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1.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
2.
Gene ; 753: 144802, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32454178

RESUMO

Synchronous and timely regulation of multiple genes results in an effective defense response that decides the fate of the host when challenged with pathogens or unexpected changes in environmental conditions. One such gene, which is downregulated in response to multiple bacterial pathogens, is a putative nonspecific lipid transfer protein (nsLTP) of unknown function that we have named DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 (DRN1). We show that upon pathogen challenge, DRN1 is strongly downregulated, while a putative DRN1-targeting novel microRNA (miRNA) named DRN1 Regulating miRNA (DmiR) is reciprocally upregulated. Furthermore, we provide evidence that DRN1 is required for defense against bacterial and fungal pathogens as well as for normal seedling growth under salinity stress. Although nsLTP family members from different plant species are known to be a significant source of food allergens and are often associated with antimicrobial properties, our knowledge on the biological functions and regulation of this gene family is limited. Our current work not only sheds light on the mechanism of regulation but also helps in the functional characterization of DRN1, a putative nsLTP family member of hitherto unknown function.


Assuntos
Arabidopsis/genética , Proteínas de Transferência de Fosfolipídeos/genética , Estresse Salino/genética , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Resistência à Doença/genética , Secas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Transferência de Fosfolipídeos/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Patologia Vegetal , Plantas Geneticamente Modificadas , Salinidade , Tolerância ao Sal/genética , Plântula/genética , Estresse Fisiológico/genética
3.
Proc Natl Acad Sci U S A ; 117(16): 9101-9111, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245810

RESUMO

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fotossíntese , Tilacoides/metabolismo , Proteínas de Arabidopsis/genética , Microscopia Eletrônica de Transmissão , Mutação , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Plântula , Homologia de Sequência de Aminoácidos , Tilacoides/ultraestrutura
4.
Exp Cell Res ; 389(1): 111879, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32017928

RESUMO

Coordinated regulation of autophagy and apoptosis helps to enhance the antitumor effects of sodium selenite. However, the potential molecules that act as switch nodes in the crosstalk between autophagy and apoptosis is still elusive. Phospholipid scramblase 1 (PLSCR1) has been shown to regulate leukocyte differentiation, while its role in autophagy/apoptosis toggle switch remains unexplored. In this study, we showed that sodium selenite switched protective autophagy to apoptosis in p53-wild type NB4 cells without obvious caspase-8/apoptosis-inducing factor (AIF) axis activation, while induced autophagy-dependent caspase-8/AIF axis activation in p53-mutant Jurkat cells. Additionally, p53 was demonstrated as a positive regulator of PLSCR1. p53-dependent up-regulation of PLSCR1 accounted for the differential regulation of autophagy and apoptosis induced by sodium selenite. Furthermore, sodium selenite induced the release of AIF from mitochondria to cytosol with the facilitation of caspase-8 in Jurkat cells, while not in NB4 cells. The released AIF further enhanced autophagy flux through interacting with PLSCR1, which hereby resulting in the disassociation of PLSCR1 from Atg5-Atg12 complex. Our results indicate that PLSCR1 plays a critical role in p53-dependent regulation of autophagy and apoptosis in sodium selenite-treated leukemia cells. Manipulation of p53-PLSCR1 cascade might be beneficial to enhance the anti-tumor effects of sodium selenite.


Assuntos
Apoptose , Autofagia , Leucemia/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Selenito de Sódio/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia/genética , Camundongos
5.
Arterioscler Thromb Vasc Biol ; 40(3): 611-623, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31941380

