Energy-based cold tolerance in genetically tractable eukaryotic systems

遗传易处理的真核系统中基于能量的耐寒性

• 批准号: 7922472
• 负责人: DANIEL H SHAIN
• 金额: $ 38.54万
• 依托单位: RUTGERS THE STATE UNIV OF NJ CAMDEN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922472
• 关键词: 5' -AMP-activated protein kinase; 5' -Nucleotidase; AMP Deaminase; ATP Hydrolysis; Adenosine Triphosphate; Animal Organ; Animals; Antarctic; Bacteria; Biological Assay; Biological Models; Cell Survival; Cells; Cessation of life; Cods; Cultured Cells; Data; Drosophila genus; Drosophila melanogaster; Energy Metabolism; Engineering; Enzymes; Equilibrium; Escherichia coli; Eukaryota; Eukaryotic Cell; Free Energy; Genetic; Genetic Crosses; Glacier; Goals; Hela Cells; Homeostasis; Human; Ice; Knock-out; Knowledge; Laboratories; Lead; Life; Literature; Maintenance; Mammalian Cell; Mammals; Medical; Modeling; Molecular; Monitor; Motion; Nucleotidases; Nucleotides; Organ; Organ Transplantation; Organism; Physiological; Prokaryotic Cells; RNA Interference; Research; Role; Small Interfering RNA; System; Temperature; Testing; Time; Tissues; Translational Research; Transplantation; United States National Institutes of Health; adenylate; analog; base; cold temperature; combinatorial; falls; fly; human disease; mutant; nucleotidase; public health relevance; research study; response

DESCRIPTION (provided by applicant): Cold-adapted organisms ranging from psychrophilic bacteria to glacier ice worms respond to declining temperature by raising intracellular energy levels (i.e., ATP; 5' adenosine triphosphate), in an apparent mechanism to off-set inherent reductions in molecular motion (and thus lethargy and death) associated with cold temperature. This response is distinctively opposite to that observed in mesophilic animals, including mammals, which rapidly lose adenylates as temperatures fall making them vulnerable to irreversible cellular damage. Cold tolerance in disparate cells (e.g., hibernating animals, organs acclimated to low physiological temperatures) has been correlated with the maintenance of adenylate nucleotides, and recently we engineered a genetically modified bacterial strain with ~30% higher ATP levels than wild type that displayed enhanced cold tolerance. This proposal seeks to investigate the protective role of elevated ATP levels in tractable eukaryotic systems, namely the fruit fly, Drosophila melanogaster, and cultured mammalian cells (e.g., HeLa, NIH 3T3). We will target two AMP degradative enzymes, AMP phosphatase (5'-nucleotidase) and AMP deaminase, by siRNA and genetic knockout approaches, and introduce a panel of non-hydrolyzable AMP analogs in a collective effort to increase steady-state intracellular ATP levels. Experimental cells will be tested for their ability to survive at low physiological temperatures (0-4¿C) as a function of time. Based on circumstantial evidence throughout the literature and our own experimental results, we propose that cells/animals whose ATP "thermostat" is shifted to sustain higher intracellular energy levels will be significantly more cold tolerant, thus offering potential to enhance organ storage/transplantation capabilities. PUBLIC HEALTH RELEVANCE: The ability to store mammalian cells and tissues over long time periods (i.e., days to weeks) offers great potential for treating human disease, and facilitating cell and organ transplantation. This research will investigate the acquired ability of diverse metazoan cells to resist cold temperature exposure after manipulating intracellular energy levels. Knowledge gained from this study will be inherently applicable to translational research in mammals.

描述（由申请人提供）：从嗜冷细菌到冰川冰虫的冷适应生物体通过提高细胞内能量水平（即 ATP；5' 三磷酸腺苷）来应对温度下降，其明显机制是抵消体内固有的减少。与低温相关的分子运动（从而导致昏睡和死亡）与在嗜温动物（包括哺乳动物）中观察到的反应截然不同，后者会迅速失去活性。随着温度下降，腺苷酸容易受到不可逆的细胞损伤，不同细胞（例如冬眠的动物、适应低生理温度的器官）的耐冷性与腺苷酸核苷酸的维持有关，最近我们设计了一种转基因细菌菌株ATP 水平比表现出增强的耐冷性的野生型高约 30% 该提案旨在研究升高的 ATP 水平在易处理的真核系统（即果蝇）中的保护作用。我们将通过 siRNA 和基因敲除方法，针对两种 AMP 降解酶：AMP 磷酸酶（5'-核苷酸酶）和 AMP 脱氨酶，并引入一组非-果蝇和培养的哺乳动物细胞。将测试可水解 AMP 类似物，共同提高细胞内稳态 ATP 水平。他们在低生理温度（0-4°）下生存的能力C) 作为时间的函数，根据文献中的间接证据和我们自己的实验结果，我们提出，ATP“恒温器”被转变以维持更高的细胞内能量水平的细胞/动物将显着更耐冷，从而提供了潜力。提高器官储存/移植能力。 公共健康相关性：长期（即数天至数周）储存哺乳动物细胞和组织的能力为治疗人类疾病和促进细胞和器官移植提供了巨大潜力。这项研究将调查多种后生动物细胞获得的能力。从这项研究中获得的知识将本质上适用于哺乳动物的转化研究。