Genetic Control of Vacuolar H+-ATPase Expression

描述（由申请人提供）：液泡质子转运ATP酶或V-ATP酶是一种多亚基复合物，利用ATP水解产生的能量将质子转运穿过细胞膜。在大多数真核生物中，V-ATP酶仅存在于胞吞网络的膜中，在那里它们用于酸化内体、溶酶体、高尔基体和其他液泡区室。然而，一些专门的细胞类型，包括肾上皮细胞、破骨细胞、巨噬细胞和神经元，在高水平和专门的亚细胞区室中表达V-ATP酶，其中该酶对于诸如尿酸化、骨吸收、细胞内pH调节和神经递质的调节囊泡摄取等多种功能至关重要。V-ATP酶在哺乳动物细胞中发挥的多种功能可归因于多种亚基同种型的表达、总体表达水平的差异以及调节靶向特定膜区室的能力。人类中V-ATP酶的不适当表达已显示导致严重的疾病状态，包括肾小管酸中毒、耳聋和骨硬化症。 调节V-ATP酶表达的遗传控制必须是多种多样的，以满足许多专门细胞类型的特定质子运输需求，以及其在胞吞网络中的组成性作用。这些包括转录和转录后机制，以及V-ATP酶mRNA和蛋白质的细胞内靶向。该项目的长期目标是了解V-ATP酶在适当水平和适当膜隔室中表达的机制。为此，具体目标是：（1）检查质子分泌细胞（如巨噬细胞和肾上皮细胞）中V-ATP酶mRNA稳定性调节的机制;（2）检查V-ATP酶mRNA内介导特定细胞类型细胞内运输的决定簇;（3）定义控制参与细胞内运输的独特调节V-ATP酶亚基表达的遗传元件。这些目标将通过在细胞培养模型中对V-ATP酶mRNA进行遗传操作的实验来实现;将确定这些操作对V-ATP酶表达和细胞功能的影响。

DESCRIPTION (provided by applicant): The vacuolar proton-translocating ATPase, or V-ATPase, is a multi-subunit complex that uses energy from ATP hydrolysis to transport protons across cellular membranes. In most eukaryotes, V-ATPases reside solely in membranes of the endocytic network, where they serve to acidify endosomes, lysosomes, the trans-Golgi, and other vacuolar compartments. However, some specialized cell types, including kidney epithelia, osteoclasts, macrophages, and neurons, express the V-ATPase at high levels and in specialized subcellular compartments, where the enzyme is critical for such diverse functions as urinary acidification, bone resorption, regulation of intracellular pH, and regulated vesicular uptake of neurotransmitters. The variety of functions performed by V-ATPases among mammalian cells is attributable to expression of multiple subunit isoforms, differences in overall expression levels, and the capacity for regulated targeting to specialized membrane compartments. Inappropriate expression of V-ATPase in humans has been shown to lead to serious disease states, including renal tubular acidosis, deafness, and osteopetrosis. The genetic controls that regulate V-ATPase expression must be varied and diverse to meet the specific proton-transport needs of many specialized cell types, as well as its constitutive role in the endocytic network. These include both transcriptional and post-transcriptonal mechansims, as well as intracellular targeting of both V-ATPase mRNA and proteins. The long-term goal of this project is to understand the mechanisms by which V-ATPases are expressed at appropriate levels and in appropriate membrane compartments. To this end, the specific aims are directed toward (1) examining mechanisms by which V-ATPase mRNA stability is regulated in proton-secreting cells such as macrophages and kidney epithelia; (2) examining determinants within V-ATPase mRNAs that mediate intracellular trafficking in specialized cell types; and (3) defining genetic elements that control expression of a uniquely regulated V-ATPase subunit involved in intracellular trafficking. These aims will be achieved through experiments in which V-ATPase mRNAs are genetically manipulated in cell culture models; the effects of these manipulations of V-ATPase expression and cell function will be determined.