RESUMO

OBJECTIVE: We tested the hypothesis that enlarged, dysfunctional HDL (high-density lipoprotein) particles contribute to the augmented atherosclerosis susceptibility associated with SR-BI (scavenger receptor BI) deficiency in mice. Approach and Results: We eliminated the ability of HDL particles to fully mature by targeting PLTP (phospholipid transfer protein) functionality. Particle size of the HDL population was almost fully normalized in male and female SR-BI×PLTP double knockout mice. In contrast, the plasma unesterified cholesterol to cholesteryl ester ratio remained elevated. The PLTP deficiency-induced reduction in HDL size in SR-BI knockout mice resulted in a normalized aortic tissue oxidative stress status on Western-type diet. Atherosclerosis susceptibility was-however-only partially reversed in double knockout mice, which can likely be attributed to the fact that they developed a metabolic syndrome-like phenotype characterized by obesity, hypertriglyceridemia, and a reduced glucose tolerance. Mechanistic studies in chow diet-fed mice revealed that the diminished glucose tolerance was probably secondary to the exaggerated postprandial triglyceride response. The absence of PLTP did not affect LPL (lipoprotein lipase)-mediated triglyceride lipolysis but rather modified the ability of VLDL (very low-density lipoprotein)/chylomicron remnants to be cleared from the circulation by the liver through receptors other than SR-BI. As a result, livers of double knockout mice only cleared 26% of the fractional dose of [14C]cholesteryl oleate after intravenous VLDL-like particle injection. CONCLUSIONS: We have shown that disruption of PLTP-mediated HDL maturation reduces SR-BI deficiency-driven atherosclerosis susceptibility in mice despite the induction of proatherogenic metabolic complications in the double knockout mice.


Assuntos
Aterosclerose/prevenção & controle , HDL-Colesterol/sangue , Metabolismo Energético , Fígado/metabolismo , Síndrome Metabólica/sangue , Proteínas de Transferência de Fosfolipídeos/deficiência , Receptores Depuradores Classe B/deficiência , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Ésteres do Colesterol/administração & dosagem , Ésteres do Colesterol/sangue , Modelos Animais de Doenças , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Hipertrigliceridemia/sangue , Hipertrigliceridemia/genética , Masculino , Síndrome Metabólica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Proteínas de Transferência de Fosfolipídeos/genética , Placa Aterosclerótica , Receptores Depuradores Classe B/genética
6.
Cell Rep ; 30(4): 1129-1140.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995754

RESUMO

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.


Assuntos
Anoctaminas/metabolismo , Toxinas Bacterianas/toxicidade , Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Timócitos/metabolismo , Animais , Anoctaminas/genética , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Membrana Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Fígado/citologia , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Neutrófilos/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Baço/citologia , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Timócitos/efeitos dos fármacos , Timócitos/ultraestrutura
7.
Artigo em Inglês | MEDLINE | ID: mdl-31786280

RESUMO

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a ß-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a ß-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Mutação com Ganho de Função , Proteínas de Membrana Transportadoras/genética , Mutagênese , Proteínas de Transferência de Fosfolipídeos/genética , Mutação Puntual , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
8.
Nat Commun ; 10(1): 3956, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477691

RESUMO

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Lipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Anoctaminas/química , Anoctaminas/genética , Células COS , Cálcio/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Cristalografia por Raios X , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera
9.
Proc Natl Acad Sci U S A ; 116(33): 16332-16337, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31371510

RESUMO

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.


Assuntos
Adenosina Trifosfatases/genética , Transporte Biológico/genética , Lipídeos de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Ataxia Cerebelar/genética , Citoplasma/genética , Citoplasma/metabolismo , Fenômenos Eletrofisiológicos/genética , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Deficiência Intelectual/genética , Lipídeos de Membrana/metabolismo , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/genética , Especificidade por Substrato/genética
10.
Hum Mutat ; 40(12): 2353-2364, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31397519

RESUMO

ATP8A2 is a P4-ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease-associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi-recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native-like conformation, but lack key amino acid residues required for ATP-dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Leupeptinas/farmacologia , Doenças do Sistema Nervoso/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Dobramento de Proteína , Proteólise
11.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(10): 1412-1421, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31302248

RESUMO

Yeast phosphatidylinositol transfer protein (PITP) Pdr17 is an essential component of the complex required for decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) at a non-mitochondrial location. According to current understanding, this process involves the transfer of PS from the endoplasmic reticulum to the Golgi/endosomes. We generated a Pdr17E237A, K269A mutant protein to better understand the mechanism by which Pdr17p participates in the processes connected to the decarboxylation of PS to PE. We show that the Pdr17E237A, K269A mutant protein is not capable of binding phosphatidylinositol (PI) using permeabilized human cells, but still retains the ability to transfer PI between two membrane compartments in vitro. We provide data together with molecular models showing that the mutations E237A and K269A changed only the lipid binding cavity of Pdr17p and not its surface properties. In contrast to Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its major cellular function in the inter-membrane transfer of PS. We hypothesize that these two closely related yeast PITPs, Pdr16p and Pdr17p, have evolved from a common ancestor. Pdr16p fulfills those role(s) in which the ability to bind and transfer PI is required, while Pdr17p appears to have adapted to a different role which does not require the high affinity binding of PI, although the protein retains the capacity to transfer PI. Our results indicate that PITPs function in complex ways in vivo and underscore the need to consider multiple PITP parameters when studying these proteins in vitro.


