Lipid phosphatases as dynamic regulators of protein conformation

脂质磷酸酶作为蛋白质构象的动态调节剂

基本信息

批准号：
10005416
负责人：
Bethany S Strunk
金额：
$ 24.89万
依托单位：
TRINITY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10005416
关键词：
Active Sites Alanine Alleles Amino Acids Amyotrophic Lateral Sclerosis Binding Binding Sites Biological Assay C-terminal Catalytic Domain Cells Charcot-Marie-Tooth Disease Complex Cryoelectron Microscopy Disease Distal Dominant-Negative Mutation Engineering Enzymes Failure Fluorescence Resonance Energy Transfer Future Genetic Screening Impairment International Journals Laboratories Link Lipids Malignant Neoplasms Mammals Maps Mentors Michigan Modeling Molecular Conformation Mus Mutagenesis Mutate Mutation Neurodegenerative Disorders Oranges PTEN gene PTEN protein Paper Pathology Pathway interactions Peripheral Nervous System Diseases Phase Phenotype Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Point Mutation Protein Conformation Protein Dephosphorylation Protein phosphatase Proteins Proteomics Recombinant Proteins Regulation Research Roentgen Rays Role Rotation Scaffolding Protein Scanning Signal Pathway Site Stimulus Structure Surface System Tail Testing Training Variant Writing Yeasts base crosslink disease-causing mutation enzyme model human disease improved in vivo meetings mouse development mutant novel phosphatidylinositol 3,4,5-triphosphate phosphatidylinositol 3,5-diphosphate prevent protein protein interaction student participation tissue culture tumor undergraduate student unnatural amino acids yeast genetics

项目摘要

Abstract: Mutations in phosphatidylinositol (PI) phosphatases cause diseases including cancer and neurodegenerative disorders. A common assumption is that failure to dephosphorylate a specific lipid substrate underlies these diseases. Emerging evidence raises the possibility that PI phosphatases have lipid- phosphatase-independent functions that contribute to disease pathology when these enzymes are mutated. In support of this hypothesis, mutations that inactivate some PI phosphatases result in phenotypes that are different from a total loss of the protein. This proposal will test the hypothesis that conformational changes that accompany the regulation of the activity of PI phosphatases influence their interactions with protein partners. The disease related PI phosphatases PTEN and Fig4 will be used as model enzymes. The mentored phase of this proposal will take place in the laboratory of Dr. Lois Weisman, a leading expert in the phosphatidylinositol 3,5-bisphosphate (PI3,5P2) signaling pathway. In pursuit of the proposed research, the applicant will be trained in large scale genetic screens in yeast, genetic incorporation of unnatural amino acids for probing dynamic protein-protein interactions (with Dr. Ann Mapp, U. Michigan), and mammalian tissue culture. In addition to research, the mentored phase will include: writing scientific papers, presentations at national and/or international meetings, mentoring of undergraduate and graduate rotation students, and participation in local journal and research clubs. In the independent phase, the applicant will establish an academic laboratory where the applicant proposes to use both yeast and mammalian systems to elucidate principles of cross-talk between cellular pathways via lipid-phosphatase-independent functions of PI phosphatases. Aim 1 will characterize unpublished Fig4 mutants that either activate or inhibit Fab1 kinase activity and screen for new mutants. In Aim 2 these mutants will be used to determine how Fig4 regulates PI3,5P2 via regulation of the Fab1/Vac14/Fig4 complex. Fig4 domains involved in protein-protein interactions will be probed through incorporation of photo-activatable amino acid cross-linkers in Fig4 using nonsense suppression. The potential for Fig4 to act as a protein phosphatase will be tested by identifying phosphorylation sites altered in cells expressing wild-type versus catalytically inactive Fig4. These and future studies will test the hypothesis that specific stimuli, which induce conformational changes in yeast Fig4, in turn activate or inhibit Fab1. Aim 3 will determine the roles of the PTEN active site in a regulatory intramolecular interaction with the C-terminal tail. Binding assays between PTEN active site mutants and variants of the C-terminal tail will be performed both in intact cells and with recombinant proteins. In addition, proteins that specifically interact with active or inactive PTEN conformations will be identified via purification of native complexes associated with TAP-tagged mutants from intact cells. These studies will test the hypothesis that conformational changes regulating PTEN lipid phosphatase activity serve in parallel to regulate downstream pathways through protein-protein interactions.

摘要：磷脂酰肌醇（PI）磷酸酶的突变引起癌症和疾病神经退行性疾病。一个常见的假设是，未能去磷酸化特定的脂质底物这些疾病是基础。新兴证据提高了PI磷酸酶的脂质 - 当这些酶突变时，磷酸酶独立的功能会导致疾病病理。在支持这一假设，使某些PI磷酸酶失活的突变导致表型与蛋白质的总损失不同。该提议将检验以下假设，即构象变化伴随着PI磷酸酶活性的调节会影响其与蛋白质伴侣的相互作用。与疾病相关的PI磷酸酶PTEN和FIF4将用作模型酶。指导的阶段该建议将在磷脂酰肌醇的领先专家Lois Weisman博士的实验室中进行。 3,5-双磷酸（PI3,5P2）信号通路。为了追求拟议的研究，申请人将接受培训在酵母中的大规模遗传筛选中，非天然氨基酸的遗传掺入用于探测动态蛋白质 - 蛋白质相互作用（与密歇根州的Ann Mapp博士）和哺乳动物组织培养。此外研究，指导阶段将包括：撰写科学论文，国家和/或国际会议，指导本科生和研究生轮换学生以及参加当地期刊和研究俱乐部。在独立阶段，申请人将建立一个学术实验室申请人建议同时使用酵母菌和哺乳动物系统来阐明交叉原理 PI磷酸酶的脂质磷酸酶独立于脂质磷酸酶的功能之间的细胞途径之间。目标1意志表征未发表的FIG4突变体，该突变体激活或抑制Fab1激酶活性和新的筛选突变体。在AIM 2中，这些突变体将用于确定FIF4如何通过调节来调节PI3,5P2 Fab1/vac14/fig4复合物。涉及蛋白质 - 蛋白质相互作用的FIG4结构域将通过使用胡说八道的抑制在FIG4中掺入光活化氨基酸交联。潜力为了使图4充当蛋白质磷酸酶，将通过鉴定细胞中改变的磷酸化位点进行测试表达野生型与催化无活性的无效图4。这些和未来的研究将检验以下假设诱导酵母FIG4构象变化的特定刺激反过来激活或抑制Fab1。目标3意志确定PTEN活性位点与C末端尾部分子内相互作用中的作用。 PTEN活性位点突变体之间的结合测定和C末端的变体将在完整的细胞和重组蛋白。另外，专门与活动或非活性相互作用的蛋白质将通过纯化与Tapged突变体相关的天然复合物来识别PTEN构象来自完整的细胞。这些研究将检验以下假设：构象变化调节PTEN脂质磷酸酶的活性并联，可以通过蛋白质蛋白质相互作用来调节下游途径。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Multiple pathways for Fig4 contributions to cellular homeostasis in yeast.

Fig4 对酵母细胞稳态的贡献有多种途径。

DOI：
发表时间：
2022
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Khan,Imran;Reeves,HannahE;Darbro,CarolineS;Hall,HarrisonS;Strunk,BethanyS
通讯作者：
Strunk,BethanyS