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1.
Immunol Rev ; 317(1): 166-186, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37144896

RESUMO

The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.


Assuntos
COVID-19 , Surfactantes Pulmonares , Animais , Humanos , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/uso terapêutico , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Receptor 2 Toll-Like , SARS-CoV-2 , Pulmão/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fosfatidilgliceróis/uso terapêutico , Fosfatidilgliceróis/farmacologia
2.
Annu Rev Biochem ; 78: 827-56, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19489735

RESUMO

Robust lipid traffic within and among membranes is essential for cell growth and membrane biogenesis. Many of these transport reactions occur by nonvesicular pathways, and the genetic and biochemical details of these processes are now beginning to emerge. Intramembrane lipid transport reactions utilize P-type ATPases, ABC transporters, scramblases, and Niemann-Pick type C (NPC) family proteins. The intramembrane processes regulate the establishment and elimination of membrane lipid asymmetry, the cellular influx and efflux of sterols and phospholipids, and the egress of lysosomally deposited lipids. The intermembrane lipid transport processes play important roles in membrane biogenesis, sterol sequestration, and steroid hormone formation. The roles of soluble lipid carriers and membrane-bound lipid-transporting complexes, as well as the mechanisms for regulation of their targeting and assembly, are now becoming apparent. Elucidation of the details of these systems is providing new perspectives on the regulation of lipid traffic within cells.


Assuntos
Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos , Animais , Proteínas de Transporte/química , Humanos , Lipídeos/química , Transdução de Sinais
3.
J Biol Chem ; 299(5): 104659, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36997087

RESUMO

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.


Assuntos
Carboxiliases , Malária , Fosfolipídeos , Plasmodium , Motivos de Aminoácidos , Cálcio/metabolismo , Cálcio/farmacologia , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Carboxiliases/metabolismo , Precursores Enzimáticos/metabolismo , Lipossomos , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Fosfatidilgliceróis/metabolismo , Fosfatidilgliceróis/farmacologia , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosfolipídeos/farmacologia , Ligação Proteica , Malária/parasitologia , Proteólise/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Plasmodium/enzimologia
4.
J Immunol ; 204(10): 2829-2839, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32245819

RESUMO

The lung surfactant proteins are recognized as critical not only for their role in lowering lung surface tension but also in innate host defense. Reports have shown that some asthmatic patients have decreased levels of one member of this protein family in particular, surfactant protein-A (SP-A). Our studies set out to determine the contribution of SP-A to the response of a key effector cytokine in asthma, IL-13. Our studies employ both animal models sufficient and deficient in SP-A challenged with IL-13 and primary epithelial cells from participants with asthma that are exogenously treated with SP-A in the context of IL-13 challenge. The inflammatory response and mucin production were assessed in both model systems. As compared with WT mice, we show that the activity of IL-13 is dramatically augmented in SP-A-/- mice, which have significantly increased neutrophil and eosinophil recruitment, mucin production and asthma-associated cytokines in the bronchoalveolar lavage fluid. In parallel, we show asthma-associated factors are attenuated in human cells from asthma subjects when exogenous SP-A is added during IL-13 challenge. Although many of these phenotypes have previously been associated with STAT6 signaling, SP-A inhibited IL-13-induced STAT3 phosphorylation in mice and in human epithelial cells while having little effect on STAT6 phosphorylation. In addition, when either STAT3 or IL-6 were inhibited in mice, the phenotypes observed in SP-A-/- mice were significantly attenuated. These studies suggest a novel mechanism for SP-A in asthma as a modulator of IL-13-induced inflammation via mediating downstream IL-6/STAT3 signaling.


Assuntos
Eosinófilos/imunologia , Inflamação/metabolismo , Interleucina-13/metabolismo , Neutrófilos/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Mucosa Respiratória/metabolismo , Animais , Asma , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína A Associada a Surfactante Pulmonar/genética , Mucosa Respiratória/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
5.
J Biol Chem ; 295(27): 9211-9222, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32430397

RESUMO

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.


