Regulation of V-ATPases by Phosphoinositides

磷酸肌醇对 V-ATP 酶的调节

基本信息

批准号：
10162616
负责人：
PATRICIA M KANE
金额：
$ 27.54万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10162616
关键词：
Address Affinity Binding Binding Sites Biochemical Genetics Biological Models C-terminal Cell Compartmentation Cells Characteristics Code Complex Cytosol Defect Disease Disseminated Malignant Neoplasm Drug Targeting Endosomes Engineering Environment Eukaryotic Cell Functional disorder Future Golgi Apparatus Homology Modeling Human In Vitro Individual Intracellular Membranes Length Link Lipid Binding Lipids Lysosomes Malignant Neoplasms Membrane Metastatic to Molecular Conformation Mycobacterium Infections Mycobacterium tuberculosis N-terminal Nerve Degeneration Neurodegenerative Disorders Organelles Osteoporosis Pathway interactions Peripheral Phosphatidylinositols Physiology Play Process Property Protein Isoforms Proteins Proton Pump Protons Regulation Role Route Secretory Vesicles Signal Transduction Signaling Molecule Sodium Chloride Specificity Stress Structural Models Structure System Testing Toxin Transmembrane Domain Vacuole Virus Virus Diseases Work Yeasts base biological adaptation to stress developmental disease experimental study genetic approach in vivo insight loss of function mutation mutant novel novel therapeutics pH Homeostasis pH gradient pathogen preference public health relevance residence response therapeutic target vacuolar H+-ATPase

项目摘要

PROJECT SUMMARY V-ATPases are highly conserved, multisubunit proton pumps that acidify organelles including lysosomes, endosomes, Golgi apparatus, and secretory granules in all eukaryotic cells. Each of these organelles requires tuning of its luminal pH to a narrow range for its function. However, it is not fully understood how these pH ranges are maintained or how V-ATPases in different organelles respond when environmental conditions challenge local pH gradients. Our central hypothesis is that PI lipids provide organelle-specific inputs to locally regulate V-ATPase activity, and thus can impact individual subsets of the many functions dependent on V-ATPase activity and organelle acidification. In Aim 1, we use the well- characterized yeast V-ATPase system to characterize binding of the cytosolic domains of the membrane- bound a-subunit isoforms, Vph1 and Stv1, to phosphoinositide (PI) lipids. We have found that vacuole-resident Vph1 is able to bind the vacuolar signaling lipid PI(3,5)P2 and Golgi-resident Stv1 prefers the Golgi-enriched lipid PI(4)P. We will quantify and characterize these binding preferences and probe how binding of specific lipids activates V-ATPases containing individual a-subunit isoforms. Based on recent structures and homology modeling, we will then develop lipid-binding mutants for each isoform. Finally, we will evaluate PI binding specificities of the four human a-subunit isoforms. We have tested three of the four human isoforms and found evidence of PI-specific interactions in vitro. We will characterize specificity and affinities of PI binding and identify potential binding sites. In Aim 2, we address the spatial and temporal consequences of PI lipid binding to intact V-ATPases containing full-length Vph1 and Stv1. We hypothesize that PI lipids exert spatial control by activating V-ATPases in their organelles of residence. Specifically, we propose that: 1) PI(3,5)P2 binding to Vph1-containing V-ATPases induces a conformation that stabilizes interactions between the peripheral V1 sector and the integral membrane Vo sector of the V-ATPase, increasing activity, 2) PI(4)P binding to Stv1- containing V-ATPases promotes retention in the Golgi apparatus, and 3) PI(3)P promotes acidification of the endocytic pathway by interacting with Vph1-containing V-ATPases. In contrast, hyperosmotic stress promotes a rapid and transient rise in PI(3,5)P2 that reversibly activates Vph1-containing V-ATPases in the vacuole. We will analyze the mechanisms of this temporal V-ATPase activation and its role in providing short-term adaptation to salt stress. These experiments link PI and V-ATPase isoform content, two fundamental features of organelle identity. They promise to provide novel insights into the many diseases linked to defective organelle acidification, including neurodegeneration, cancer, and mycobacterial infection, and could suggest new therapeutic routes.

项目摘要 V-ATP酶是高度保守的多亚基质子泵所有真核细胞中的溶酶体，内体，高尔基体和分泌颗粒。每个细胞器需要将其腔内pH调整为狭窄的范围。但是，它不是完全了解这些pH范围如何保持或不同细胞器中的V-ATP酶的响应环境条件挑战局部pH梯度。我们的中心假设是Pi脂质提供细胞器特异性输入以局部调节V-ATPase活性，因此可能影响许多功能取决于V-ATPase活性和细胞器酸化。在AIM 1中，我们使用了表征酵母V-ATPase系统以表征膜的胞质结构域的结合结合a-亚基同工型VPH1和STV1与磷酸肌醇（PI）脂质。我们发现液态居民 VPH1能够结合液泡信号脂质Pi（3,5）P2和Golgi-endent STV1更喜欢Golgi-engeded 脂质pi（4）p。我们将量化和表征这些结合偏好，并探测特定的结合脂质激活含有单个A-亚基同工型的V-ATP酶。基于最近的结构和同源性然后，我们将为每种同工型开发脂质结合突变体。最后，我们将评估PI绑定四个人类A-亚基同工型的特异性。我们已经测试了四种人类同工型中的三个，并发现体外PI特异性相互作用的证据。我们将表征PI结合的特异性和亲和力，识别潜在的结合位点。在AIM 2中，我们解决了PI脂质结合的空间和时间后果到包含全长VPH1和STV1的完整V-ATP酶。我们假设PI脂质通过在其居住细胞器中激活V-ATP酶。具体而言，我们建议：1）PI（3,5）P2结合到含VPH1的V-ATP酶诱导构象，使外围V1之间的相互作用稳定 V-ATPase的扇形和整体膜VO部门，活动的增加，2）PI（4）P与STV1-结合含有V-ATP酶会促进高尔基体的保留率，3）PI（3）P促进了酸化的酸化。通过与含VPH1的V-ATP酶相互作用的内吞途径。相反，高渗性应力促进 PI（3,5）P2的快速瞬时上升可逆地激活液泡中含有VPH1的V-ATP酶。我们将分析这种时间V-ATPase激活的机制及其在提供短期的作用适应盐胁迫。这些实验链接PI和V-ATPase同工型含量，两个基本特征细胞器的身份。他们承诺将提供与有缺陷有关的许多疾病的新颖见解细胞器酸化，包括神经变性，癌症和分枝杆菌感染，可以建议新的治疗路线。