A systems biology approach to elucidating mechanisms underlying amino acid toxicities

阐明氨基酸毒性机制的系统生物学方法

• 批准号: 10680272
• 负责人: Kylie Jacobs
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680272
• 关键词: Affect; Amino Acid Permease; Amino Acid Transporter; Amino Acids; Arrestins; Biological Models; Branched-Chain Amino Acids; Buffers; Carbon; Cardiovascular Diseases; Cell membrane; Cell physiology; Cell surface; Cells; Communities; Cysteine; Data; Diabetes Mellitus; Disease; Dose; Drug Targeting; Essential Amino Acids; Exposure to; Genetic; Genetic Screening; Genetic Transcription; Growth; Health; Heart Diseases; Homeostasis; Human; Image; Impairment; Inborn Errors of Metabolism; Insulin Resistance; Knowledge; Lysosomes; Maintenance; Malignant Neoplasms; Mammalian Cell; Membrane Transport Proteins; Metabolic; Metabolic Diseases; Metabolism; Methionine; Mutate; Neutral Amino Acids; Nitrogen; Nucleotides; Organelles; Organism; Pathway interactions; Patients; Phenylketonurias; Play; Production; Proliferating; Protein Biosynthesis; Proteins; Regulation; Research; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Serum; Signal Transduction; Signaling Molecule; Source; Specificity; Starvation; System; Systems Biology; Toxic effect; Translations; Vacuole; Yeasts; cell growth; deprivation; design; experimental study; genetic manipulation; insight; lipidomics; metabolomics; mutant; overexpression; permease; response; sensor; transcriptome sequencing; transcriptomics; uptake; yeast genetics

PROJECT SUMMARY Amino acids are an intrinsic part of protein biosynthesis, nucleotide production, and provide sources of carbon and nitrogen for the cell. The cell keeps amino acid (AA) levels balanced by increasing AA transporters on the cell surface, catabolizing AAs to form essential metabolites, protein translation, and storage of surplus AAs in the lysosome. An autophagic response is initiated when the cell is starved of AAs. Exposure to high concentrations of AAs is also problematic, but the mechanisms underlying that toxicity are not yet fully understood. AA levels are tightly regulated and when this regulation is disrupted, toxic intermediates can buildup and diseases such as phenylketonuria and cancer can occur. Previous research in our lab has studied amino acid toxicity through vacuole impairment, which is imprecise and cannot always provide a detailed understanding of the effects of single amino acids. To circumvent these issues, I have designed a yeast strain that overexpresses a mutated copy of Gap1, a high-capacity, low-specificity amino acid permease. Because of the two mutated Gap1 residues, this permease remains on the plasma membrane instead of being recycled to the vacuole in high AA conditions. This causes the tight AA regulation to be broken and initiate continuous AA uptake, which will allow us to study the effects of specific AAs on cellular processes. The aim of this project is to elucidate the role each AA plays in toxicity and cellular function using Saccharomyces cerevisiae as a model system. Aim 1 will identify transcriptional, metabolic, and organellar changes resulting from AA toxicity. Aim 2 aims to understand the cell’s capacity to buffer excess amino acids and adapt to their toxicity. Finally, the Aim 3 will characterize the mammalian plasma membrane transporter, L-type amino acid transporter (LAT1). LAT1 and its regulation are poorly characterized, and this aim will begin to fill this knowledge gap. Together, these proposed aims will help to uncover how AAs affect cellular function using the power of yeast genetics and it will begin to elucidate amino acid transporter regulation in mammalian cells.

项目概要 氨基酸是蛋白质生物合成、核苷酸生产的内在组成部分，并提供碳源 通过增加细胞上的 AA 转运蛋白来平衡细胞的氨基酸 (AA) 水平。 细胞表面、分解代谢 AA 形成必需代谢物、蛋白质翻译以及剩余 AA 的储存 当细胞缺乏 AA 时，就会启动自噬反应。 AA 的浓度也存在问题，但其毒性的机制尚未完全阐明。 AA 水平受到严格监管，当这种监管被破坏时，有毒中间体就会积聚。 我们实验室之前的研究研究了氨基，可能会导致苯丙酮尿症和癌症等疾病。 通过真空损伤产生的酸毒性，这是不精确的并且不能总是提供详细的理解 为了避免这些问题，我设计了一种酵母菌株。 过度表达 Gap1 的突变副本，Gap1 是一种高容量、低特异性的氨基酸通透酶。 由于两个突变的 Gap1 残基，该渗透酶保留在质膜上，而不是被回收到 高 AA 条件下的真空会导致严格的 AA 调节被打破并引发连续的 AA 吸收， 这将使我们能够研究特定氨基酸对细胞过程的影响。该项目的目的是阐明。 使用酿酒酵母作为模型系统，了解每种 AA 在毒性和细胞功能中的作用。 目标 1 将识别 AA 毒性导致的转录、代谢和细胞器变化。 最后，Aim 3 将 描述哺乳动物质膜转运蛋白 L 型氨基酸转运蛋白 (LAT1) 及其特征。 监管的特点很差，而这一目标将开始填补这一知识空白。 目标将有助于揭示 AA 如何利用酵母遗传学的力量影响细胞功能，并将开始 阐明哺乳动物细胞中的氨基酸转运蛋白调节。