Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 272
Filtrar
1.
Biochemistry ; 63(5): 699-710, 2024 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-38386885

RESUMEN

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C. jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C. jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C. jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.


Asunto(s)
Campylobacter jejuni , Animales , Humanos , Sorbitol/metabolismo , Pollos/metabolismo , Polisacáridos/metabolismo , Citidina Difosfato/metabolismo , Fructosa/metabolismo , Polisacáridos Bacterianos/metabolismo
2.
Trends Plant Sci ; 28(12): 1344-1346, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37648632

RESUMEN

Lesion mimic mutations (LMMs) often confer broad-spectrum resistance (BSR) in plants, but with significant yield penalties. Sha et al. recently demonstrated that genome editing of the rice BSR gene RESISTANCE TO BLAST1 (RBL1), encoding a cytidine diphosphate diacylglycerol (CDP-DAG) synthase involved in phospholipid biosynthesis, confers multipathogen resistance without an obvious trade-off in yield.


Asunto(s)
Diacilglicerol Colinafosfotransferasa , Oryza , Diacilglicerol Colinafosfotransferasa/genética , Oryza/genética , Citidina Difosfato , Diglicéridos , Mutación/genética
3.
Plant J ; 114(2): 338-354, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36789486

RESUMEN

Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.


Asunto(s)
Arabidopsis , Arabidopsis/metabolismo , Glucógeno Sintasa/metabolismo , Citidina Difosfato/metabolismo , Diglicéridos/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Mitocondrias/metabolismo , Fosfatidilgliceroles/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
EMBO J ; 41(23): e110771, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36300838

RESUMEN

Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.


Asunto(s)
Autofagosomas , Fosfolípidos , Autofagosomas/metabolismo , Fosfolípidos/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Colina/metabolismo , Citidina Difosfato/metabolismo
5.
J Bacteriol ; 204(10): e0024722, 2022 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-36094307

RESUMEN

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.


Asunto(s)
Enterococcus faecalis , Nisina , Humanos , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Nisina/genética , Muramidasa/metabolismo , Detergentes/metabolismo , Polimixina B , Acetilgalactosamina , Glicerofosfatos , Difosfatos/metabolismo , Glicerol/metabolismo , Polisacáridos/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Fenotipo , Citidina , Citidina Difosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
6.
Arch Biochem Biophys ; 729: 109376, 2022 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-36007576

RESUMEN

Selenoprotein I (selenoi) is a unique selenocysteine (Sec)-containing protein widely expressed throughout the body. Selenoi belongs to two different protein families: the selenoproteins that are characterized by a redox reactive Sec residue and the lipid phosphotransferases that contain the highly conserved cytidine diphosphate (CDP)-alcohol phosphotransferase motif. Selenoi catalyzes the third reaction of the CDP-ethanolamine branch of the Kennedy pathway within the endoplasmic reticulum membrane. This is not a redox reaction and does not directly involve the Sec residue, making selenoi quite distinct among selenoproteins. Selenoi is also unique among lipid phosphotransferases as the only family member containing a Sec residue near its C-terminus that serves an unknown function. The reaction catalyzed by selenoi involves the transfer of the ethanolamine phosphate group from CDP-ethanolamine to one of two lipid donors, 1,2-diacylglycerol (DAG) or 1-alkyl-2-acylglycerol (AAG), to produce PE or plasmanyl PE, respectively. Plasmanyl PE is subsequently converted to plasmenyl PE by plasmanylethanolamine desaturase. Both PE and plasmenyl PE are critical phospholipids in the central nervous system (CNS), as demonstrated through clinical studies involving SELENOI mutations as well as studies in cell lines and mice. Deletion of SELENOI in mice is embryonic lethal, while loss-of-function mutations in the human SELENOI gene have been found in rare cases leading to a form of hereditary spastic paraplegia (HSP). HSP is an upper motor disease characterized by spasticity of the lower limbs, which is often manifested with other symptoms such as impaired vision/hearing, ataxia, cognitive/intellectual impairment, and seizures. This article will summarize the current understanding of selenoi as a metabolic enzyme and discuss its role in the CNS physiology and pathophysiology.


