RESUMO
Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer.
Assuntos
Fosfolipídeos , Fatores de Transcrição , Feminino , Humanos , Fosfolipídeos/genética , Ligantes , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismo , Fatores de Transcrição/metabolismo , Receptores Citoplasmáticos e Nucleares , DNARESUMO
The translocon SecYEG and the associated ATPase SecA form the primary protein secretion system in the cytoplasmic membrane of bacteria. The secretion is essentially dependent on the surrounding lipids, but the mechanistic understanding of their role in SecA : SecYEG activity is sparse. Here, we reveal that the unsaturated fatty acids (UFAs) of the membrane phospholipids, including tetraoleoyl-cardiolipin, stimulate SecA : SecYEG-mediated protein translocation up to ten-fold. Biophysical analysis and molecular dynamics simulations show that UFAs increase the area per lipid and cause loose packing of lipid head groups, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced up to fivefold at elevated ionic strength. Tight SecA : lipid interactions convert into the augmented translocation. Our results identify the fatty acid structure as a notable factor in SecA : SecYEG activity, which may be crucial for protein secretion in bacteria, which actively change their membrane composition in response to their habitat.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Canais de Translocação SEC/genética , Proteínas SecA/genética , Cardiolipinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos Insaturados/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/genética , Oleandomicina/metabolismo , Fosfolipídeos/genética , Transporte Proteico/genética , Tetraciclina/metabolismoRESUMO
Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-ß 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Amiloide/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Proteínas Amiloidogênicas/antagonistas & inibidores , Proteínas Amiloidogênicas/genética , Catequina/análogos & derivados , Catequina/farmacologia , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Polifenóis/farmacologia , alfa-Sinucleína/ultraestruturaRESUMO
Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.
Assuntos
Fosfolipases A2/genética , Éteres Fosfolipídicos/metabolismo , Fosfolipídeos/genética , Compostos de Vinila/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Citosol/metabolismo , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Humanos , Hidrólise , Cinética , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Especificidade por Substrato/genética , Compostos de Vinila/químicaRESUMO
BACKGROUND: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. METHODS: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr-/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2V617F). RESULTS: Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk-/- platelets promoted increased NETosis when incubated with Lnk-/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk-/- platelets, as well as increased priming and response of Lnk-/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk-/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ controls. CONCLUSIONS: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Plaquetas/metabolismo , Neutrófilos/metabolismo , Fosfolipídeos/metabolismo , Agregação Plaquetária , Trombose/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Artérias/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Fosfolipídeos/genética , Trombose/genéticaRESUMO
Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Autofagossomos/genética , Autofagossomos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Humanos , Metabolismo/genética , Fosfatos de Fosfatidilinositol/genética , Fosfolipídeos/genética , Transdução de Sinais/genéticaRESUMO
Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously identified the Xkr family protein Xkr4 as a phospholipid-scrambling protein, but its activation mechanisms remain unknown. Here we show that Xkr4 is activated in two steps: dimer formation by caspase-mediated cleavage and structural change caused by activating factors. To identify the factors, we developed a new screening system, "revival screening," using a CRISPR sgRNA library. Applying this system, we identified the nuclear protein XRCC4 as the single candidate for the Xkr4 activator. Upon apoptotic stimuli, XRCC4, contained in the DNA repair complex, is cleaved by caspases, and its C-terminal fragment with an intrinsically disordered region is released into the cytoplasm. Protein interaction screening showed that the fragment interacts directly with the Xkr4 dimer to activate it. This study demonstrates that caspase-mediated cleavage releases a nuclear protein fragment for direct regulation of lipid dynamics on the plasma membrane.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteólise , Animais , Proteínas Reguladoras de Apoptose/genética , Caspases/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfolipídeos/genética , Multimerização ProteicaRESUMO
Lysophosphatidic acid (LPA) is an abundant bioactive phospholipid, with multiple functions both in development and in pathological conditions. Here, we review the literature about the differential signaling of LPA through its specific receptors, which makes this lipid a versatile signaling molecule. This differential signaling is important for understanding how this molecule can have such diverse effects during central nervous system development and angiogenesis; and also, how it can act as a powerful mediator of pathological conditions, such as neuropathic pain, neurodegenerative diseases, and cancer progression. Ultimately, we review the preclinical and clinical uses of Autotaxin, LPA, and its receptors as therapeutic targets, approaching the most recent data of promising molecules modulating both LPA production and signaling. This review aims to summarize the most update knowledge about the mechanisms of LPA production and signaling in order to understand its biological functions in the central nervous system both in health and disease.
