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1.
BMC Plant Biol ; 21(1): 377, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399687

RESUMO

BACKGROUND: C. panzhihuaensis is more tolerant to freezing than C. bifida but the mechanisms underlying the different freezing tolerance are unclear. Photosynthesis is one of the most temperature-sensitive processes. Lipids play important roles in membrane structure, signal transduction and energy storage, which are closely related to the stress responses of plants. In this study, the chlorophyll fluorescence parameters and lipid profiles of the two species were characterized to explore the changes in photosynthetic activity and lipid metabolism following low-temperature exposure and subsequent recovery. RESULTS: Photosynthetic activity significantly decreased in C. bifida with the decrease of temperatures and reached zero after recovery. Photosynthetic activity, however, was little affected in C. panzhihuaensis. The lipid composition of C. bifida was more affected by cold and freezing treatments than C. panzhihuaensis. Compared with the control, the proportions of all the lipid categories recovered to the original level in C. panzhihuaensis, but the proportions of most lipid categories changed significantly in C. bifida after 3 d of recovery. In particular, the glycerophospholipids and prenol lipids degraded severely during the recovery period of C. bifida. Changes in acyl chain length and double bond index (DBI) occurred in more lipid classes immediately after low-temperature exposure in C. panzhihuaensis compare with those in C. bifida. DBI of the total main membrane lipids of C. panzhihuaensis was significantly higher than that of C. bifida following all temperature treatments. CONCLUSIONS: The results of chlorophyll fluorescence parameters confirmed that the freezing tolerance of C. panzhihuaensis was greater than that of C. bifida. The lipid metabolism of the two species had differential responses to low temperatures. The homeostasis and plastic adjustment of lipid metabolism and the higher level of DBI of the main membrane lipids may contribute to the greater tolerance of C. panzhihuaensis to low temperatures.


Assuntos
Aclimatação , Cycas/fisiologia , Lipídeos de Membrana/metabolismo , China , Clorofila/metabolismo , Cycas/metabolismo , Congelamento , Glicerofosfatos/metabolismo , Homeostase , Especificidade da Espécie , Temperatura
2.
ACS Chem Biol ; 16(8): 1344-1349, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255482

RESUMO

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.


Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Epitopos/metabolismo , Glicerofosfatos/metabolismo , Ácidos Teicoicos/metabolismo , Animais , Anticorpos Monoclonais Murinos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Glicerofosfatos/química , Glicerofosfatos/imunologia , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia
3.
mBio ; 12(3): e0089721, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34076489

RESUMO

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.


Assuntos
Actinobacillus pleuropneumoniae/química , Cápsulas Bacterianas/química , Glicerofosfatos/biossíntese , Neisseria meningitidis/química , Pasteurellaceae/química , Polímeros/química , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Vacinas Bacterianas/química , Bovinos , Glicerofosfatos/análise , Glicerofosfatos/metabolismo , Ovinos , Suínos
4.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799449

RESUMO

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and ß-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.


Assuntos
Aterosclerose/genética , Transdiferenciação Celular/genética , Difosfatos/metabolismo , Calcificação Vascular/genética , Trifosfato de Adenosina , Fosfatase Alcalina/genética , Animais , Aorta/citologia , Aorta/metabolismo , Ácido Ascórbico/farmacologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Glicerofosfatos/genética , Glicerofosfatos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fosfatos/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
5.
Protein Sci ; 30(3): 543-557, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33314435

RESUMO

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.


Assuntos
Triptofano Sintase , Regulação Alostérica/genética , Catálise , Domínio Catalítico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicerofosfatos/química , Glicerofosfatos/metabolismo , Indóis/química , Indóis/metabolismo , Conformação Proteica , Triptofano Sintase/química , Triptofano Sintase/genética , Triptofano Sintase/metabolismo
6.
EMBO J ; 39(20): e104231, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32882062

RESUMO

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.


Assuntos
Bile/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glicerofosfatos/metabolismo , Chaperonas Moleculares/metabolismo , Ampicilina/farmacologia , Proteínas de Transporte/genética , Dicroísmo Circular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Concentração de Íons de Hidrogênio , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Conformação Molecular , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteoma/metabolismo
7.
J Biol Chem ; 295(38): 13250-13266, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32723868

RESUMO

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.