Asunto(s)
Fosfolípidos , Selenocisteína , Animales , Sistema Nervioso Central/metabolismo , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Etanolaminas/metabolismo , Humanos , Ratones , Fosfolípidos/metabolismo , Fosfotransferasas , Selenoproteínas/metabolismo
7.
Poult Sci ; 101(6): 101893, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35504066

RESUMEN

The cytidine diphosphate diacylglycerol synthases (CDSs) gene encodes the cytidine diphosphate-diacylglycerol (CDP-DAG) synthase enzyme that catalyzes the formation of CDP-diacylglycerol from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT-PCR amplification, 5' rapid amplification of cDNA ends (RACE), and 3' RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on the mRNA level of total CDS2 via quantitative RT-PCR. Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3'-untranslated region (UTR). Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue, and exhibited low expression in liver and pectoralis of 49-day-old chickens. Regarding the spatio-temporal expression patterns of CDS2 in chicken, total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 d in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout the development of the chicken. Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Exogenous insulin significantly down-regulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). In conclusion, chicken CDS2 isoforms with variation at the 3'-UTR were identified, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, that is they were differentially regulated with age in brain, liver, and pectoralis. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.


Asunto(s)
Pollos , Insulina , Animales , Pollos/genética , Pollos/metabolismo , Citidina Difosfato , Diglicéridos , Isoformas de Proteínas , ARN Mensajero/metabolismo
8.
Int J Pharm ; 619: 121691, 2022 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-35331830

RESUMEN

Liposomes (Lip) are useful nanocarriers for drug delivery and cancer nanomedicine because of their ability to efficiently encapsulate drugs with different physical and chemical properties. The pH gradient between normal and tumoral tissues, and their rapid metabolism that induces hyperthermia encourage the development of pH- and thermo-sensitive Lip for delivering anticancer drugs. Nucleolipids have been studied as scaffolding material to prepare Lip, mainly for cancer therapy. Herein, we report for the first time the use of 1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) (DG-CDP) to develop pH/thermo-sensitive nucleolipid-containing stealth Lip stabilized by combination with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol, anchored with NH2-PEGylated gold nanoparticles (PEG-AuNPs, 15 nm) for triggering delivery of doxorubicin (Dox). The optimal composition of DPPC, DG-CDP and cholesterol (94:3:3) was established by Langmuir isotherms. Unloaded and Dox-loaded Lip and AuNPs-Lip exhibited nano-scale sizes (415-650 nm), acceptable polydispersity indexes (<0.33), spherical shapes, and negative Z-potential (-23 to -6.6 mV) due to the phosphate groups of DG-CDP, which allowed the anchoring with positively charged AuNPs. High EE% were achieved (>78%) and although efficient control in the Dox release towards different receptor media was observed, the release of Dox from PEG-AuNPs-Lip-Dox was significantly triggered at acidic pH and hyperthermia conditions, demonstrating its responsiveness to both stimuli. Dox-loaded Lip showed high cytotoxic activity against MDA-MB-231 breast cancer cells and SK-OV-3 ovarian cancer cells, suggesting that Dox was released from these nanocarriers over time. Overall, the liposomal formulations showed promising properties as stimuli-responsive nanocarriers for cancer nanomedicine, with prospects for hyperthermia therapy.


Asunto(s)
Antineoplásicos , Hipertermia Inducida , Nanopartículas del Metal , Neoplasias , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Colesterol/química , Citidina Difosfato/uso terapéutico , Doxorrubicina , Oro/uso terapéutico , Humanos , Concentración de Iones de Hidrógeno , Liposomas/química , Neoplasias/tratamiento farmacológico , Polietilenglicoles/química , Temperatura
9.
J Biochem ; 171(4): 429-441, 2022 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-34964897

RESUMEN

Translocator assembly and maintenance 41 (Tam41) catalyses the synthesis of cytidine diphosphate diacylglycerol (CDP-DAG), which is a high-energy intermediate phospholipid critical for generating cardiolipin in mitochondria. Although Tam41 is present almost exclusively in eukaryotic cells, a Firmicutes bacterium contains the gene encoding Tam41-type CDP-DAG synthase (FbTam41). FbTam41 converted phosphatidic acid (PA) to CDP-DAG using a ternary complex mechanism in vitro. Additionally, FbTam41 functionally substituted yeast Tam41 in vivo. These results demonstrate that Tam41-type CDP-DAG synthase functions in some prokaryotic cells. We determined the crystal structure of FbTam41 lacking the C-terminal 18 residues in the cytidine triphosphate (CTP)-Mg2+ bound form at a resolution of 2.6 Å. The crystal structure showed that FbTam41 contained a positively charged pocket that specifically accommodated CTP-Mg2+ and PA in close proximity. By using this structure, we constructed a model for the full-length structure of FbTam41 containing the last a-helix, which was missing in the crystal structure. Based on this model, we propose a molecular mechanism for CDP-DAG synthesis in bacterial cells and mitochondria.