Assuntos
Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Mutação Puntual , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Esteróis/metabolismo
12.
Elife ; 82019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31318330

RESUMO

TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.


Assuntos
Ânions/metabolismo , Anoctaminas/química , Anoctaminas/metabolismo , Cátions/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas/genética , Análise Mutacional de DNA , Camundongos , Proteínas de Transferência de Fosfolipídeos/genética , Especificidade por Substrato
13.
Cancer Biomark ; 26(1): 79-91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31322545

RESUMO

Non-small cell lung cancer (NSCLC) , as the most prevalent type of lung carcinoma with high severity, is of urgent necessity to be investigated for novel therapeutic strategies. Long non-coding RNAs (lncRNAs) are notable for their participation in cancer regulation, and lncRNA long intergenic non-protein coding RNA 641 (LINC00641) has been found to have an inhibitory influence on bladder cancer, but its role in NSCLC has not yet been studied. In this research, we launched an investigation into the biological functions and the underlying molecular mechanisms of LINC00641 in NSCLC. At first, downregulation of LINC00641 was identified in NSCLC cells. Functionally, LINC00641 suppressed cell proliferation and induced cell apoptosis in NSCLC, indicating that LINC00641 exerted tumor-suppressive role in NSCLC. Through mechanism investigation, we determined that LINC00641 acted as a competing endogenous RNA (ceRNA) in NSCLC by sponging miR-424-5p to upregulate phospholipid scramblase (PLSCR4) expression. Further rescue assays indicated that miR-424-5p and PLSCR4 could reverse LINC00641-mediated cellular processes. Taken together, it is demonstrated in our study that LINC00641 can function as a tumor suppressor in NSCLC via a ceRNA network.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , RNA Longo não Codificante/genética , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/genética , RNA Longo não Codificante/biossíntese , Transfecção , Regulação para Cima
14.
Genome Biol ; 20(1): 133, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287004

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells. RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA. CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Elementos Reguladores de Transcrição , Transcriptoma
15.
Exp Eye Res ; 185: 107703, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31211954

RESUMO

Glaucoma is a neurodegenerative disease with elevated intraocular pressure as one of the major risk factors. Glaucoma leads to irreversible loss of vision and its progression involves optic nerve head cupping, axonal degeneration, retinal ganglion cell (RGC) loss, and visual field defects. Despite its high global prevalence, glaucoma still remains a major neurodegenerative disease. Introduction of mouse models of experimental glaucoma has become integral to glaucoma research due to well-studied genetics as well as ease of manipulations. Many established inherent and inducible mouse models of glaucoma are used to study the molecular and physiological progression of the disease. One such model of spontaneous mutation is the nee model, which is caused by mutation of the Sh3pxd2b gene. In both humans and mice, mutations disrupting function of the SH3PXD2B adaptor protein cause a developmental syndrome including secondary congenital glaucoma. The purpose of this study was to characterize the early onset nee glaucoma phenotype on the C57BL/6J background and to evaluate the pattern of RGC loss and axonal degeneration in specific RGC subtypes. We found that the B6.Sh3pxd2bnee mutant animals exhibit glaucoma phenotypes of elevated intraocular pressure, RGC loss and axonal degeneration. Moreover, the non-image forming RGCs survived longer than the On-Off direction selective RGCs (DSGC), and the axonal death in these RGCs was independent of their respective RGC subtype. In conclusion, through this study we characterized an experimental model of early onset glaucoma on a C57BL/6J background exhibiting key glaucoma phenotypes. In addition, we describe that RGC death has subtype-specific sensitivities and follows a specific pattern of cell death under glaucomatous conditions.


Assuntos
Modelos Animais de Doenças , Glaucoma/fisiopatologia , Hipertensão Ocular/fisiopatologia , Células Ganglionares da Retina/patologia , Animais , Axônios/patologia , Contagem de Células , Sobrevivência Celular , Feminino , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico , Fenótipo , Proteínas de Transferência de Fosfolipídeos/genética , Microscopia com Lâmpada de Fenda , Tonometria Ocular
16.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(10): 1305-1313, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31220615

RESUMO

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the PLTP deficiency induced significantly more GLUT4 protein in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP-deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increase in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulating lipid levels in the plasma membrane, especially in the lipid rafts.


