Regulation and Cellular Functions of V-ATPases

V-ATP酶的调节和细胞功能

• 批准号: 10593953
• 负责人: PATRICIA M KANE
• 金额: $ 57.05万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593953
• 关键词: Acute; Aging; Albers-Schonberg disease; Alzheimer' s Disease; Binding; Cell membrane; Cell physiology; Cells; Chronic; Complex; Defect; Disease; Distal; Endocytosis; Endosomes; Environment; Enzymes; Epilepsy; Eukaryota; Eukaryotic Cell; Functional disorder; In Vitro; Infertility; Link; Lipids; Location; Lysosomes; Malignant Neoplasms; Mammalian Cell; Membrane; Mutation; Neoplasm Metastasis; Nerve Degeneration; Organelles; Osteoporosis; Peripheral; Phosphatidylinositols; Phospholipids; Play; Process; Protein Isoforms; Proteins; Proton Pump; Pump; Regulation; Renal tubular acidosis; Role; Structure; Therapeutic; Vacuole; Virus; Yeasts; deafness; environmental stressor; extracellular; fungus; pH Homeostasis; public health relevance; vacuolar H+-ATPase; virtual

PROJECT SUMMARY V-ATPases are versatile, highly conserved, multi-subunit proton pumps responsible for organelle acidification in virtually all eukaryotic cells. Complete loss of V-ATPase activity is lethal in all eukaryotes except fungi, but mutations in subunit isoforms are linked to distal renal tubule acidosis, infertility, deafness, and osteopetrosis. V-ATPase activity is subverted in cancer to support additional demands on cellular pH homeostasis and promote metastasis by creating an acidic extracellular environment. Endosomal acidification by V-ATPases also promotes entry of many viruses. There is a crucial need to understand the roles and regulation of V- ATPase subunit isoforms and enzyme subpopulations in order to target of V-ATPases in specific locations therapeutically. V-ATPases are regulated by reversible disassembly of the peripheral V1 subcomplex from the integral membrane Vo subcomplex, and RAVE/Rabconnectin-3 complexes play a critical role in this process. We will investigate the structure, mechanism, and subunit composition of the yeast RAVE and mammalian Rabconnectin-3 complexes, which appear to target specific V-ATPase subunit isoforms as part of their activity. Importantly, mutations in Rabconnectin-3 complexes have been associated with epilepsy and neurodegeneration but the underlying disease mechanism is unclear. We previously demonstrated in yeast that organelle-enriched phosphoinositide phospholipids bind differentially to the two a-subunit isoforms of the Vo subcomplex, providing organelle-specific inputs into V-ATPase localization and activity. Similar lipid interactions are observed with mammalian a-subunit isoforms in vitro, and we will extend these studies to characterizing the effects of the interactions in cultured mammalian cells. Finally, although V-ATPases must function in concert with other cellular mechanisms of pH homeostasis, the underlying mechanisms of this coordination are not understood. We will address this question in yeast, where we have discovered that acute or chronic loss of V-ATPase activity triggers endocytosis of a portion of the major H+ export pump, Pma1, from the plasma membrane. We will determine the mechanism of this vacuole to plasma membrane pH crosstalk. Reduced vacuole/lysosome acidification is an early step in aging in both yeast and mammalian cells. We will assess whether aging yeast cells display a loss in coordinated pH homeostasis or emerging defects in the V- ATPase itself and determine whether these processes can be manipulated.

项目摘要 V-ATP酶是用途广泛的，高度保守的多含量质子泵，负责细胞器酸化 在几乎所有真核细胞中。除真菌以外的所有真核生物中，V-ATPase活性的完全丧失都是致命的，但是 亚基同工型中的突变与肾小管远端小管酸中毒，不育症，耳聋和骨质骨术有关。 V-ATPase活性在癌症中被颠覆，以支持对细胞pH稳态和 通过创建酸性细胞外环境来促进转移。 V-ATP酶的内体酸化 还促进了许多病毒的进入。了解V-的作用和调节至关重要 ATPase亚基同工型和酶亚群，以便在特定位置靶向V-ATPase 在治疗上。 V-ATP酶受外围V1亚复合物的可逆拆卸调节 整体膜VO子复合物和Rave/RabConnectin-3复合物在此过程中起着关键作用。 我们将研究酵母狂欢和哺乳动物的结构，机制和亚基组成 RabConnectin-3复合物似乎是针对特定的V-ATPase亚基同工型作为其活性的一部分。 重要的是，RabConnectin-3复合物中的突变与癫痫和 神经变性但潜在的疾病机制尚不清楚。我们以前在酵母中证明 富含细胞器的磷酸肌醇磷脂与两种A-亚基同工型的结合 VO子复合物，将特定于Organle的输入提供到V-ATPase定位和活性中。类似的脂质 在体外观察到与哺乳动物A-亚基同工型的相互作用，我们将将这些研究扩展到 表征相互作用在培养的哺乳动物细胞中的影响。最后，尽管V-atpases必须 与其他pH稳态的其他细胞机制一致的功能，这是基本机制 协调不了解。我们将在酵母中解决这个问题，在那里我们发现了急性 或V-ATPase活性的慢性丧失触发了主要H+出口泵PMA1的一部分内吞作用 质膜。我们将确定该液泡对质膜pH串扰的机制。 液泡/溶酶体酸化减少是酵母和哺乳动物细胞衰老的早期一步。我们将 评估衰老的酵母细胞是在V-中表现出的pH稳态损失还是新兴缺陷 ATPase本身并确定是否可以操纵这些过程。