Identifying and characterizing pathways of amino acid metabolism and signaling

氨基酸代谢和信号传导途径的识别和表征

• 批准号: RGPIN-2014-03687
• 负责人: Pulinilkunnil, Thomas
• 金额: $ 2.55万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629523
• 关键词: Identifying; characterizing; pathways; amino; acid

In muscle cells a balanced usage of nutrients such as glucose, fatty acids, ketones, amino acids and lactate is a prerequisite for optimal muscle growth. The longstanding view is that generation of energy within the power house of the cells (mitochondria) primarily from glucose and fatty acids stimulates muscle growth and survival. Amino acids particularly branched-chain amino acids (BCAA) such as leucine, isoleucine, and valine are mostly used for manufacturing proteins for growth within muscle but are also used for generating energy. However our understanding of how BCAA are utilized for generating energy within muscle cells is still lacking. Goals of our research are to determine the enzymes mediating this process, how these enzymes are regulated and the impact on the cell when BCAA are used as an energy source. We want to know whether BCAA compete with other nutrients for being consumed by the cell for energy and which pathways regulate the competition process. What is known so far is that, once inside the cell, BCAA are chemically converted to branch chain alpha ketoacids (BCKA) by virtue of the enzymes branch chain amino transferase (BCAT), branch chain ketodehydrogenase (BCKDH) and ketodehydrogenase kinase (BCKDK) to allow BCKA to enter mitochondria for use as an energy source. Despite the widespread presence of amino acid metabolizing enzymes in different tissues their impact on mitochondrial energy generation and function remains unexplored. Using cell biology, genetic and whole body approaches in zebrafish and mouse my research program will focus specifically on addressing two conceptual puzzles. Puzzle 1) to uncover how the nutrient triad glucose, fatty acid and BCAA biochemically communicate with each other resulting in nutrient transport and utilization within the mitochondria; Puzzle 2) to decipher whether within the cell different biomolecules such as ions, hormones, lipids, sugars and neurotransmitters employ the services of amino acids to govern cellular growth and survival. For puzzle 1, studies will be undertaken in zebrafish and mouse evaluating the effect of alterations in concentration and types of BCAA and BCKA on mitochondrial function in skeletal and cardiac muscle tissues. A method for analysing breakdown products of BCAA will be developed. In addition, conventional cell and molecular biology methodologies will be employed to evaluate the effects of altered BCAA and BCKA on murine and zebrafish amino acid metabolic enzymes (BCAT, BCKDH and BCKDK). To establish the contribution of these enzymes to cellular homeostasis, genetically mutated versions of these enzymes tagged with light emitting probes (fluorescence) will be engineered and expressed in murine cells and zebrafish. For resolving puzzle 2, cellular organelles from murine and zebrafish cells and/or tissues subjected to BCAA and BCKA treatment will be collected and subjected to discovery of genes and proteins influencing cellular size, proliferation and survival. To address how amino acids exert physiological effects of hormones, ions, sugars, and neuronal factors, mice and zebrafish will be administered biomolecules to mirror biological effects and cells and tissues harvested for molecular and physiological analysis. This research program will identify and characterize previously unrecognized proteins and novel molecular networks of cellular amino acid signaling, metabolism and physiology. The research will provide opportunities for graduate and undergraduate trainees to directly contribute to expanding our knowledge of amino acid biology, knowledge that will have direct impacts in the fields of mitochondrial biology, food science and human nutrition offering tremendous benefit to Canadian discovery science, education and innovation.

在肌肉细胞中，平衡使用葡萄糖、脂肪酸、酮、氨基酸和乳酸等营养物质是肌肉最佳生长的先决条件。长期以来的观点是，在细胞(线粒体)的能量屋内产生能量，主要来自葡萄糖和脂肪酸，可以刺激肌肉的生长和存活。氨基酸，特别是支链氨基酸(BCAA)，如亮氨酸、异亮氨酸和缬氨酸，主要用于制造肌肉内生长所需的蛋白质，但也用于产生能量。然而，我们对支链氨基酸是如何在肌肉细胞内产生能量的了解仍然不足。我们研究的目标是确定介导这一过程的酶，这些酶是如何调节的，以及支链氨基酸作为能源时对细胞的影响。我们想知道支链氨基酸是否与其他营养物质竞争被细胞消耗的能量，以及哪些途径调节竞争过程。目前已知的是，支链氨基酸一旦进入细胞，就可以通过支链氨基转移酶(BCAT)、支链酮基脱氢酶(BCKDK)和支链脱氢酶(BCKDK)等酶化学转化为支链α-酮酸(BCKA)，从而使BCKA进入线粒体作为能量来源。尽管氨基酸代谢酶在不同的组织中广泛存在，但它们对线粒体能量产生和功能的影响仍未被研究。利用斑马鱼和老鼠的细胞生物学、遗传学和全身研究方法，我的研究计划将专门专注于解决两个概念难题。谜题1)揭示营养三联体葡萄糖、脂肪酸和支链氨基酸是如何以生物化学方式相互通讯，导致线粒体内的养分运输和利用；谜题2)解开细胞内不同的生物分子，如离子、激素、脂类、糖和神经递质是否利用氨基酸的服务来控制细胞的生长和生存。对于谜题1，将在斑马鱼和小鼠身上进行研究，评估BCAA和BCKA浓度和类型的变化对骨骼肌和心肌组织线粒体功能的影响。将开发一种分析支链氨基酸分解产物的方法。此外，还将使用传统的细胞和分子生物学方法来评估改变的支链氨基酸和支链氨基酸对小鼠和斑马鱼氨基酸代谢酶(BCAT、BCKDH和BCKDK)的影响。为了确定这些酶对细胞动态平衡的贡献，这些酶的基因突变版本将被设计并在小鼠细胞和斑马鱼中表达，这些基因突变版本标记有发光探针(荧光)。为了解开谜题2，将收集小鼠和斑马鱼细胞和/或接受支链氨基酸和支链氨基酸处理的组织的细胞器，并对影响细胞大小、增殖和存活的基因和蛋白质进行发现。为了解决氨基酸如何发挥激素、离子、糖和神经因子的生理效应，将向小鼠和斑马鱼注射生物分子，以反映生物效应以及收集的细胞和组织进行分子和生理分析。这项研究计划将识别和表征以前未被识别的蛋白质和细胞氨基酸信号、新陈代谢和生理的新分子网络。这项研究将为研究生和本科生提供机会，直接为扩大我们的氨基酸生物学知识做出贡献，这些知识将对线粒体生物学、食品科学和人类营养领域产生直接影响，为加拿大的发现科学、教育和创新带来巨大好处。