Assuntos
Lisofosfolipídeos/genética , Neovascularização Patológica/genética , Fosfolipídeos/genética , Humanos , Lisofosfolipídeos/metabolismo , Terapia de Alvo Molecular , Neovascularização Patológica/tratamento farmacológico , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/uso terapêutico , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/uso terapêutico , Transdução de Sinais/genéticaRESUMO
Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , ATPases do Tipo-P/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Transporte Biológico/genética , Membrana Celular/enzimologia , Proliferação de Células/genética , Células Eucarióticas/enzimologia , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Numerous studies have reported an association between genetic variants in fatty acid desaturases (FADS1 and FADS2) and plasma or erythrocyte long chain polyunsaturated fatty acid (PUFA) levels. Increased levels of n-6 PUFAs have been associated with inflammation and several chronic diseases, including diabetes and cancer. We hypothesized that genetic variants of FADS that more efficiently convert precursor n-6 PUFA to arachidonic acid (AA) may explain the higher burden of chronic diseases observed in African Americans. To test this hypothesis, we measured the level of n-6 and n-3 PUFAs in erythrocyte membrane phospholipids and genotyped the rs174537 FADS variants associated with higher AA conversion among African American and European American populations. We included data from 1,733 individuals who participated in the Tennessee Colorectal Polyp Study, a large colonoscopy-based case-control study. Erythrocyte membrane PUFA percentages were measured using gas chromatography. Generalized linear models were used to estimate association of race and genotype on erythrocyte phospholipid membrane PUFA levels while controlling for self-reported dietary intake. We found that African Americans have higher levels of AA and a higher prevalence of GG allele compared to whites, 81% vs 43%, respectively. Homozygous GG genotype was negatively associated with precursor PUFAs (linoleic [LA], di-homo-γ-linolenic [DGLA]), positively associated with both product PUFA (AA, docosahexaenoic acid [DHA]), product to precursor ratio (AA to DGLA), an indirect measure of FADs efficiency and increased urinary isoprostane F2 (F2-IsoP) and isoprostane F3 (F3-IsoP), markers of oxidative stress. Increased consumption of n-6 PUFA and LA resulting in increased AA and subsequent inflammation may be fueling increased prevalence of chronic diseases especially in African descent.
Assuntos
Negro ou Afro-Americano/genética , Membrana Eritrocítica , Ácidos Graxos Dessaturases , Ácidos Graxos Insaturados , Fosfolipídeos , Polimorfismo de Nucleotídeo Único , População Branca/genética , Dessaturase de Ácido Graxo Delta-5 , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
Glioblastoma multiforme is the most aggressive type of tumor of the CNS with an overall survival rate of approximately one year. Since this rate has not changed significantly over the last 20 years, the development of new therapeutic strategies for the treatment of these tumors is peremptory. The over-expression of the proto-oncogene c-Fos has been observed in several CNS tumors including glioblastoma multiforme and is usually associated with a poor prognosis. Besides its genomic activity as an AP-1 transcription factor, this protein can also activate phospholipid synthesis by a direct interaction with key enzymes of their metabolic pathways. Given that the amino-terminal portion of c-Fos (c-Fos-NA: amino acids 1-138) associates to but does not activate phospholipid synthesizing enzymes, we evaluated if c-Fos-NA or some shorter derivatives are capable of acting as dominant-negative peptides of the activating capacity of c-Fos. The over-expression or the exogenous administration of c-Fos-NA to cultured T98G cells hampers the interaction between c-Fos and PI4K2A, an enzyme activated by c-Fos. Moreover, it was observed a decrease in tumor cell proliferation rates in vitro and a reduction in tumor growth in vivo when a U87-MG-generated xenograft on nude mice is intratumorally treated with recombinant c-Fos-NA. Importantly, a smaller peptide of 92 amino acids derived from c-Fos-NA retains the capacity to interfere with tumor proliferation in vitro and in vivo. Taken together, these results support the use of the N-terminal portion of c-Fos, or shorter derivatives as a novel therapeutic strategy for the treatment of glioblastoma multiforme.