Assuntos
Adipócitos/metabolismo , Proteínas de Drosophila/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Lipogênese , Transdução de Sinais , Células 3T3-L1 , Animais , Drosophila melanogaster , Glicerofosfatos/metabolismo , Camundongos , NADP/metabolismo
8.
J Mol Med (Berl) ; 98(7): 985-997, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32488546

RESUMO

In chronic kidney disease, hyperphosphatemia is a key pathological factor promoting medial vascular calcification, a common complication associated with cardiovascular events and mortality. This active pathophysiological process involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs) via complex intracellular mechanisms that are still incompletely understood. Little is known about the effects of phosphate on the bioenergetic profile of VSMCs during the onset of this process. Therefore, the present study explored the effects of the phosphate donor ß-glycerophosphate on cellular bioenergetics of VSMCs. Mitochondrial and glycolytic functions were determined utilizing extracellular flux analysis in primary human aortic VSMCs following exposure to ß-glycerophosphate. In VSMCs, ß-glycerophosphate increased basal respiration, mitochondrial ATP production as well as proton leak and decreased spare respiratory capacity and coupling efficiency, but did not modify non-mitochondrial or maximal respiration. ß-Glycerophosphate-treated VSMCs had higher ability to increase mitochondrial glutamine and long-chain fatty acid usage as oxidation substrates to meet their energy demand. ß-Glycerophosphate did not modify glycolytic function or basal and glycolytic proton efflux rate. In contrast, ß-glycerophosphate increased non-glycolytic acidification. ß-Glycerophosphate-treated VSMCs had a more oxidative and less glycolytic phenotype, but a reduced ability to respond to stressed conditions via mitochondrial respiration. Moreover, compounds targeting components of mitochondrial respiration modulated ß-glycerophosphate-induced oxidative stress, osteo-/chondrogenic signalling and mineralization of VSMCs. In conclusion, ß-glycerophosphate modifies key parameters of mitochondrial function and cellular bioenergetics in VSMCs that may contribute to the onset of phenotypical transdifferentiation and calcification. These observations advance the understanding of the role of energy metabolism in VSMC physiology and pathophysiology of vascular calcification during hyperphosphatemia. KEY MESSAGES: ß-Glycerophosphate modifies key parameters of mitochondrial respiration in VSMCs. ß-Glycerophosphate induces changes in mitochondrial fuel choice in VSMCs. ß-Glycerophosphate promotes a more oxidative and less glycolytic phenotype of VSMCs. ß-Glycerophosphate triggers mitochondrial-dependent oxidative stress in VSMCs. Bioenergetics impact ß-glycerophosphate-induced VSMC calcification.


Assuntos
Metabolismo Energético/fisiologia , Glicerofosfatos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Aorta/metabolismo , Transdiferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/fisiologia , Humanos , Hiperfosfatemia/metabolismo , Osteogênese/fisiologia , Fosfatos/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/fisiologia , Calcificação Vascular/metabolismo
9.
Cancer Res ; 80(11): 2150-2162, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32179514

RESUMO

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.


Assuntos
Glicerolfosfato Desidrogenase/metabolismo , Glicerofosfatos/metabolismo , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Células A549 , Trifosfato de Adenosina/biossíntese , Animais , Antineoplásicos/farmacologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Respiração Celular/fisiologia , Sinergismo Farmacológico , Glicerolfosfato Desidrogenase/biossíntese , Glicerolfosfato Desidrogenase/genética , Células HCT116 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
Sci Rep ; 10(1): 1728, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015442

RESUMO

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.


Assuntos
Plaquetas/fisiologia , Glicerofosfatos/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Rim/metabolismo , Megacariócitos/fisiologia , Proteína ORAI1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Idoso , Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Proteínas Imediatamente Precoces/genética , Rim/patologia , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Regulação para Cima
11.
J Biol Chem ; 295(12): 4024-4034, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32047114

RESUMO

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.


