RESUMO
Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane.
Assuntos
Adenosina Trifosfatases , Membrana Celular , Homeostase , Fosfatidilinositóis , Humanos , Membrana Celular/metabolismo , Células HeLa , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Fosfatidilinositóis/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genéticaRESUMO
Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.
Assuntos
Anoctaminas , Cálcio , Proteínas de Transferência de Fosfolipídeos , Humanos , Anoctaminas/metabolismo , Anoctaminas/genética , Células HEK293 , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismoRESUMO
The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.
Assuntos
Trifosfato de Adenosina , Toxinas Bacterianas , Cálcio , Canais de Cloreto , Vesículas Extracelulares , Oócitos , Xenopus laevis , Animais , Oócitos/metabolismo , Oócitos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Canais de Cloreto/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Feminino , Clostridium perfringens/metabolismoRESUMO
Cells establish the asymmetrical distribution of phospholipids and alter their distribution by phospholipid scrambling (PLS) to adapt to environmental changes. Here, we demonstrate that a protein complex, consisting of the ion channel Tmem63b and the thiamine transporter Slc19a2, induces PLS upon calcium (Ca2+) stimulation. Through revival screening using a CRISPR sgRNA library on high PLS cells, we identify Tmem63b as a PLS-inducing factor. Ca2+ stimulation-mediated PLS is suppressed by deletion of Tmem63b, while human disease-related Tmem63b mutants induce constitutive PLS. To search for a molecular link between Ca2+ stimulation and PLS, we perform revival screening on Tmem63b-overexpressing cells, and identify Slc19a2 and the Ca2+-activated K+ channel Kcnn4 as PLS-regulating factors. Deletion of either of these genes decreases PLS activity. Biochemical screening indicates that Tmem63b and Slc19a2 form a heterodimer. These results demonstrate that a Tmem63b/Slc19a2 heterodimer induces PLS upon Ca2+ stimulation, along with Kcnn4 activation.
Assuntos
Cálcio , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos , Humanos , Cálcio/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Células HEK293 , Canais Iônicos/metabolismo , Canais Iônicos/genética , Animais , Sistemas CRISPR-CasRESUMO
To facilitate our understanding of proteome dynamics during signaling events, robust workflows affording fast time resolution without confounding factors are essential. We present Surface-exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide-derivative (SLAPSHOT) to label extracellularly exposed proteins in a rapid, specific, and sensitive manner. Simple and flexible SLAPSHOT utilizes recombinant soluble APEX2 protein applied to cells, thus circumventing the engineering of tools and cells, biological perturbations, and labeling biases. We applied SLAPSHOT and quantitative proteomics to examine the TMEM16F-dependent plasma membrane remodeling in WT and TMEM16F KO cells. Time-course data ranging from 1 to 30 min of calcium stimulation revealed co-regulation of known protein families, including the integrin and ICAM families, and identified proteins known to reside in intracellular organelles as occupants of the freshly deposited extracellularly exposed membrane. Our data provide the first accounts of the immediate consequences of calcium signaling on the extracellularly exposed proteome.
Assuntos
Anoctaminas , Cálcio , Membrana Celular , Proteoma , Proteoma/metabolismo , Membrana Celular/metabolismo , Cálcio/metabolismo , Anoctaminas/metabolismo , Anoctaminas/genética , Animais , Proteômica/métodos , Humanos , Camundongos , Fosfolipídeos/metabolismo , Sinalização do Cálcio , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Peróxido de Hidrogênio/metabolismoRESUMO
TMEM16 proteins, which function as Ca2+activated Cl channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl channels can be used for the moleculebased treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca2+ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulatorbased treatment methods.
Assuntos
Anoctaminas , Proteínas de Transferência de Fosfolipídeos , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/metabolismo , Anoctaminas/genética , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Terapia de Alvo Molecular , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Calcium ions (Ca2+) play crucial roles in sperm motility and fertilization. The copine (CPNE) family comprises several Ca2+-dependent phospholipid-binding proteins. Of these, CPNE1 is extensively expressed in mammalian tissues; however, its precise role in testicular development and spermatogenesis is yet to be fully characterized. In this study, we used proteomics to analyze testicular biopsies and found that levels of CPNE1 were significantly reduced in patients with non-obstructive azoospermia (defective spermatogenesis) compared to those in patients with obstructive azoospermia (physiological spermatogenesis). In mice, CPNE1 is expressed at various stages of germ cell development and is associated with the Golgi apparatus. Ultimately, CPNE1 is expressed in the flagella of mature sperms. To further examine the role of CPNE1, we developed a Cpne1 knockout mouse model. Analysis showed that the loss of Cpne1 did not impair testicular development, spermatogenesis, or sperm morphology and motility in physiological conditions. When treated with gadolinium (III) chloride or 2-aminoethoxydiphenyl borate, known inhibitors of store-operated Ca2+ entry, Ca2+ signals and sperm motility were significantly compromised in wild-type mice; however, both mechanisms were conserved in KO mice. These results suggested that CPNE1 is dispensable for testicular development, spermatogenesis or sperm motility in physiological conditions. In addition, CPNE1 may represent a target of Ca2+ channel inhibitors and may therefore be implicated in the regulation of Ca2+ signaling and sperm motility.
