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.

项目摘要 氨基酸是蛋白质生物合成、核苷酸生产的固有部分，并提供碳源 和氮气。细胞通过增加细胞膜上的氨基酸转运蛋白来保持氨基酸（AA）水平的平衡。 细胞表面，分解代谢AA以形成必需代谢物，蛋白质翻译，以及剩余AA在细胞中的储存。 溶酶体当细胞缺乏AA时，启动自噬反应。暴露于高 浓度的AA也是有问题的，但潜在的机制，毒性还没有完全 明白AA水平受到严格的调节，当这种调节被破坏时，有毒的中间体会积聚 并且可能发生诸如苯丙酮尿症和癌症的疾病。我们实验室以前的研究已经研究了氨基 酸毒性通过液泡损伤，这是不精确的，并不能总是提供详细的了解 单一氨基酸的影响。为了解决这些问题，我设计了一种酵母菌株， 过表达Gap1的突变拷贝，Gap1是一种高容量、低特异性的氨基酸通透酶。因为 两个突变的Gap1残基，这种通透酶保留在质膜上，而不是再循环到细胞膜上。 高AA条件下的液泡。这导致AA的严格调节被打破，并开始连续的AA摄取， 这将使我们能够研究特定AA对细胞过程的影响。这个项目的目的是阐明 使用酿酒酵母作为模型系统，每个AA在毒性和细胞功能中所起的作用。目的 1将确定转录，代谢和细胞器的变化所造成的AA毒性。目标2旨在 了解细胞缓冲过量氨基酸并适应其毒性的能力。最后，Aim 3将 表征哺乳动物质膜转运蛋白，L型氨基酸转运蛋白（LAT1）。LAT1及其 监管的特点很差，这一目标将开始填补这一知识空白。总之，这些建议 目的将有助于揭示氨基酸如何影响细胞功能使用酵母遗传学的力量，它将开始， 阐明哺乳动物细胞中氨基酸转运蛋白的调节。