Genetic Control of Vacuolar H+-ATPase Expression

描述(申请人提供)：液泡质子转移ATPase，或V-ATPase，是一种多亚单位复合体，利用三磷酸腺苷水解产生的能量通过细胞膜运输质子。在大多数真核生物中，V-ATPase只存在于内吞网络的膜上，在那里它们用来酸化内小体、溶酶体、反高尔基体和其他空泡室。然而，一些特殊类型的细胞，包括肾上皮细胞、破骨细胞、巨噬细胞和神经元，高水平表达V-ATPase，并在特殊的亚细胞间隔中表达V-ATPase，这种酶在尿酸、骨吸收、调节细胞内pH和调节神经递质的囊泡摄取等多种功能中起关键作用。V-ATPase在哺乳动物细胞中执行的不同功能归因于多种亚基异构体的表达，总体表达水平的差异，以及调节靶向特化膜间隔的能力。V-ATPase在人类中的不适当表达已被证明会导致严重的疾病状态，包括肾小管酸中毒、耳聋和骨化症。 调节V-ATPase表达的遗传控制必须多种多样，以满足许多特殊细胞类型的特定质子运输需求，以及它在内吞网络中的构成作用。这些机制包括转录和转录后机制，以及V-ATPase mRNA和蛋白质的细胞内靶向。这个项目的长期目标是了解V-ATPase在适当的水平和适当的膜间隔中表达的机制。为此，具体目标是：(1)研究在质子分泌细胞，如巨噬细胞和肾上皮细胞中调节V-ATPase mRNA稳定性的机制；(2)检测V-ATPase mRNAs中调节特定细胞类型的细胞内转运的决定因素；以及(3)确定控制参与细胞内转运的独特调节的V-ATPase亚单位表达的遗传元件。这些目标将通过在细胞培养模型中对V-ATPase mRNAs进行遗传操作的实验来实现；这些操作对V-ATPase表达和细胞功能的影响将被确定。

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.