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.

项目摘要 V-ATP酶是一种多功能、高度保守的多亚基质子泵，负责细胞器的酸化 几乎所有的真核细胞。V-ATP酶活性的完全丧失在除真菌外的所有真核生物中是致命的，但 亚单位同种型的突变与远端肾小管酸中毒、不育、耳聋和骨硬化症有关。 V-ATP酶活性在癌症中被破坏，以支持对细胞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 也促进了许多病毒的进入。有一个关键需要了解的作用和调节V- ATP酶亚基亚型和酶亚群，以便在特定位置靶向V-ATP酶 治疗上V-ATP酶受外周V1亚复合物从细胞膜上可逆分解的调节。 完整的膜Vo亚复合物和RAVE/Rabconnectin-3复合物在此过程中起关键作用。 我们将研究酵母RAVE和哺乳动物RAVE的结构、机制和亚基组成。 Rabconnectin-3复合物，其似乎靶向特定的V-ATP酶亚基亚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相关， 神经变性，但潜在的疾病机制尚不清楚。我们之前在酵母中证明了 细胞器富集的磷脂酰肌醇磷脂与两种α-亚单位亚型的结合差异， Vo亚复合物，为V-ATP酶定位和活性提供细胞器特异性输入。相似脂质 在体外观察到与哺乳动物a亚基同种型的相互作用，我们将把这些研究扩展到 表征在培养的哺乳动物细胞中的相互作用的影响。最后，虽然V-ATP酶必须 与pH稳态的其他细胞机制一起发挥作用，这种作用的潜在机制 协调不被理解。我们将在酵母中解决这个问题，在那里我们发现， 或V-ATP酶活性的慢性丧失触发主要H+输出泵Pma 1的一部分的内吞作用， 质膜。我们将确定这种液泡对质膜pH串扰的机制。 减少的液泡/溶酶体酸化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显示协调pH稳态的丧失或V- ATP酶本身，并确定这些过程是否可以操纵。