Assuntos
Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ácidos Teicoicos/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Glicerofosfatos/química , Glicerofosfatos/metabolismo , Glicosilação , Lipopolissacarídeos/biossíntese , Diester Fosfórico Hidrolases/genética , Polímeros/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Compostos de Sódio/química , Estereoisomerismo , Especificidade por Substrato , Ácidos Teicoicos/biossíntese
12.
Genes (Basel) ; 11(2)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32093268

RESUMO

Two genes, Bx1 and Igl, both encoding indole-3-glycerol phosphate lyase (IGL), are believed to control the conversion of indole-3-glycerol phosphate (IGP) to indole. The first of these has generally been supposed to be regulated developmentally, being expressed at early stages of plant development with the indole being used in the benzoxazinoid (BX) biosynthesis pathway. In contrast, it has been proposed that the second one is regulated by stresses and that the associated free indole is secreted as a volatile. However, our previous results contradicted this. In the present study, we show that the ScIgl gene takes over the role of ScBx1 at later developmental stages, between the 42nd and 70th days after germination. In the majority of plants with silenced ScBx1 expression, ScIgl was either expressed at a significantly higher level than ScBx1 or it was the only gene with detectable expression. Therefore, we postulate that the synthesis of indole used in BX biosynthesis in rye is controlled by both ScBx1 and ScIgl, which are both regulated developmentally and by stresses. In silico and in vivo analyses of the promoter sequences further confirmed our hypothesis that the roles and modes of regulation of the ScBx1 and ScIgl genes are similar.


Assuntos
Liases/genética , Secale/crescimento & desenvolvimento , Secale/genética , Benzoxazinas/metabolismo , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/genética , Glicerofosfatos/genética , Glicerofosfatos/metabolismo , Indóis/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas
13.
J Clin Invest ; 130(3): 1513-1526, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32065590

RESUMO

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin D3 [1,25(OH)2D] production. Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography-mass spectrometry-based (LC-MS-based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase-mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury.


Assuntos
Injúria Renal Aguda/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicerofosfatos/metabolismo , Rim/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Linhagem Celular , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Rim/patologia , Masculino , Metabolômica , Camundongos , Camundongos Knockout , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo
14.
Artigo em Inglês | MEDLINE | ID: mdl-30959116

RESUMO

The successive acylation of glycerol-3-phosphate (G3P) by glycerol-3-phosphate acyltransferases and acylglycerol-3-phosphate acyltransferases produces phosphatidic acid (PA), a precursor for CDP-diacylglycerol-dependent phospholipid synthesis. PA is further dephosphorylated by LIPINs to produce diacylglycerol (DG), a substrate for the synthesis of triglyceride (TG) by DG acyltransferases and a precursor for phospholipid synthesis via the CDP-choline and CDP-ethanolamine (Kennedy) pathways. The channeling of fatty acids into TG for storage in lipid droplets and secretion in lipoproteins or phospholipids for membrane biogenesis is dependent on isoform expression, activity and localization of G3P pathway enzymes, as well as dietary and hormonal and tissue-specific factors. Here, we review the mechanisms that control partitioning of substrates into lipid products of the G3P pathway.


Assuntos
Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Glicerofosfatos/metabolismo , Glicerofosfolipídeos/metabolismo , Lipogênese , Transdução de Sinais , Acilação , Animais , Ácidos Graxos/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Triglicerídeos/metabolismo
15.
Nat Commun ; 10(1): 5303, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757957

RESUMO

Glycerol-3-phosphate (G3P) is a well-known mobile regulator of systemic acquired resistance (SAR), which provides broad spectrum systemic immunity in response to localized foliar pathogenic infections. We show that G3P-derived foliar immunity is also activated in response to genetically-regulated incompatible interactions with nitrogen-fixing bacteria. Using gene knock-down we show that G3P is essential for strain-specific exclusion of non-desirable root-nodulating bacteria and the associated foliar pathogen immunity in soybean. Grafting studies show that while recognition of rhizobium incompatibility is root driven, bacterial exclusion requires G3P biosynthesis in the shoot. Biochemical analyses support shoot-to-root transport of G3P during incompatible rhizobia interaction. We describe a root-shoot-root signaling mechanism which simultaneously enables the plant to exclude non-desirable nitrogen-fixing rhizobia in the root and pathogenic microbes in the shoot.