Assuntos
Carboxiliases/análise , Corantes Fluorescentes/síntese química , Espectrometria de Fluorescência/métodos , Acetofenonas/química , Candida albicans/metabolismo , Carboxiliases/metabolismo , Membrana Celular/metabolismo , Etanolamina , Fluorescência , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mercaptoetanol/química , Mitocôndrias , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Estirenos/química
6.
J Biol Chem ; 295(6): 1704-1715, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31882535

RESUMO

The influenza A (H1N1)pdm09 outbreak in 2009 exemplified the problems accompanying the emergence of novel influenza A virus (IAV) strains and their unanticipated virulence in populations with no pre-existing immunity. Neuraminidase inhibitors (NAIs) are currently the drugs of choice for intervention against IAV outbreaks, but there are concerns that NAI-resistant viruses can transmit to high-risk populations. These issues highlight the need for new approaches that address the annual influenza burden. In this study, we examined whether palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI) effectively antagonize (H1N1)pdm09 infection. POPG and PI markedly suppressed cytopathic effects and attenuated viral gene expression in (H1N1)pdm09-infected Madin-Darby canine kidney cells. POPG and PI bound to (H1N1)pdm09 with high affinity and disrupted viral spread from infected to noninfected cells in tissue culture and also reduced (H1N1)pdm09 propagation by a factor of 102 after viral infection was established in vitro In a mouse infection model of (H1N1)pdm09, POPG and PI significantly reduced lung inflammation and viral burden. Of note, when mice were challenged with a typically lethal dose of 1000 plaque-forming units of (H1N1)pdm09, survival after 10 days was 100% (14 of 14 mice) with the POPG treatment compared with 0% (0 of 14 mice) without this treatment. POPG also significantly reduced inflammatory infiltrates and the viral burden induced by (H1N1)pdm09 infection in a ferret model. These findings indicate that anionic phospholipids potently and efficiently disrupt influenza infections in animal models.


Assuntos
Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Fosfatidilgliceróis/uso terapêutico , Fosfatidilinositóis/uso terapêutico , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Cães , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Fosfatidilgliceróis/farmacologia , Fosfatidilinositóis/farmacologia , Surfactantes Pulmonares/farmacologia , Surfactantes Pulmonares/uso terapêutico
7.
Am J Respir Cell Mol Biol ; 62(3): 354-363, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31545652

RESUMO

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Evasão da Resposta Imune/fisiologia , Lipídeos de Membrana/fisiologia , Micobactérias não Tuberculosas/patogenicidade , Fosfolipídeos/fisiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/imunologia , Cromatografia em Camada Fina , Escherichia coli/efeitos dos fármacos , Humanos , Macrófagos/microbiologia , Macrófagos Alveolares/microbiologia , Lipídeos de Membrana/isolamento & purificação , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/fisiologia , Fosfolipídeos/isolamento & purificação , Filogenia , Especificidade da Espécie , Células THP-1 , Virulência , Catelicidinas
8.
J Biol Chem ; 294(12): 4282-4289, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30733339

RESUMO

Toll-like receptors (TLRs) coupled to intracellular signaling cascades function as central elements of innate immunity that control transcription of numerous pro-inflammatory genes. Two minor anionic phospholipids present in the pulmonary surfactant complex, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), antagonize the cognate ligand activation of TLRs 2 and 4. The lipids block recognition of activating ligands by the TLRs, either directly or via the TLR4 coreceptors CD14 and MD2. Antagonism of TLR activation results in inhibition of the initiating step of the pro-inflammatory signaling pathways. Evidence for this mechanism of action comes from direct binding studies between CD14 and MD2 with POPG and PI. Additional evidence for this mechanism of antagonism also comes from monitoring the reduction of protein phosphorylation events that characterize the intracellular signaling by activated TLRs. The pathogenesis of respiratory syncytial virus (RSV) and influenza A virus (IAV) have been linked to TLR4 activation, and we examined the action of POPG and PI as potential antagonists of the pathology of these viruses. Surprisingly, POPG and PI dramatically curtail infection, in addition to inhibiting inflammatory sequelae associated with RSV and IAV infections. The mechanism of action by the lipids is disruption of virus particle binding to host cell plasma membrane receptors, required for viral uptake. The antagonism of activation of TLRs and virus binding to the alveolar epithelium by resident constituents of the pulmonary surfactant system suggests that POPG and PI function in homeostasis, to prevent inflammatory processes that result in reductions in gas exchange within the alveolar compartment.