Assuntos
Proliferação de Células , Glioblastoma/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfolipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Antígenos de Histocompatibilidade Menor/genética , Fosfolipídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
P4 ATPase lipid flippases are ATP-driven transporters that translocate specific lipids from the exoplasmic to the cytosolic leaflet of biological membranes, thus establishing a lipid gradient between the two leaflets that is essential for many cellular processes. While substrate specificity, subcellular and tissue-specific expression, and physiological functions have been assigned to a number of these transporters in several organisms, the mechanism of lipid transport has been a topic of intense debate in the field. The recent publication of a series of structural models based on X-ray crystallography and cryo-EM studies has provided the first glimpse into how P4 ATPases have adapted the transport mechanism used by the cation-pumping family members to accommodate a substrate that is at least an order of magnitude larger than cations.
Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Membrana Celular/genética , Humanos , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/genéticaRESUMO
Cancer cells need to modulate the biosynthesis of membrane lipids and fatty acids to adapt themselves to an accelerated rate of cell division and survive into an extracellular environment characterised by a low pH. To gain insight this crucial survival process, we investigated the lipid composition of Mel 501 melanoma cells cultured at either physiological or acidic pH and observed the remodelling of phospholipids towards longer and more unsaturated acyl chains at low pH. This modification was related to changes in gene expression profile, as we observed an up-regulation of genes involved in acyl chain desaturation, elongation and transfer to phospholipids. PC3 prostate and MCF7 breast cancer cells adapted at acidic pH also demonstrated phospholipid fatty acid remodelling related to gene expression changes. Overall findings clearly indicate that low extracellular pH impresses a specific lipid signature to cells, associated with transcriptional reprogramming.
Assuntos
Ácidos Graxos/metabolismo , Lipidômica , Lipídeos/genética , Modelos Biológicos , Neoplasias/metabolismo , Fosfolipídeos/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos/genética , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Células MCF-7 , Estrutura Molecular , Neoplasias/genética , Células PC-3 , Fosfolipídeos/genética , Relação Estrutura-Atividade , Transcriptoma , Células Tumorais CultivadasRESUMO
Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteína Forkhead Box O1/metabolismo , Cálculos Biliares/metabolismo , Resistência à Insulina , Transdução de Sinais , Animais , Ácidos e Sais Biliares/genética , Colesterol/genética , Colesterol/metabolismo , Feminino , Proteína Forkhead Box O1/genética , Cálculos Biliares/genética , Deleção de Genes , Regulação da Expressão Gênica , Fígado , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because VDR deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including MOGAT1, LPGAT1, AGPAT2, and DGAT1 (glycerolipid metabolism); CDS1, PCTP, and MAT1A (phospholipid metabolism); and FATP2, SLC6A12, and AQP3 (uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4-6 h) upon VDR activation by 10 nM VitD or 100 µM lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Fosfolipídeos/biossíntese , Receptores de Calcitriol/metabolismo , Triglicerídeos/biossíntese , Animais , Células Hep G2 , Humanos , Camundongos , Camundongos Knockout para ApoE , Fosfolipídeos/genética , Receptores de Calcitriol/genética , Triglicerídeos/genéticaRESUMO
Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Here, we focus on P1, a particularly membrane-active isoform, and investigate how metallating it and making OxPL available influence its membrane activity. First, we quantify through dye leakage experiments the permeabilization of the apo- and holo-forms of P1 on model membranes containing a fixed ratio of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) but varying amounts of Aldo-PC, an OxPL derived from the degradation of several UFAs. Remarkably, metallating P1 increases membranolysis by a factor of five in each lipid system. Conversely, making Aldo-PC available improves permeabilization by a factor of two for each peptide form. Second, we demonstrate through CD-monitored titrations that the strength of the peptide-membrane interactions is similar in PC/PG and PC/PG/Aldo-PC. Thus, peptide-induced membrane activity is boosted by properties intrinsic to the peptide (e.g., charge and structural changes associated with metallation) and bilayer (e.g., reversal of sn-2 chain due to oxidation). Third, we show using oriented-sample 15N solid-state NMR that the helical portion of P1 lies parallel to the bilayer surface in both lipid systems. 31P NMR experiments show that both the apo- and holo-states interact more readily with PC in PC/PG. However, the presence of Aldo-PC renders the holo-, but not the apo-state, more specific to PG. Hence, the membrane disruptive effects of P1 and its specificity for the anionic lipids found on pathogenic cell membrane surfaces are simultaneously optimized when it is metallated and the OxPL is present. Overall, this study deepens our insights into how OxPLs affect peptide-lipid interactions and how host defense metallopeptides could help integrate the effects of antimicrobial agents.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Ácidos Graxos Insaturados/química , Proteínas de Peixes/genética , Metais/química , Animais , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Membrana Celular , Cobre/química , Ácidos Graxos Insaturados/genética , Proteínas de Peixes/química , Histidina/química , Histidina/genética , Humanos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Níquel/química , Fosfolipídeos/química , Fosfolipídeos/genéticaRESUMO
Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2- system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR-/- mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.
Assuntos
Apolipoproteína A-I/genética , Lipoproteínas HDL3/genética , Fosfolipídeos/genética , Receptores de LDL/genética , Animais , Aorta/metabolismo , Cromatografia Líquida , Humanos , Peróxido de Hidrogênio/metabolismo , Lipoproteínas HDL/genética , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Oxirredução , Fosforilação Oxidativa , Fosfolipídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.
Assuntos
Endocanabinoides/biossíntese , Engenharia Metabólica/métodos , Ácidos Oleicos/biossíntese , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Coenzima A Ligases/genética , Endocanabinoides/genética , Enzimas/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Microrganismos Geneticamente Modificados , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Ácidos Oleicos/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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/genéticaRESUMO
Viral infections universally rely on numerous hijacked host factors to be successful. It is therefore possible to control viral infections by manipulating host factors that are critical for viral replication. Given that host genes may play essential roles in certain cellular processes, any successful manipulations for virus control should cause no or mild effects on host fitness. We previously showed that a group of positive-strand RNA viruses enrich phosphatidylcholine (PC) at the sites of viral replication. Specifically, brome mosaic virus (BMV) replication protein 1a interacts with and recruits a PC synthesis enzyme, phosphatidylethanolamine methyltransferase, Cho2p, to the viral replication sites that are assembled on the perinuclear endoplasmic reticulum (ER) membrane. Deletion of the CHO2 gene inhibited BMV replication by 5-fold; however, it slowed down host cell growth as well. Here, we show that an engineered Cho2p mutant supports general PC synthesis and normal cell growth but blocks BMV replication. This mutant interacts and colocalizes with BMV 1a but prevents BMV 1a from localizing to the perinuclear ER membrane. The mislocalized BMV 1a fails to induce the formation of viral replication complexes. Our study demonstrates an effective antiviral strategy in which a host lipid synthesis gene is engineered to control viral replication without comprising host growth.