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.
抽象的
糖酵解是真核生物和生物中最重要的代谢途径之一。
原核生物。在该途径中,葡萄糖被分解形成小的 3-碳磷酸盐代谢物,这是维持生命所必需的。
细胞生长和存活。在微生物中,正确维持糖酵解对于微生物的发育非常重要
细菌感染和毒力以及抗生素耐药性。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 的功能阐明糖酵解的新调节机制
细菌。 PgpA 是一种普遍存在于革兰氏阴性细菌中的整合膜蛋白。我们发现
PgpA 充当兼职酶;即 PgpA 不仅参与磷脂生物合成,还参与
通过水解大肠杆菌中关键的 3-碳磷酸糖酵解代谢物,充当重要的代谢调节剂。
大肠杆菌。大肠杆菌中PgpA的突变失活极大地促进了细菌的代谢和生长。我们还有
确定了 PgpA 的一种新型氧化还原调节机制,这对于维持细菌代谢非常重要
体内平衡。我们的研究结果提出了氧化还原介导的调节机制的假设,其中 PgpA
通过控制基于外部和内部的谷胱甘肽介导的氧化还原平衡来调节细菌糖酵解
代谢信号。这种调节机制是新颖的,尚未在任何细胞类型中被报道。为了进一步
了解了这种调节机制,我们将研究PgpA如何控制细菌葡萄糖摄取并调节
结合生物化学、微生物学和代谢组学方法来检测糖酵解活性。
了解 PgpA 如何调节细胞内氧化还原平衡,我们将检查谷胱甘肽生物合成和
监测 PgpA 膜表面的氧化还原变化,以演示 PgpA 如何使用综合“Ying-
Yang”机制实现代谢稳态和氧化还原平衡。我们还发现了氧化还原介导的
PgpA 的调节是由 PgpA 二聚体内的二聚体二硫键交联介导的。获得结构性见解
为了研究这种新颖的氧化还原调节催化机制,我们将研究 PgpA 和辅因子的催化活性
使用生化测定在体外响应氧化还原变化而结合 Mg2+。我们还将研究这个分子
使用 FRET 的机制来演示二聚体交联如何将蛋白质构象改变为变构
改变活性位点构象以控制 PgpA 催化。由于没有可用的结构
PgpA 家族,我们将使用以下方法确定两种不同氧化还原(活性/失活)状态下的 PgpA 结构:
X 射线晶体学和单粒子冷冻电镜方法为氧化还原-建立结构基础
PgpA 的调控催化机制。这种机制在许多革兰氏阴性病原体中是保守的。我们的
研究将揭示理解微生物代谢调节的重要机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Lei Zheng其他文献
Lei Zheng的其他文献
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{{ truncateString('Lei Zheng', 18)}}的其他基金
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:
10408084 - 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:
10280369 - 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:
10652472 - 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:
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
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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:
8579467 - 财政年份:2013
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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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9764752 - 财政年份:2013
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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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- 批准号:
10358637 - 财政年份:2013
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