Asunto(s)
Citidina Difosfato , Diacilglicerol Colinafosfotransferasa , Cardiolipinas , Diacilglicerol Colinafosfotransferasa/genética , Diacilglicerol Colinafosfotransferasa/metabolismo , Diglicéridos , Firmicutes/metabolismo
10.
Nat Commun ; 12(1): 6982, 2021 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-34848707

RESUMEN

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.


Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/química , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Methanocaldococcus/enzimología , Sitios de Unión , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Cristalografía por Rayos X , Citidina Difosfato , Escherichia coli , Lípidos de la Membrana/química , Fosfatidilserinas , Fosfolípidos , Fosfotransferasas , Transferasas
11.
J Biochem ; 170(2): 183-194, 2021 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-34255834

RESUMEN

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.


Asunto(s)
Distroglicanos/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Animales , Cromatografía Liquida/métodos , Citidina Difosfato/metabolismo , Glicerol/metabolismo , Glicosilación , Células HEK293 , Humanos , Masculino , Mamíferos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Pentosiltransferasa/metabolismo , Fosfatidiletanolaminas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Polisacáridos/metabolismo , Espectrometría de Masas en Tándem/métodos
12.
Nature ; 595(7869): 724-729, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34234346

RESUMEN

T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.


Asunto(s)
Inmunidad Humoral , Fosfatidiletanolaminas/metabolismo , Receptores CXCR5/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/inmunología , Sistemas CRISPR-Cas , Diferenciación Celular , Citidina Difosfato , Femenino , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfotransferasas (Aceptor de Grupo Alcohol) , ARN Nucleotidiltransferasas , Transducción de Señal
14.
PLoS Genet ; 16(10): e1009070, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33064773

RESUMEN

The major glycerophospholipid phosphatidylethanolamine (PE) in the nervous system is essential for neural development and function. There are two major PE synthesis pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway in mitochondria. However, the role played by mitochondrial PE synthesis in maintaining cellular PE homeostasis is unknown. Here, we show that Drosophila pect (phosphoethanolamine cytidylyltransferase) mutants lacking the CDP-ethanolamine pathway, exhibited alterations in phospholipid composition, defective phototransduction, and retinal degeneration. Induction of the PSD pathway fully restored levels and composition of cellular PE, thus rescued the retinal degeneration and defective visual responses in pect mutants. Disrupting lipid exchange between mitochondria and ER blocked the ability of PSD to rescue pect mutant phenotypes. These findings provide direct evidence that the synthesis of PE in mitochondria contributes to cellular PE homeostasis, and suggest the induction of mitochondrial PE synthesis as a promising therapeutic approach for disorders associated with PE deficiency.


Asunto(s)
Carboxiliasas/genética , Citidina Difosfato/análogos & derivados , Retículo Endoplásmico/genética , Degeneración Retiniana/genética , Animales , Carboxiliasas/metabolismo , Citidina Difosfato/deficiencia , Citidina Difosfato/genética , Citidina Difosfato/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Retículo Endoplásmico/metabolismo , Etanolaminas/metabolismo , Homeostasis/genética , Humanos , Metabolismo de los Lípidos/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Transducción de Señal/genética
15.
Gastric Cancer ; 23(6): 974-987, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32388635