Assuntos
Imunidade Inata/fisiologia , Vírus da Influenza A/isolamento & purificação , Fosfolipídeos/fisiologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Animais , Humanos , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo
9.
J Biol Chem ; 294(32): 12146-12156, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31227523

RESUMO

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.


Assuntos
Carboxiliases/antagonistas & inibidores , Inibidores Enzimáticos/análise , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Estirenos/química , Candida albicans/efeitos dos fármacos , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Etanolamina/farmacologia , Humanos , Concentração Inibidora 50 , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
10.
Nat Rev Mol Cell Biol ; 9(2): 112-24, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18216768

RESUMO

Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Animais , Bioquímica/métodos , Biofísica/métodos , Citosol/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lipídeos/química , Modelos Biológicos , Fosfatidilcolinas/química , Transporte Proteico
11.
J Allergy Clin Immunol ; 144(4): 962-971, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30930175

RESUMO

BACKGROUND: Research in transformed immortalized cell lines indicates the cadherin-related family member 3 (CDHR3) protein serves as a receptor for human rhinovirus (HRV)-C. Similar experiments indicate that the CDHR3 coding variant rs6967330 increases CDHR3 protein surface expression. OBJECTIVE: We sought to determine whether CDHR3 is necessary for HRV-C infection of primary airway epithelial cells (AECs) and to identify molecular mechanisms by which CDHR3 variants confer risk for asthma exacerbations. METHODS: CDHR3 function and influence on HRV-C infection were investigated by using single-cell transcriptomics, CRISPR-Cas9 gene knockout, and genotype-specific donor experiments performed in primary AECs. Nasal airway epithelium cis-expression quantitative trait locus (eQTL) analysis of CDHR3 was performed, followed by association testing for asthma hospitalization in minority children. RESULTS: CDHR3 lung expression is exclusive to ciliated AECs and associated with basal bodies during and after motile ciliogenesis. Knockout of CDHR3 in human AECs did not prevent ciliated cell differentiation but was associated with a decrease in transepithelial resistance and an 80% decrease in HRV-C infection of the mucociliary epithelium. AECs from subjects homozygous for the risk-associated rs6967330 single nucleotide polymorphism (SNP) exhibited greater HRV-C infection compared with cells homozygous for the nonrisk allele. AEC cis-eQTL analysis indicated that rs6967330 and other SNPs are eQTLs for CDHR3. Only the eQTL block containing the rs6967330 SNP showed a significant association with childhood asthma hospitalization. CONCLUSIONS: Genetic deletion and genotype-specific studies in primary AECs indicate CDHR3 is critical to HRV-C infection of ciliated cells. The rs6967330 SNP confers risk of severe childhood asthma exacerbations, likely through increasing HRV-C infection levels and protein surface localization.


Assuntos
Asma/genética , Caderinas/genética , Infecções por Enterovirus/genética , Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas Relacionadas a Caderinas , Caderinas/metabolismo , Criança , Enterovirus , Infecções por Enterovirus/metabolismo , Feminino , Genótipo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Polimorfismo de Nucleotídeo Único , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia
12.
Am J Respir Cell Mol Biol ; 60(1): 58-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30156431

RESUMO

Human rhinovirus (RV), the major cause of the common cold, triggers the majority of acute airway exacerbations in patients with asthma and chronic obstructive pulmonary disease. Nitric oxide, and the related metabolite S-nitrosoglutathione, are produced in the airway epithelium via nitric oxide synthase (NOS) 2 and have been shown to function in host defense against RV infection. We hypothesized that inhibitors of the S-nitrosoglutathione-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), might potentiate the antiviral properties of airway-derived NOS2. Using in vitro models of RV-A serotype 16 (RV-A16) and mNeonGreen-H1N1pr8 infection of human airway epithelial cells, we found that treatment with a previously characterized GSNOR inhibitor (4-[[2-[[(3-cyanophenyl)methyl]thio]-4-oxothieno-[3,2-d]pyrimidin-3(4H)-yl]methyl]-benzoic acid; referred to as C3m) decreased RV-A16 replication and expression of downstream proinflammatory and antiviral mediators (e.g., RANTES [regulated upon activation, normal T cell expressed and secreted], CXCL10, and Mx1), and increased Nrf2 (nuclear factor erythroid 2-related factor 2)-dependent genes (e.g., SQSTM1 and TrxR1). In contrast, C3m had no effect on influenza virus H1N1pr8 replication. Moreover, a structurally dissimilar GSNOR inhibitor (N6022) did not alter RV replication, suggesting that the properties of C3m may be specific to rhinovirus owing to an off-target effect. Consistent with this, C3m antiviral effects were not blocked by either NOS inhibition or GSNOR knockdown but appeared to be mediated by reduced intercellular adhesion molecule 1 transcription and increased shedding of soluble intercellular adhesion molecule 1 protein. Collectively these data show that C3m has novel antirhinoviral properties that may synergize with, but are unrelated to, its GSNOR inhibitor activity.