Assuntos
Azoospermia , Camundongos Knockout , Motilidade dos Espermatozoides , Espermatogênese , Espermatozoides , Masculino , Animais , Motilidade dos Espermatozoides/efeitos dos fármacos , Camundongos , Espermatogênese/efeitos dos fármacos , Azoospermia/metabolismo , Azoospermia/genética , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Humanos , Testículo/metabolismo , Testículo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Sinalização do Cálcio/efeitos dos fármacosRESUMO
Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.
Assuntos
Caspase 8 , Fusão Celular , Osteoclastos , Fosfatidilserinas , Proteínas de Transferência de Fosfolipídeos , Caspase 8/metabolismo , Caspase 8/genética , Animais , Osteoclastos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/genética , Diferenciação Celular , Ligante RANK/metabolismoRESUMO
The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD ß6-ß7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Escherichia coli , Periplasma , Fosfolipídeos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transporte Biológico , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana , Modelos Moleculares , Periplasma/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases - including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.
Assuntos
Neoplasias , Fosfatidilinositóis , Proteínas de Transferência de Fosfolipídeos , Transdução de Sinais , Humanos , Neoplasias/metabolismo , Animais , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfatidilinositóis/metabolismoRESUMO
BACKGROUND: Phospholipid transfer protein (PLTP) transfers surface phospholipids between lipoproteins and as such plays a role in lipoprotein metabolism, but with unclear effects on coronary artery disease (CAD) risk. We aimed to investigate the associations of genetically-influenced PLTP activity with 1-H nuclear magnetic resonance (1H-NMR) metabolomic measures and with CAD. Furthermore, using factorial Mendelian randomization (MR), we examined the potential additional effect of genetically-influenced PLTP activity on CAD risk on top of genetically-influenced low-density lipoprotein-cholesterol (LDL-C) lowering. METHODS: Using data from UK Biobank, genetic scores for PLTP activity and LDL-C were calculated and dichotomised based on the median, generating four groups with combinations of high/low PLTP activity and high/low LDL-C levels for the factorial MR. Linear and logistic regressions were performed on 168 metabolomic measures (N = 58,514) and CAD (N = 318,734, N-cases=37,552), respectively, with results expressed as ß coefficients (in standard deviation units) or odds ratios (ORs) and 95% confidence interval (CI). RESULTS: Irrespective of the genetically-influenced LDL-C, genetically-influenced low PLTP activity was associated with a higher high-density lipoprotein (HDL) particle concentration (ß [95% CI]: 0.03 [0.01, 0.05]), smaller HDL size (-0.14 [-0.15, -0.12]) and higher triglyceride (TG) concentration (0.04 [0.02, 0.05]), but not with CAD (OR 0.99 [0.97, 1.02]). In factorial MR analyses, genetically-influenced low PLTP activity and genetically-influenced low LDL-C had independent associations with metabolomic measures, and genetically-influenced low PLTP activity did not show an additional effect on CAD risk. CONCLUSIONS: Low PLTP activity associates with higher HDL particle concentration, smaller HDL particle size and higher TG concentration, but no association with CAD risk was observed.
Assuntos
Doença da Artéria Coronariana , Proteínas de Transferência de Fosfolipídeos , Humanos , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/sangue , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Masculino , Feminino , LDL-Colesterol/sangue , LDL-Colesterol/metabolismo , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Lipoproteínas/metabolismo , Lipoproteínas/sangueRESUMO
The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.
Assuntos
Proteínas de Escherichia coli , Fosfolipídeos , Fosfolipídeos/metabolismo , Fosfolipídeos/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transporte Biológico/genética , Cardiolipinas/metabolismo , Cardiolipinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Baixa , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismoRESUMO
Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.