RESUMEN

BACKGROUND: Increasing evidence indicates that angiogenesis plays an important role in tumor progression. The function of cathepsin L (CTSL), an endosomal proteolytic enzyme, in promoting tumor metastasis is well recognized. The mechanisms by which CTSL has promoted the angiogenesis of gastric cancer (GC), however, remains unclear. METHODS: The nuclear expression levels of CTSL were assessed in GC samples. The effects of CTSL on GC angiogenesis were determined by endothelial tube formation analysis, HUVEC migration assay, and chick embryo chorioallantoic membrane (CAM) assay. The involvement of the CDP/Cux/VEGF-D pathway was analyzed by angiogenesis antibody array, Western blot, co-immunoprecipitation (Co-IP) and dual-luciferase reporter assay. RESULTS: In this study, we found that the nuclear CTSL expression level in GC was significantly higher than that in adjacent nontumor gastric tissues and was a potential important clinical prognostic factor. Loss- and gain-of-function assays indicated that CTSL promotes the tubular formation and migration of HUVEC cells in vitro. The CAM assay also showed that CTSL promotes angiogenesis of GC in vivo. Mechanistic analysis demonstrated that CTSL can proteolytically process CDP/Cux and produce the physiologically relevant p110 isoform, which stably binds to VEGF-D and promotes the transcription of VEGF-D, thus contributing to the angiogenesis of GC. CONCLUSION: The findings of the present study suggested that CTSL plays a constructive role in the regulation of angiogenesis in human GC and could be a potential therapeutic target for GC.


Asunto(s)
Inductores de la Angiogénesis/metabolismo , Catepsina L/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Transducción de Señal/genética , Neoplasias Gástricas/genética , Animales , Embrión de Pollo , Citidina Difosfato/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo
16.
Sci Adv ; 6(20): eaaz8041, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32440549

RESUMEN

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a ß sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.


Asunto(s)
Legionella , Proteínas Bacterianas/metabolismo , Citidina Difosfato , GTP Fosfohidrolasas/metabolismo , Legionella/metabolismo , Fosforilcolina/metabolismo
17.
PLoS One ; 15(5): e0233689, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32469932

RESUMEN

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.


Asunto(s)
Proteínas Bacterianas/química , Nucleósido-Fosfato Quinasa/química , Thermus thermophilus/enzimología , Adenosina Difosfato/química , Cristalografía por Rayos X , Citidina Difosfato/química , Dominios Proteicos
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 4): 160-167, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32254049

RESUMEN

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05 Šresolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.


Asunto(s)
Liasas de Carbono-Oxígeno/química , Dominio Catalítico , Cristalografía por Rayos X , Citidina Difosfato/análogos & derivados , Citidina Difosfato/química , Etanolaminas/química , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Fosfato de Piridoxal/química
19.
Pak J Pharm Sci ; 33(1(Supplementary)): 241-244, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32122854

RESUMEN

Cerebrovascular diseases are known as serious public health problem worldwide, which can be addressed more precisely through molecular imaging of non-functional brain cells. CDP-choline is an active cerebrovascular chemotherapeutic agent that can be used for diagnosis of cerebrovascular diseases post radiolabeling with γ-emitter radioisotopes. In this study we developed 99mTc labeled CDP-choline for imaging of cerebrovascular diseases particularly alzheimer, stroke, and parkinson's diseases. The radiosynthesis reaction resulted 97.47±2.34% radiochemical with promising stability, that is, >95% up to 6 h in blood serum. The biodistribution study in healthy mice revealed non-accumulated uptake of radiochemical in key body organs; in brain it was 8.59±1.11% ID/g at 1h post-injection which washed-out leaving behind 0.87±0.61% ID/g at 24 h post-injection. The over-all data revealed the 99mTc-CDP-choline could be a good candidate for further imaging investigations in diseased animal model.


Asunto(s)
Trastornos Cerebrovasculares/metabolismo , Colina/metabolismo , Citidina Difosfato/metabolismo , Compuestos de Organotecnecio/metabolismo , Tomografía Computarizada de Emisión de Fotón Único/métodos , Trastornos Cerebrovasculares/diagnóstico por imagen , Humanos , Distribución Tisular/fisiología , Tomografía Computarizada de Emisión de Fotón Único/tendencias
20.
J Biol Chem ; 295(51): 17877-17886, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33454021

RESUMEN

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.


Asunto(s)
Citidina Difosfato Colina/metabolismo , Citidina Difosfato/análogos & derivados , Etanolaminas/metabolismo , Animales , Colina Quinasa/química , Colina Quinasa/genética , Citidililtransferasa de Colina-Fosfato/química , Citidililtransferasa de Colina-Fosfato/genética , Citidina Difosfato/metabolismo , Estudios de Asociación Genética , Humanos , Distrofias Musculares/congénito , Distrofias Musculares/genética , Distrofias Musculares/patología , Osteocondrodisplasias/congénito , Osteocondrodisplasias/genética , Osteocondrodisplasias/patología , Polimorfismo de Nucleótido Simple
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...