Studies of Yeast BTN1P and Human CLN3P in Yeast

酵母中酵母BTN1P和人CLN3P的研究

• 批准号: 7348290
• 负责人: DAVID A. PEARCE
• 金额: $ 33.69万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-28 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7348290
• 关键词: Adolescent; Amino Acids; Arginine; Biochemical; Biochemistry; Biological Assay; Biology; CLN3 gene; Cell physiology; Cells; Child; Codon Nucleotides; Collection; Complement; Coupled; Coupling; Data; Defect; Disease; Disruption; Electrophoresis; Equilibrium; Functional disorder; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Screening; Genotype; Goals; Grant; Green Fluorescent Proteins; Growth; Homologous Gene; Human; Incidence; Individual; Lead; Live Birth; Localized; MALDI-TOF Mass Spectrometry; Mammalian Cell; Measures; Mediating; Membrane; Molecular; Mutation; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Oligonucleotide Microarrays; Organism; Pathogenesis; Pathway interactions; Phase; Phenotype; Phosphorylation Site; Polyamines; Polyphosphates; Principal Investigator; Process; Protein Overexpression; Proteins; Proton Pump; Protons; Regulation; Reporting; Research; Role; Screening procedure; Sorting - Cell Movement; Spielmeyer-Vogt Disease; Staging; Translations; Trehalose; Vacuole; Variant; Work; Yeast Model System; Yeasts; base; disease-causing mutation; enzyme activity; extracellular; glycosylation; intracellular protein transport; pH Homeostasis; prenylation; programs; protein transport; small molecule; sugar; trafficking; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): The neuronal ceroid-lipofuscinoses (NCL) are possibly the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births, and with about 440,000 carriers in the USA. Juvenile NCL/Batten disease is the most common of these disorders and the subject of this proposal. Individuals with the disease were found to harbor a 1 kb deletion, which introduces a frameshift that leads to a predicted translation product of 181 amino acids, of which only the first 153 residues correspond to the first 153 of the normal 438 amino acid CLN3 gene product. The yeast homolog to CLN3 was identified and designated BTN1. We had previously shown that Btn1 p may be involved in maintaining pH homeostasis. Importantly, CLN3 is able to complement the alteration in vacuolar pH in the yeast model lacking Btn1 p, indicating that they have similar, if not the same cellular functions. Our more recent studies indicated that lacking Btnlp resulted in a defect in vacuolar transport of arginine, and again CLN3 is able to complement the defect in arginine transport. Overall our studies indicate that yeast cells work to maintain pH homeostasis, and that Btn1 p is an integral part of the biology of this process. This proposal sets out to investigate bfn1-/l-mediated disruption of pH homeostasis within the single cell of yeast. By elucidating the mechanisms by which this single celled organism balances intracellular pH and by uncovering the specific function of Btnlp and other proteins in the BTN-pathway we will establish a basis for understanding pH homeostasis in mammalian cells. We propose to further characterize the biochemistry of Btn1 p-dependent regulation of vacuolar pH. Moreover by exploiting assays that correlate to Btn1 p function such as vacuolar arginine transport and vacuolar proton pumping we will further define the structural requirements of Btn1p/CLN3. Concomitant to these studies we will identify components of the BTN1- pathway through use of a variety of genetic screens such as phenotypic suppression and synthetic lethality. Finally we will characterize the pathway of trafficking Btnlp to the vacuole. Further understanding of Btnlp (and ClnSp) in yeast will provide valuable information on the pathogenesis of Batten disease.

描述（由申请人提供）：神经元蜡样质脂褐素沉积症 (NCL) 可能是儿童中最常见的进行性神经退行性疾病，发病率高达 12,500 名活产儿中就有 1 人，在美国约有 440,000 名携带者。青少年 NCL/Batten 病是这些疾病中最常见的，也是本提案的主题。发现患有该疾病的个体存在 1 kb 缺失，这引入了移码，导致预测的 181 个氨基酸翻译产物，其中只有前 153 个残基对应于正常 438 个氨基酸 CLN3 基因产物的前 153 个残基。鉴定出 CLN3 的酵母同源物并命名为 BTN1。我们之前已经证明 Btn1 p 可能参与维持 pH 稳态。重要的是，CLN3 能够补充缺乏 Btn1 p 的酵母模型中液泡 pH 的变化，这表明它们具有相似的细胞功能，即使不是相同的。我们最近的研究表明，缺乏 Btnlp 会导致精氨酸的液泡运输缺陷，而 CLN3 又能够弥补精氨酸运输的缺陷。总体而言，我们的研究表明酵母细胞致力于维持 pH 稳态，并且 Btn1 p 是该过程的生物学不可或缺的一部分。该提案旨在研究 bfn1-/l 介导的酵母单细胞内 pH 稳态破坏。通过阐明这种单细胞生物平衡细胞内 pH 值的机制，并揭示 Btnlp 和 BTN 途径中其他蛋白质的特定功能，我们将为理解哺乳动物细胞中 pH 稳态奠定基础。我们建议进一步表征 Btn1 p 依赖性液泡 pH 调节的生物化学特征。此外，通过利用与 Btn1 p 功能相关的测定（例如液泡精氨酸转运和液泡质子泵），我们将进一步定义 Btn1p/CLN3 的结构要求。伴随这些研究，我们将通过使用各种遗传筛选（例如表型抑制和合成致死率）来鉴定 BTN1- 途径的组成部分。最后，我们将描述 Btnlp 运输到液泡的途径。进一步了解酵母中的 Btnlp（和 ClnSp）将为巴顿病的发病机制提供有价值的信息。