Assuntos
Aldeído Oxirredutases/antagonistas & inibidores , Brônquios/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Infecções por Picornaviridae/tratamento farmacológico , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Benzamidas/farmacologia , Brônquios/metabolismo , Brônquios/virologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Inflamação/metabolismo , Inflamação/virologia , Óxido Nítrico Sintase Tipo II/metabolismo , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/virologia , Pirróis/farmacologia
13.
J Biol Chem ; 293(45): 17308-17316, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30287688

RESUMO

The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.


Assuntos
Estágios do Ciclo de Vida/fisiologia , Malária Falciparum/genética , Malária Falciparum/metabolismo , Fosfolipídeos , Plasmodium falciparum , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Desenvolvimento de Medicamentos , Humanos , Malária Falciparum/tratamento farmacológico , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo
14.
J Biol Chem ; 293(5): 1493-1503, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29247006

RESUMO

Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.


Assuntos
Carboxiliases/química , Fluorescência , Proteínas Ligantes de Maltose/química , Fosfatidiletanolaminas/química , Plasmodium knowlesi/enzimologia , Proteínas de Protozoários/química , Carboxiliases/genética , Proteínas Ligantes de Maltose/genética , Fosfatidiletanolaminas/análise , Plasmodium knowlesi/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
15.
Biophys J ; 115(8): 1509-1517, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30266319

RESUMO

Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.


Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimologia , Membrana Celular/química , Lipossomos/metabolismo , Fosfatidiletanolaminas/metabolismo
16.
Mol Microbiol ; 104(3): 412-427, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28142188

RESUMO

Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.


Assuntos
Carboxiliases/metabolismo , Transferases de Grupos Nitrogenados/metabolismo , Trypanosoma brucei brucei/enzimologia , Descarboxilação , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética
17.
Traffic ; 16(2): 123-34, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25355612

RESUMO

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.


Assuntos
Carboxiliases/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilserinas/metabolismo , Leveduras/metabolismo , Transporte Biológico , Carboxiliases/genética , Leveduras/genética
18.
Mol Microbiol ; 99(6): 999-1014, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26585333

RESUMO

Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity.


Assuntos
Antimaláricos/farmacologia , Carboxiliases/antagonistas & inibidores , Carboxiliases/metabolismo , Inibidores Enzimáticos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/enzimologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/genética , Clonagem Molecular , Feminino , Malária Falciparum/microbiologia , Camundongos , Testes de Sensibilidade Parasitária , Fosfatidilserinas/metabolismo , Plasmodium falciparum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
19.
J Immunol ; 194(12): 6123-32, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25957169

RESUMO

Mycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases.


Assuntos
Variação Genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mycoplasma pneumoniae/imunologia , Mycoplasma pneumoniae/metabolismo , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/imunologia , Proteína A Associada a Surfactante Pulmonar/genética , Animais , Membrana Celular/imunologia , Membrana Celular/metabolismo , Quimiotaxia/imunologia , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Muco/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fosforilação , Pneumonia por Mycoplasma/metabolismo , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar/metabolismo
20.
J Lipid Res ; 57(6): 993-1005, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27095543

RESUMO

The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.


Assuntos
Inflamação/tratamento farmacológico , Fosfatidilgliceróis/administração & dosagem , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Animais , Membrana Celular/efeitos dos fármacos , Eicosanoides/administração & dosagem , Eicosanoides/química , Endotoxinas/administração & dosagem , Endotoxinas/química , Humanos , Inflamação/genética , Inflamação/patologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fosfatidilgliceróis/química , Surfactantes Pulmonares/administração & dosagem , Surfactantes Pulmonares/química , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores
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