PLCgamma regulates HNE3 activity through direct binding and dynamic complexes

PLCgamma 通过直接结合和动态复合物调节 HNE3 活性

• 批准号: 7920788
• 负责人: Nicholas Constantine Zachos
• 金额: $ 14.85万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-20 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7920788
• 关键词: Accounting; Affect; Amino Acids; Bacterial Toxins; Binding; Biochemical; Bioinformatics; Biological Assay; Brush Border; C-terminal; Calcium; Calcium Signaling; Colon; Complex; Data; Density Gradient Centrifugation; Diarrhea; Digestion; Disease; Endocytosis; Epithelial Cells; Gastrointestinal tract structure; Gene Family; In Vitro; Intestines; Intracellular Second Messenger; Lead; Lipid Binding; Mediating; Membrane Microdomains; Mutagenesis; Mutation; Nutrient; PH Domain; Phospholipids; Play; Principal Investigator; Protein Binding Domain; Regulation; Research Personnel; Role; SRC gene; Scaffolding Protein; Second Messenger Systems; Signaling Molecule; Signaling Protein; Small Intestines; Sodium; Sodium-Hydrogen Antiporter; Sucrose; Testing; Water; Yeasts; absorption; base; design; human PLCG2 protein; in vivo; insight; novel; phospholipase C gamma; platelet protein P47; prevent; protein complex; protein protein interaction; public health relevance; scaffold; src Homology Region 2 Domain; yeast two hybrid system

DESCRIPTION (provided by applicant): In the gastrointestinal tract, the absorption of water and nutrients occurs as normal part of digestion. In the intestine, impaired sodium absorption results in decreased water absorption and diarrhea. In diarrheal diseases, elevation of intracellular second messengers, such as calcium (Ca2+), by neurohumoral substances and bacterial toxins results in decreased sodium absorption in the intestine and colon. The sodium/hydrogen exchanger (NHE) gene family plays an integral role in sodium absorption in the mammalian intestine. Regulation of brush border NHE3 accounts for most of the recognized digestive changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular Ca2+ may involve the participation of phospholipase C gamma (PLCg), however the mechanism responsible for Ca2+ regulation of NHE3 activity is not well understood. Bioinformatics analysis has suggested PLCg may regulate NHE3 through a direct protein-protein interaction and results in the formation of an intermolecular PH domain. Therefore, the current study is designed to test the hypothesis that (1) NHE3 and PLCg directly interact; (2) PLCg contributes to basal activity and is necessary forCa2+ inhibition of NHE3 activity; (3) PLCg scaffolds NHE3 to signaling proteins, such as c-Src, which are required for proper function and regulation of NHE3 activity; (4) NHE3 and PLCg binding results in the formation of an intermolecular PH domain which regulates NHE3 activity by binding specific phospholipids in lipid raft microdomains of intestinal epithelial cells. To answer these questions, the principal investigator will utilize in vitro binding assays(e.g. pull-down, yeast two-hybrid) to determine the amino acids within NHE3, by mutagenesis, that are responsible for direct binding to PLCg. Investigator will determine the consequence of preventing PLCg binding to NHE3 on basal andCa2+ inhibition of NHE3 activity. Biochemical studies, including sucrose density gradient centrifugation, will be performed to analyze the contribution of PLCg on formation of NHE3 complexes. Finally, the investigator will perform lipid binding assays to uncover novel interactions between phospholipids and NHE3 as predicted by bioinformatics analysis. The results of this study will provide novel insights into the role of PLCg in regulation of NHE3 activity to further understand sodium absorption in digestion and diarrheal diseases. PUBLIC HEALTH RELEVANCE: In the small intestine and colon, regulation of NHE3 activity accounts for most of the recognized changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular calcium may involve the participation of PLCg, however, the mechansim [sic] responsible for the regulation is not well understood. The current study will provide novel insights into understanding the role of PLCg in regulating NHE3 activity in digestion and diarrhea.

描述（由申请人提供）： 在胃肠道中，水的吸收和养分是消化的正常部分。在肠中，钠吸收受损会导致吸水和腹泻降低。在腹泻疾病中，通过神经肿瘤物质和细菌毒素的细胞内第二信使（例如钙（Ca2+））的升高导致肠和结肠中的钠吸收降低。钠/氢交换剂（NHE）基因家族在哺乳动物肠中钠吸收中起着不可或缺的作用。刷子边界NHE3的调节是消化过程中发生的大多数公认的钠吸收消化变化以及腹泻期间发生的钠吸收的抑制作用。细胞内Ca2+对NHE3活性的抑制可能涉及磷脂酶C伽马（PLCG）的参与，但是尚不清楚负责Ca2+调节NHE3活性的机制。生物信息学分析表明，PLCG可以通过直接蛋白质 - 蛋白质相互作用来调节NHE3，并导致形成分子间pH结构域。因此，当前的研究旨在检验（1）NHE3和PLCG直接相互作用的假设。 （2）PLCG有助于基础活性，并且是对NHE3活性的forca2+抑制作用； （3）PLCG支架NHE3对信号蛋白，例如C-SRC，这是适当功能和调节NHE3活性所必需的； （4）NHE3和PLCG结合导致形成分子间pH结构域，该结构域通过结合肠上皮细胞脂质筏微区域中特定磷脂来调节NHE3活性。为了回答这些问题，主要研究者将通过诱变，利用诱变，以直接与PLCG结合来确定NHE3中NHE3内的氨基酸的体外结合测定法（例如下拉，酵母菌两杂化）。研究者将确定防止PLCG与NHE3结合对基底和CA2+ NHE3活性抑制的结果。将进行包括蔗糖密度梯度离心在内的生化研究，以分析PLCG对NHE3复合物形成的贡献。最后，研究者将执行脂质结合测定，以发现磷脂与NHE3之间的新型相互作用，如生物信息学分析所预测。这项研究的结果将为PLCG在调节NHE3活性中的作用提供新的见解，以进一步了解消化和腹泻疾病中的钠吸收。 公共卫生相关性：在小肠和结肠中，NHE3活性的调节是消化过程中发生的大多数公认的钠吸收变化，以及腹泻期间发生的钠吸收的抑制。细胞内钙对NHE3活性的抑制可能涉及PLCG的参与，但是，对调节负责的Mechansim [SIC]尚不清楚。当前的研究将为了解PLCG在调节NHE3活性在消化和腹泻中的作用方面提供新的见解。