Assuntos
Glicerofosfatos/imunologia , Imunidade Vegetal/imunologia , Proteínas de Plantas/genética , Raízes de Plantas/imunologia , Brotos de Planta/imunologia , Rhizobium/imunologia , Soja/imunologia , Simbiose/imunologia , Técnicas de Silenciamento de Genes , Glicerofosfatos/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Rhizobium/metabolismo , Transdução de Sinais , Soja/metabolismo
16.
Sci Rep ; 9(1): 12735, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484941

RESUMO

It is well documented that four gene families, including the glycerophosphate acyltransferases (GPATs), acylglycerophosphate acyltransferases (AGPATs), lipid phosphate phosphohydrolases (LPINs) and diacylglycerol acyltransferases (DGATs), are involved in the glycerophosphate pathway of de novo triglyceride (TG) biosynthesis in mammals. However, no systematic analysis has been conducted to characterize the gene families in poultry. In this study, the sequences of gene family members in the glycerophosphate pathway were obtained by screening the public databases. The phylogenetic tree, gene structures and conserved motifs of the corresponding proteins were evaluated. Dynamic expression changes of the genes at different developmental stages were analyzed by qRT-PCR. The regulatory characteristics of the genes were analyzed by in vivo experiments. The results showed that the GPAT, AGPAT and LPIN gene families have 2, 7 and 2 members, respectively, and they were classified into 2, 4 and 2 cluster respectively based on phylogenetic analysis. All of the genes except AGPAT1 were extensively expressed in various tissues. Estrogen induction upregulated the expression of GPAM and AGPAT2, downregulated the expression of AGPAT3, AGPAT9, LPIN1 and LPIN2, and had no effect on the expression of the other genes. These findings provide a valuable resource for further investigation of lipid metabolism in liver of chicken.


Assuntos
Galinhas/genética , Galinhas/metabolismo , Glicerofosfatos/metabolismo , Triglicerídeos/biossíntese , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Vias Biossintéticas , Galinhas/classificação , Glicerol-3-Fosfato O-Aciltransferase/genética , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Filogenia
17.
Nat Commun ; 10(1): 3813, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444353

RESUMO

Salicylic acid (SA)-mediated innate immune responses are activated in plants perceiving volatile monoterpenes. Here, we show that monoterpene-associated responses are propagated in feed-forward loops involving the systemic acquired resistance (SAR) signaling components pipecolic acid, glycerol-3-phosphate, and LEGUME LECTIN-LIKE PROTEIN1 (LLP1). In this cascade, LLP1 forms a key regulatory unit in both within-plant and between-plant propagation of immunity. The data integrate molecular components of SAR into systemic signaling networks that are separate from conventional, SA-associated innate immune mechanisms. These networks are central to plant-to-plant propagation of immunity, potentially raising SAR to the population level. In this process, monoterpenes act as microbe-inducible plant volatiles, which as part of plant-derived volatile blends have the potential to promote the generation of a wave of innate immune signaling within canopies or plant stands. Hence, plant-to-plant propagation of SAR holds significant potential to fortify future durable crop protection strategies following a single volatile trigger.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Lectinas de Plantas/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Retroalimentação Fisiológica , Glicerofosfatos/imunologia , Glicerofosfatos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Monoterpenos/imunologia , Monoterpenos/metabolismo , Ácidos Pipecólicos/imunologia , Ácidos Pipecólicos/metabolismo , Doenças das Plantas/microbiologia , Lectinas de Plantas/genética , Plantas Geneticamente Modificadas , Pseudomonas syringae/imunologia , Ácido Salicílico/imunologia , Ácido Salicílico/metabolismo , Transdução de Sinais/imunologia , Compostos Orgânicos Voláteis/imunologia
18.
Insect Biochem Mol Biol ; 114: 103226, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31446033

