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（适用提供）：从精神分裂细菌到冰川冰蠕虫的冷适应生物通过提高细胞内能级（即ATP; 5'5'Odenosine triphospophate）的温度降低，从而以明显的机制来抵消与分子运动和死亡相关的固有的固有机制。这种反应明显与在嗜嗜动物（包括哺乳动物）中观察到的反应明显相反，包括哺乳动物，随着温度降低，哺乳动物迅速失去腺苷酸盐，使其容易受到不可逆转的细胞损伤的影响。不同的细胞（例如，冬眠的动物，适应低体温温度的器官）的冷耐受性与维持腺苷酸核唯一的维持相关，最近我们设计了一种普遍改良的细菌菌株，其ATP水平高约30％，比野生类型高约30％，以表现出增强的冷耐受性。我们将通过siRNA和遗传敲除方法靶向两种AMP降解酶，AMP光光疗（5'-核苷酸酶）和AMP脱氨酶，并引入了一组非氢化氧化AMP类似物，以集体努力，以增加稳态内静脉内ATP水平。实验细胞将测试其在低体温温度（0-4¿C）随时间的函数下生存的能力。基于整个文献中的间接证据和我们自己的实验结果，我们建议将ATP“恒温器”转移以维持较高细胞内能级的细胞/动物将显着冷耐受性，从而增强器官的存储/移植能力。 公共卫生相关性：长期以来存储哺乳动物细胞和组织的能力（即天到几周）为治疗人类疾病以及支持细胞和器官移植提供了巨大的潜力。这项研究将调查潜水员后生细胞在操纵细胞内能级后抵抗冷温暴露的能力。从这项研究中获得的知识将固有地适用于哺乳动物的转化研究。