Phospholipid Metabolism in Cell Membrane

细胞膜中的磷脂代谢

• 批准号: 8653971
• 负责人: Lei Zheng
• 金额: $ 28.88万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8653971
• 关键词: Binding; Biochemical; Biochemical Pathway; Biological; Cardiovascular Diseases; Catalysis; Catalytic Domain; Cell Cycle Arrest; Cell membrane; Cell physiology; Cells; Communication; Complex; Detergents; Development; Energy Transfer; Environment; Enzymes; Escherichia coli; Extracellular Domain; Family member; Fluorescence; Head; Homeostasis; Homologous Gene; Hormonal; Integral Membrane Protein; Label; Lateral; Lipid Bilayers; Lipids; Malignant Neoplasms; Measurement; Measures; Mediating; Membrane; Metabolic; Metabolism; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Mutagenesis; Mutation; Neck; Neoplasm Metastasis; Orthovanadate; Pathway interactions; Phospholipid Metabolism; Phospholipids; Phosphoric Monoester Hydrolases; Play; Protein Conformation; Protein Dephosphorylation; Protein Dynamics; Protein Family; Protein phosphatase; Proteins; Regulation; Roentgen Rays; Role; Route; Scanning; Site; Solutions; Solvents; Spin Labels; Structural Models; Structure; Substrate Interaction; Testing; Therapeutic; Time; Tissues; Transmembrane Domain; Tryptophan; Vanadates; analog; base; carcinogenesis; cardiovascular injury; cell growth; cell type; crosslink; design; essential phospholipids; inhibitor/antagonist; inorganic phosphate; lipid metabolism; lipid phosphate phosphatase; meetings; member; membrane synthesis; nanodisk; novel; protein complex; protein structure function; reconstitution; response; signal processing; stopped-flow fluorescence

DESCRIPTION (provided by applicant): Phospholipid metabolism is fundamental in cells. It not only generates basic biological membranes, but also plays important roles in cellular signaling processes in nearly all tissues. In addition, many proteins, both globular and membrane bound, require specific phospholipids to fulfill their functions. Cells maintain a complicated and regulated metabolic network to synthesize a great diversity of phospholipids and degrade them in a time fashion to meet cellular requirements. Many steps of phospholipid metabolism take place on the cell membrane and are catalyzed by membrane-embedded enzymes. Their molecular mechanisms are poorly understood largely due to the paucity of structural information. In particular, how these enzymes select their substrates from the lipid membrane bilayer and carry out catalysis in a hydrophobic membrane environment is a central question still unanswered for general phospholipid metabolic mechanisms. To understand this important question, we study lipid phosphate phosphatases (LPPs) as model. LPPs, members of an intramembrane phosphatase protein family, play important roles in phospholipid synthesis and homeostasis. LPPs also catalyze dephosphorylation of several important phospholipid hormonal messengers regulating numerous phospholipids-mediated signaling processes. Based on our recent apo form crystal structure of the PgpB protein, an LPP homolog from E.coli, we proposed a novel hypothesis for the intramembrane dephosphorylation mechanism of PgpB, in which a) phospholipid substrates access an conserved intramembrane tunnel from the membrane bilayer to reach the catalytic site and b) a large conformational change of TM3 is essential for substrate binding and catalysis. To test this important hypothesis, in this project w will focus on two key aspects using a combination of biochemical, biophysical and X-ray structural approaches. 1) To demonstrate the substrate binding conformation and substrate-induced protein conformational changes, we will determine PgpB complex structures bound with a metabolism-stabilized phospholipid substrate analog or vanadate, a phosphate product analog, to gain structural details of a catalytic cycle. 2) To functionally characterize the intramembrane substrate access tunnel, we have designed several mutagenesis and crosslinking strategies to elucidate how the substrate passes through the intramembrane tunnel to reach the catalytic site. We will also explore the product release pathway using similar approaches to understand how the dephosphorylated product is delivered to the membrane bilayer after catalysis. 3) To further demonstrate the protein conformational changes, we will apply EPR and fluorescence stopped-flow approaches to catch the protein motions in response to the substrate analog binding in atomic detail in detergent solutions or in different lipid-defind nanodiscs. These structural and functional studies will not only confirm our hypothesis and reveal the catalytic mechanism of intramembrane phospholipid dephosphorylation, but also establish a structural basis to understand phospholipid metabolism in the cell membrane in general.

描述（由申请人提供）：磷脂代谢是细胞的基础。它不仅产生基本的生物膜，而且在几乎所有组织的细胞信号传导过程中起重要作用。此外，许多蛋白质（无论是球状蛋白质还是膜结合蛋白质）都需要特定的磷脂来实现其功能。细胞维持一个复杂的和受调控的代谢网络，以合成多种多样的磷脂，并以时间的方式降解它们以满足细胞的需求。磷脂代谢的许多步骤发生在细胞膜上，并由膜包埋酶催化。其分子机制知之甚少，主要是由于缺乏结构信息。特别是，这些酶如何从脂膜双层中选择底物并在疏水膜环境中进行催化，这是一般磷脂代谢机制尚未回答的核心问题。为了理解这个重要问题，我们以脂质磷酸磷酸酶（LPP）为模型进行研究。LPPs是膜内磷酸酶蛋白家族的成员，在磷脂合成和体内平衡中起重要作用。LPPs还催化几种重要的磷脂激素信使的去磷酸化，调节许多磷脂介导的信号传导过程。基于我们最近发现的Pgp B蛋白（一种来自大肠杆菌的LPP同系物）的载脂蛋白形式晶体结构，我们提出了Pgp B的膜内去磷酸化机制的新假设，其中a）磷脂底物从膜双层进入保守的膜内隧道到达催化位点，B）TM 3的大构象变化对于底物结合和催化是必不可少的。为了验证这一重要的假设，在这个项目中，我们将集中在两个关键方面，使用生物化学，生物物理和X射线结构的方法相结合。1)为了证明底物结合构象和底物诱导的蛋白质构象变化，我们将确定与代谢稳定的磷脂底物类似物或钒酸盐（磷酸盐产物类似物）结合的PgpB复合物结构，以获得催化循环的结构细节。2)为了在功能上表征膜内底物通道，我们设计了几种诱变和交联策略来阐明底物如何通过膜内通道到达催化位点。我们还将探索使用类似的方法，以了解如何去磷酸化的产品被交付到催化后的膜双层的产品释放途径。3)为了进一步证明蛋白质的构象变化，我们将应用EPR和荧光停流方法来捕捉蛋白质运动响应于在去污剂溶液或不同的脂质定义的纳米盘中的原子细节中的底物类似物结合。这些结构和功能的研究不仅将证实我们的假设，并揭示膜内磷脂去磷酸化的催化机制，但也建立了一个结构基础，了解磷脂代谢的细胞膜一般。