Regulation and Cellular Functions of V-ATPases

V-ATP酶的调节和细胞功能

• 批准号: 10405829
• 负责人: PATRICIA M KANE
• 金额: $ 52.43万
• 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/10405829
• 关键词: Acute; Address; Aging; Albers-Schonberg disease; Alzheimer' s Disease; Binding; Cell membrane; Cell physiology; Cells; Chronic; Complex; Defect; Disease; Distal; Endocytosis; 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.

项目概要 V-ATP 酶是多功能、高度保守的多亚基质子泵，负责细胞器酸化 几乎所有真核细胞中。 V-ATP酶活性的完全丧失对于除真菌之外的所有真核生物都是致命的，但是 亚基异构体的突变与远端肾小管酸中毒、不孕症、耳聋和骨硬化症有关。 V-ATP 酶活性在癌症中被破坏，以支持细胞 pH 稳态的额外需求 通过创造酸性细胞外环境促进转移。 V-ATP 酶引起的内体酸化 还促进许多病毒的进入。迫切需要了解 V- 的作用和调节 ATP 酶亚基亚型和酶亚群，以便将 V-ATP 酶靶向特定位置 治疗上。 V-ATP 酶通过外周 V1 亚复合物从 整合膜 Vo 亚复合物和 RAVE/Rabconnectin-3 复合物在此过程中发挥着关键作用。 我们将研究酵母 RAVE 和哺乳动物的结构、机制和亚基组成 Rabconnectin-3 复合物，其活性的一部分似乎是针对特定的 V-ATP 酶亚基亚型。 重要的是，Rabconnectin-3 复合物的突变与癫痫和 神经退行性疾病，但潜在的疾病机制尚不清楚。我们之前在酵母中证明了 富含细胞器的磷酸肌醇磷脂与两个α亚基异构体的结合有差异 Vo 亚复合体，为 V-ATP 酶定位和活性提供细胞器特异性输入。类似脂质 在体外观察到与哺乳动物 a 亚基亚型的相互作用，我们将把这些研究扩展到 表征培养的哺乳动物细胞中相互作用的影响。最后，虽然 V-ATP 酶必须 与 pH 稳态的其他细胞机制协同发挥作用，其潜在机制 协调不明白。我们将在酵母中解决这个问题，我们发现，急性 或 V-ATP 酶活性的慢性丧失会触发主要 H+ 输出泵 Pma1 的一部分的内吞作用， 质膜。我们将确定该液泡与质膜 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步骤。我们将 评估老化的酵母细胞是否表现出协调 pH 稳态的丧失或 V- 中出现的缺陷 ATPase 本身并确定这些过程是否可以被操纵。