RESUMO

The huge energy demand posed by insect flight activity is met by an efficient oxidative phosphorylation process that takes place within flight muscle mitochondria. In the major arbovirus vector Aedes aegypti, mitochondrial oxidation of pyruvate, proline and glycerol 3-phosphate (G3P) represent the major energy sources of ATP to sustain flight muscle energy demand. Although adenylates exert critical regulatory effects on several mitochondrial enzyme activities, the potential consequences of altered adenylate levels to G3P oxidation remains to be determined. Here, we report that mitochondrial G3P oxidation is controlled by adenylates through allosteric regulation of cytochrome c oxidase (COX) activity in A. aegypti flight muscle. We observed that ADP significantly activated respiratory rates linked to G3P oxidation, in a protonmotive force-independent manner. Kinetic analyses revealed that ADP activates respiration through a slightly cooperative mechanism. Despite adenylates caused no effects on G3P-cytochrome c oxidoreductase activity, COX activity was allosterically activated by ADP. Conversely, ATP exerted powerful inhibitory effects on respiratory rates linked to G3P oxidation and on COX activity. We also observed that high energy phosphate recycling mechanisms did not contribute to the regulatory effects of adenylates on COX activity or G3P oxidation. We conclude that mitochondrial G3P oxidation in A. aegypti flight muscle is regulated by adenylates through the allosteric modulation of COX activity, underscoring the bioenergetic relevance of this novel mechanism and the potential consequences for mosquito dispersal.


Assuntos
Aedes/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicerofosfatos/metabolismo , Mitocôndrias/metabolismo , Miofibrilas/metabolismo , Regulação Alostérica , Animais , Respiração Celular , Feminino , Oxirredução
19.
J Mol Med (Berl) ; 97(10): 1465-1475, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31385016

RESUMO

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor ß-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, ß-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of ß-glycerophosphate on SOCE. Moreover, the ß-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.


Assuntos
Cálcio/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína ORAI1/metabolismo , Calcificação Vascular/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aorta/citologia , Transdiferenciação Celular/genética , Células Cultivadas , Expressão Gênica , Glicerofosfatos/metabolismo , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Calcificação Vascular/genética
20.
Sci Total Environ ; 692: 219-232, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31349163

RESUMO

The multi-barrier deep geological repository system is currently considered as one of the safest option for the disposal of high-level radioactive wastes. Indigenous microorganisms of bentonites may affect the structure and stability of these clays through Fe-containing minerals biotransformation and radionuclides mobilization. The present work aimed to investigate the behavior of bentonite and its bacterial community in the case of a uranium leakage from the waste containers. Hence, bentonite microcosms were amended with uranyl nitrate (U) and glycerol-2-phosphate (G2P) and incubated aerobically for 6 months. Next generation 16S rRNA gene sequencing revealed that the bacterial populations of all treated microcosms were dominated by Actinobacteria and Proteobacteria, accounting for >50% of the community. Additionally, G2P and nitrate had a remarkable effect on the bacterial diversity of bentonites by the enrichment of bacteria involved in the nitrogen and carbon biogeochemical cycles (e.g. Azotobacter). A significant presence of sulfate-reducing bacteria such as Desulfonauticus and Desulfomicrobium were detected in the U-treated microcosms. The actinobacteria Amycolatopsis was enriched in G2P­uranium amended bentonites. High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy analyses showed the capacity of Amycolatopsis and a bentonite consortium formed by Bradyrhizobium-Rhizobium and Pseudomonas to precipitate U as U phosphate mineral phases, probably due to the phosphatase activity. The different amendments did not affect the mineralogy of the bentonite pointing to a high structural stability. These results would help to predict the impact of microbial processes on the biogeochemical cycles of elements (N and U) within the bentonite barrier under repository relevant conditions and to determine the changes in the microbial community induced by a uranium release.


Assuntos
Bactérias/metabolismo , Bentonita/análise , Glicerofosfatos/metabolismo , Microbiota/efeitos dos fármacos , Resíduos Radioativos/análise , Urânio/metabolismo , Bactérias/classificação
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