Assuntos
Proteínas de Transferência de Fosfolipídeos , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Células HEK293 , Esfingomielinas/metabolismo , Esfingomielinas/biossíntese , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Isoenzimas/metabolismo , Isoenzimas/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/enzimologiaRESUMO
Background: PtdIns (3,4,5) P3-dependent Rac exchanger 1 (PREX1), also known as PREX1, a member of the Rac guanine nucleotide exchange factors (Rac-GEF) family. Studies have suggested that PREX1 plays a role in mediating oncogenic pathway activation and controlling various biological mechanisms in different types of cancer, including liver hepatocellular carcinoma (LIHC). However, the function of PREX1 in the pathogenesis of LIHC and its potential role on immunological regulation is not clearly elucidated. Methods: The expression level and the clinical role of PREX1 in LIHC was analyzed based on database from the Cancer Genome Atlas (TCGA), TNM plotter and University of Alabama Cancer Database (UALCAN). We investigated the relationship between PREX1 and immunity in LIHC by TISIDB, CIBERSORT and single cell analysis. Immunotherapy responses were assessed by the immunophenoscores (IPS). Moreover, biological functional assays were performed to further investigate the roles of PREX1 in liver cancer cell lines. Results: Higher expression of PREX1 in LIHC tissues than in normal liver tissues was found based on public datasets. Further analysis revealed that PREX1 was associated with worse clinical characteristics and dismal prognosis. Pathway enrichment analysis indicated that PREX1 participated in immune-related pathways. Through CIBERSORT and single cell analysis, we found a remarkable correlation between the expression of PREX1 and various immune cells, especially macrophages. In addition, high PREX1 expression was found to be associated with a stronger response to immunotherapy. Furthermore, in vitro assays indicated that depletion of PREX1 can suppress invasion and proliferation of LIHC cells. Conclusion: Elevated expression of PREX1 indicates poor prognosis, influences immune modulation and predicts sensitivity of immunosuppression therapy in LIHC. Our results suggested that PREX1 may be a prognostic biomarker and therapeutic target, offering new treatment options for LIHC.
Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas , Análise de Célula Única , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Prognóstico , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Transcriptoma/imunologia , Transcriptoma/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , FemininoRESUMO
TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.
Assuntos
Anoctaminas , Neurônios , Fosfatidilserinas , Tauopatias , Proteínas tau , Animais , Anoctaminas/metabolismo , Anoctaminas/genética , Fosfatidilserinas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas tau/metabolismo , Proteínas tau/genética , Camundongos , Tauopatias/metabolismo , Tauopatias/patologia , Humanos , Microglia/metabolismo , Microglia/patologia , Fosforilação , Camundongos Transgênicos , Modelos Animais de Doenças , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos KnockoutRESUMO
The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase.
Assuntos
Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transporte Biológico , Oligossacarídeos/metabolismo , Fosfatos de Dolicol/metabolismo , Fosfatos de Dolicol/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Membranas Intracelulares/metabolismo , LipopolissacarídeosRESUMO
Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.
Assuntos
Cerebelo , Proteínas de Transferência de Fosfolipídeos , Sinapses , Transmissão Sináptica , Vesículas Sinápticas , Animais , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Feminino , Masculino , Cerebelo/citologia , Sinapses/metabolismo , Sinapses/fisiologia , Células Cultivadas , Camundongos Knockout , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Endocitose/fisiologiaRESUMO
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.
Assuntos
Proteínas de Drosophila , Proteínas de Transferência de Fosfolipídeos , Podócitos , Animais , Podócitos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Microdomínios da Membrana/metabolismo , Junções Intercelulares/metabolismoRESUMO
Invasive fungal infections in humans with compromised immune systems are the primary cause of morbidity and mortality, which is becoming more widely acknowledged. Amphotericin B (AmB) is one of the antifungal drugs used to treat such infections. AmB binds with plasma membrane ergosterol, inducing cellular ions to leak and causing cell death. Reduction in ergosterol content and modification of cell walls have been described as AmB resistance mechanisms. In addition, when the sphingolipid level is decreased, the cell becomes more susceptible to AmB. Previously, PDR16, a gene that encodes phosphatidylinositol transfer protein in Saccharomyces cerevisiae, was shown to enhance AmB resistance upon overexpression. However, the mechanism of PDR16-mediated AmB resistance is not clear. Here, in this study, it was discovered that a plasma membrane proteolipid 3 protein encoded by PMP3 is essential for PDR16-mediated AmB resistance. PDR16-mediated AmB resistance does not depend on ergosterol, but a functional sphingolipid biosynthetic pathway is required. Additionally, PMP3-mediated alteration in membrane integrity abolishes PDR16 mediated AmB resistance, confirming the importance of PMP3 in the PDR16 mediated AmB resistance.
Assuntos
Anfotericina B , Antifúngicos , Farmacorresistência Fúngica , Ergosterol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Testes de Sensibilidade Microbiana , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacosRESUMO
TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined.