Structure and function of a metabolic pacemaker in bacterial cell membrane
细菌细胞膜代谢起搏器的结构和功能
基本信息
- 批准号:10457395
- 负责人:
- 金额:$ 31.98万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-08-01 至 2025-06-30
- 项目状态:未结题
- 来源:
- 关键词:Active SitesAnabolismAnaerobic BacteriaAntibiotic ResistanceAttenuatedBacteriaBacterial InfectionsBindingBinding SitesBiochemicalBiochemistryBiological AssayCalorimetryCarbonCatalysisCell SurvivalCell membraneChemicalsChemistryCommunicable DiseasesComputer ModelsCryoelectron MicroscopyCrystallizationCysteineDataDevelopmentDiffusionDisease ResistanceDisulfidesEnvironmentEnzymesEquilibriumEscherichia coliEukaryotaFamilyFermentationFluorescenceFluorescence Resonance Energy TransferGene ExpressionGenetic TranscriptionGlucoseGlucose TransporterGlutathioneGlutathione DisulfideGlycolysisGram-Negative BacteriaGrowthHomeostasisHomologous ProteinIn SituIn VitroIntegral Membrane ProteinIntracellular MembranesLabelLeadLipid BilayersLipidsMeasuresMediatingMembraneMetabolicMetabolic PathwayMetabolismMethodsMicrobiologyMolecularMolecular ConformationMonitorMutationOxidation-ReductionOxidesPacemakersPathway interactionsPhosphatidylglycerolsPhospholipidsPhosphoric Monoester HydrolasesProkaryotic CellsProtein ConformationProtein phosphataseProteinsRegulationReportingResolutionRoentgen RaysRoleSignal TransductionSiteStructureSurfaceTestingTitrationsVirulenceX-Ray CrystallographyYangaminoacid biosynthesisantimicrobialbacterial metabolismbasecell growthcell typecrosslinkdimerfamily structuregenetic approachglucose uptakeinorganic phosphateinsightmetabolomicsmicroorganismmutantnanodisknovelnovel therapeutic interventionparticlepathogenpreventresponseunnatural amino acidsvapor
项目摘要
Abstract
Glycolysis constitutes one of the most important metabolic pathways conserved in both eukaryotes and
prokaryotes. In the pathway, glucose is broken down to form small 3-carbon phosphate metabolites essential for
cell growth and survival. In microorganisms, properly maintaining glycolysis is important for the development of
bacterial infection and virulence and antibiotic resistance. In this project, we aim to study the structure and
function of phosphatidylglycerol phosphatase PgpA to elucidate a novel regulatory mechanism of glycolysis in
bacterium. PgpA is an integral membrane protein ubiquitously found in Gram-negative bacterium. We found that
PgpA functions as a moonlighting enzyme; i.e. PgpA is not only involved in phospholipid biosynthesis but also
acts as an essential metabolic regulator by hydrolyzing the key 3-carbon phosphate glycolytic metabolites in E.
coli. Mutational inactivation of PgpA in E. coli greatly facilitates bacterial metabolism and growth. We have also
identified a novel redox-regulatory mechanism of PgpA, which is important to maintain bacterial metabolic
homeostasis. Our findings raise the hypothesis for a redox-mediated regulatory mechanism in which PgpA
regulates bacterial glycolysis by controlling glutathione-mediated redox balance based on external and internal
metabolic signals. This regulatory mechanism is novel and has not yet been reported in any cell type. To further
understand this regulatory mechanism, we will study how PgpA controls bacterial glucose uptake and regulate
glycolytic activity using a combination of biochemistry, microbiology, and metabolomic approaches.To
understand how PgpA regulates intracellular redox balance, we will examine glutathione biosynthesis and
monitor redox changes on the membrane surface of PgpA to demonstrate how PgpA uses an integrative “Ying-
Yang” mechanism to achieve both metabolic homeostasis and redox balance. We also found the redox-mediated
regulation of PgpA is mediated by dimeric disulfide crosslinking within PgpA dimer. To gain structural insights
into this novel redox-regulated catalytic mechanism, we will study the catalytic activity of PgpA and co-factor
Mg2+ binding in response to redox changes in vitro using biochemical assays. We will also study this molecular
mechanism using FRET to demonstrate how dimeric crosslinking alters protein conformation to allosterically
change the active site conformation in order to control the PgpA catalysis. Since no structure is available in the
PgpA family, we will determine the structures of PgpA in two distinct redox (active/inactivated) states using the
X-ray crystallography and single-particle cryoEM approaches to establish a structural basis for the redox-
regulated catalytic mechanism of PgpA. This mechanism is conserved in many Gram-negative pathogens. Our
studies will reveal an important mechanism to understand metabolic regulation in microorganisms.
摘要
糖酵解是最重要的代谢途径之一,在真核生物和
原核生物。在这个过程中,葡萄糖被分解形成小的3-碳磷酸盐代谢物
细胞的生长和存活。在微生物中,适当地维持糖酵解对于发展
细菌感染与毒力和抗生素耐药性。在这个项目中,我们的目标是研究结构和
磷脂酰甘油磷酸酶PGPA在阐明糖酵解调节机制中的作用
细菌。PGPA是革兰氏阴性菌中普遍存在的一种完整的膜蛋白。我们发现
PGPA作为一种兼职酶起作用,即PGPA不仅参与磷脂的生物合成,而且还参与
作为一种重要的代谢调节剂,在E。
Coli.PGPA在大肠杆菌中的突变失活极大地促进了细菌的新陈代谢和生长。我们还有
发现了一种新的PGPA的氧化还原调节机制,它对维持细菌的代谢很重要
动态平衡。我们的发现提出了一种假设,即氧化还原介导的调节机制中,PGPA
通过控制谷胱甘肽介导的氧化还原平衡调节细菌糖酵解
代谢信号。这种调控机制是新颖的,目前还没有任何细胞类型的报道。为了进一步
了解了这种调节机制,我们将研究PGPA是如何控制细菌葡萄糖摄取和调节的
生物化学、微生物学和代谢组学相结合的糖酵解活性。
了解PGPA是如何调节细胞内氧化还原平衡的,我们将研究谷胱甘肽的生物合成和
监测PGPA膜表面氧化还原的变化,演示PGPA如何利用一体化的“鹰”-
“阳”机制实现代谢动态平衡和氧化还原平衡。我们还发现了氧化还原介导的
PGPA的调节是通过PGPA二聚体中的二聚体二硫键交联来实现的。获得结构性洞察力
在这一新的氧化还原调节的催化机制中,我们将研究PGPA和辅因子的催化活性。
用生化分析方法研究镁离子结合对体外氧化还原反应的影响。我们也会研究这个分子
用FRET研究二聚体交联物改变蛋白质构象的机制
通过改变活性中心构象来控制PGPA的催化作用。中没有可用的结构。
作为PGPA家族的一员,我们将使用
X射线结晶学和单粒子低温电子显微镜方法,为氧化还原建立结构基础-
调节PGPA的催化机制。这一机制在许多革兰氏阴性杆菌中都是保守的。我们的
研究将揭示了解微生物代谢调节的重要机制。
项目成果
期刊论文数量(0)
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会议论文数量(0)
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{{ truncateString('Lei Zheng', 18)}}的其他基金
Structure and function of a metabolic pacemaker in bacterial cell membrane
细菌细胞膜代谢起搏器的结构和功能
- 批准号:
10280369 - 财政年份:2021
- 资助金额:
$ 31.98万 - 项目类别:
Integration of stromal targeting agents with immune checkpoint therapy
基质靶向剂与免疫检查点疗法的整合
- 批准号:
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- 资助金额:
$ 31.98万 - 项目类别:
Structure and function of a metabolic pacemaker in bacterial cell membrane
细菌细胞膜代谢起搏器的结构和功能
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10652472 - 财政年份:2021
- 资助金额:
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Integration of stromal targeting agents with immune checkpoint therapy
基质靶向剂与免疫检查点疗法的整合
- 批准号:
10661808 - 财政年份:2021
- 资助金额:
$ 31.98万 - 项目类别:
Structure and function of a metabolic pacemaker in bacterial cell membrane
细菌细胞膜代谢起搏器的结构和功能
- 批准号:
10796719 - 财政年份:2021
- 资助金额:
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Annexin A2 as a mediator of pancreatic cancer metastases
膜联蛋白 A2 作为胰腺癌转移的介质
- 批准号:
8712421 - 财政年份:2013
- 资助金额:
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Annexin A2 as a mediator of pancreatic cancer metastases
膜联蛋白 A2 作为胰腺癌转移的介质
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Interrogate the interaction between tumor cells and nerves in the tumor microenvironment of pancreatic cancer
探究胰腺癌肿瘤微环境中肿瘤细胞与神经之间的相互作用
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Interrogate the interaction between tumor cells and nerves in the tumor microenvironment of pancreatic cancer
探究胰腺癌肿瘤微环境中肿瘤细胞与神经之间的相互作用
- 批准号:
10358637 - 财政年份:2013
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$ 31.98万 - 项目类别:
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