for patient prognosis through ROC analysis. In addition, we found that patients in the high-risk group had stronger immune cell infiltration and higher levels of immune checkpoint gene expression. Finally, cellular experiments demonstrated that when the VMP1 gene was knocked down, 786-O cells showed reduced proliferation, migration, and invasion ability and increased levels of apoptosis. Conclusion: Our study can provide a reference for the diagnosis and treatment of patients with ccRCC.