Assuntos
Resistência à Insulina , Obesidade/genética , Proteínas de Transferência de Fosfolipídeos/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Deleção de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo
17.
Structure ; 27(7): 1114-1123.e3, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31130486

RESUMO

MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile. The structure displays a large amplitude opening in the transmembrane portal, which is likely required for lipid A to pass from its site of synthesis into the protein-enclosed transport pathway. Putative lipid A density is observed further inside the transmembrane cavity, consistent with a trap and flip model. Additional electron density attributed to lipid A is observed near an outer surface cleft at the periplasmic ends of the transmembrane helices. These findings provide new structural insights into the lipid A transport pathway through comparative analysis with existing MsbA structures.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Membrana Celular/química , Lipídeo A/química , Proteínas de Transferência de Fosfolipídeos/química , Salmonella typhimurium/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lipídeo A/metabolismo , Modelos Moleculares , Periplasma/química , Periplasma/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Especificidade por Substrato , Termodinâmica
18.
Nat Commun ; 10(1): 1606, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962435

RESUMO

Vascular endothelial growth factor (VEGF) regulates vasculogenesis by using its tyrosine kinase receptors. However, little is known about whether Sec14-like phosphatidylinositol transfer proteins (PTP) are involved in this process. Here, we show that zebrafish sec14l3, one of the family members, specifically participates in artery and vein formation via regulating angioblasts and subsequent venous progenitors' migration during vasculogenesis. Vascular defects caused by sec14l3 depletion are partially rescued by restoration of VEGFR2 signaling at the receptor or downstream effector level. Biochemical analyses show that Sec14l3/SEC14L2 physically bind to VEGFR2 and prevent it from dephosphorylation specifically at the Y1175 site by peri-membrane tyrosine phosphatase PTP1B, therefore potentiating VEGFR2 signaling activation. Meanwhile, Sec14l3 and SEC14L2 interact with RAB5A/4A and facilitate the formation of their GTP-bound states, which might be critical for VEGFR2 endocytic trafficking. Thus, we conclude that Sec14l3 controls vasculogenesis in zebrafish via the regulation of VEGFR2 activation.


Assuntos
Neovascularização Fisiológica/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
19.
Nat Commun ; 10(1): 1846, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015464

RESUMO

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Anoctaminas/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/genética , Isoleucina/metabolismo , Lisina/genética , Lisina/metabolismo , Mutagênese , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Tirosina/metabolismo
20.
SLAS Discov ; 24(5): 579-586, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31017809

RESUMO

Atherogenesis has been recognized as a risk factor for lethal cardiovascular diseases. Plasma low-density lipoprotein levels are correlated to the occurrence of atherosclerosis, and their control is critical for both the prevention and treatment of these diseases. Phospholipid transfer protein (PLTP) is one of the key regulators of lipoprotein metabolism; PLTP-deficient mice exhibit decreased apolipoprotein B (apoB)-containing lipoprotein secretion and atherosclerosis, indicating the validity of PLTP as a promising therapeutic target. Here, we demonstrate a high-throughput screening (HTS) method to identify a novel chemotype of PLTP inhibitors. Instead of using recombinant proteins, we used human plasma as a source of enzymes in the first screening, so as to efficiently exclude promiscuous inhibitors. The selected compounds were further confirmed to target PLTP both biochemically and biophysically and were shown to inhibit apoB secretion from hepatic cells with no apparent toxicity. We believe that our approach is suitable for filtering out nonspecific inhibitors at an earlier stage of screening campaigns and that these compounds should have potential to be developed into drugs to treat dyslipidemia.


Assuntos
Apolipoproteínas B/genética , Cardiotônicos/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Animais , Apolipoproteínas B/sangue , Aterosclerose/sangue , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Cardiotônicos/síntese química , Cardiotônicos/química , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/patologia , Proteínas de Transporte/antagonistas & inibidores , Dislipidemias/sangue , Dislipidemias/tratamento farmacológico , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Proteínas de Transferência de Fosfolipídeos/genética , Ligação Proteica/efeitos dos fármacos , Fatores de Risco , Ressonância de Plasmônio de Superfície
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