Mechanisms of metabolic rate depression: following nature's lead

我的研究目标是发现自然界中代谢停滞现象背后的分子原理和调控机制。为了在零下温度、缺氧或干旱等恶劣环境中生存，许多动物物种利用代谢率降低（MRD）进入冬眠或休眠状态。我的实验室分析了其中的生物化学成分。利用分子工具和冬眠（地松鼠），耐寒（林蛙），耐缺氧（海龟）和休眠（非洲爪蛙）（以及选择的无脊椎动物）的脊椎动物模型，我们探索了酶和蛋白质的适应性，抑制和重新排序atp昂贵细胞功能的机制，以及控制MRD和工程长期细胞保存和延长低代谢状态下的细胞信号传导和基因表达的变化。在接下来的五年里，我们的目标是继续和建议的工作，解决低代谢调节的新概念。(A)转录抑制：我们最近对冬眠和缺氧耐受性的研究表明，在低代谢过程中，表观遗传机制参与了基因转录的全局沉默；这些包括DNA甲基化和组蛋白的翻译后修饰（PTMs）（乙酰化，磷酸化改变）。这一新的见解将通过对低代谢系统中表观遗传修饰的模式和程度的综合分析，以及对所涉及的酶（如组蛋白乙酰化酶和去乙酰化酶，DNA甲基转移酶）的活性和调控变化的量化来发展。研究还将分析SUMOylation（冬眠地松鼠核中突出的PTM）在MRD期间抑制转录因子（tf）的作用，从而帮助抑制全局基因表达。(B) MICRO-RNA：我的实验室提供了microRNA作为mRNA转录后控制的关键机制的第一个证据，它有助于抑制atp昂贵的翻译，并在MRD期间提供转录物的存储。我们的后续研究将研究microRNA作为动物系统低代谢的一个原理，并重点研究选定的microRNA在控制特定tf和其他蛋白质合成中的作用。(C)信号传导和基因/蛋白表达：我们最近的工作表明，在MRD期间，选定的信号传导途径和tf在抑制atp昂贵功能（如蛋白质合成、细胞周期）或上调细胞保护（如抗氧化剂、伴侣）中的靶向作用。拟议的研究分析了低代谢过程中其他代谢功能的重组（如抗凋亡和自噬）以及响应应激的tf（如p53， STATs, SMADs）的参与。(D)酶调节：研究将探索我们工作中发现的酶控制的新概念：例如脱氢酶的磷酸化控制，乙酰化作为影响酶活性和调节的PTM。动力学，生物信息学，建模和稳定性（使用我们新颖的差示扫描荧光法）研究将被整合，以了解低代谢系统中的酶/蛋白质适应。总的来说，这项研究将为理解自然界中低代谢的普遍生化机制做出重大贡献，这些机制不仅定义了许多加拿大动物的冬季生存策略，而且在生物医学方面也有重要的潜在应用，例如开发诱导性冬眠作为一种医疗干预策略。

The objective of my research is to discover the molecular principles and mechanisms of regulatory control that underlie the phenomenon of metabolic arrest in nature. To survive severe environmental challenges such as subzero temperatures, oxygen deprivation or arid conditions, many animal species use metabolic rate depression (MRD) to enter states of torpor or dormancy. My lab analyzes the biochemistry involved. Using molecular tools and vertebrate models of hibernation (ground squirrels), freeze tolerance (wood frogs), anoxia tolerance (turtles) and estivation (African clawed frogs) (and selected invertebrates too), we explore the adaptations of enzymes and proteins, the mechanisms that suppress and reprioritize ATP-expensive cell functions, and the changes in cell signalling and gene expression that both control MRD and engineer long term cell preservation and life extension in hypometabolic states. Goals for continuing and proposed work over the next five years address new concepts in the regulation of hypometabolism. (A) TRANSCRIPTIONAL SUPPRESSION: Our recent work on hibernation and anoxia tolerance showed involvement of epigenetic mechanisms in the global silencing of gene transcription during hypometabolism; these include DNA methylation and posttranslational modifications (PTMs) of histone proteins (altered acetylation, phosphorylation). This new insight will be developed with a comprehensive analysis of the pattern and extent of epigenetic modifications across hypometabolic systems coupled with quantification of changes in the activities and regulation of the enzymes involved (e.g. histone acetylases & deacetylases, DNA methyltransferases). Studies will also analyze the role of SUMOylation (a PTM prominent in the nucleus of hibernating ground squirrels) in inhibiting the action of transcription factors (TFs) and thereby helping to suppress of global gene expression during MRD. (B) MICRO-RNA: My lab provided the first evidence for microRNA action as a crucial mechanism of post-transcriptional control over mRNA transcripts, aiding both suppression of ATP-expensive translation and providing for storage of transcripts during MRD. Our continuing studies will investigate microRNA action as a principle of hypometabolism across animal systems and focus on the actions of selected microRNAs in controlling the synthesis of specific TFs and other proteins. (C) SIGNALING & GENE/PROTEIN EXPRESSION: Our recent work showed targeted roles for selected signalling pathways and TFs in suppression of ATP-expensive functions (e.g. protein synthesis, cell cycle) or up-regulation of cytoprotection (e.g. antioxidants, chaperones) during MRD. Proposed studies analyze the reorganization of other metabolic functions during hypometabolism (e.g. anti-apoptosis and autophagy) and the involvement of TFs (e.g. p53, STATs, SMADs) that respond to stress. (D) ENZYME REGULATION: Studies will explore new concepts in enzyme control identified by our work: e.g. phosphorylation control of dehydrogenase enzymes, acetylation as a PTM affecting enzyme activity and regulation. Kinetic, bioinformatic, modelling and stability (using our novel differential scanning fluorimetry method) studies will be integrated to understand enzyme/protein adaptation in hypometabolic systems. Overall, the research will make major contributions to understanding the universal biochemical mechanisms of hypometabolism in nature, mechanisms that not only define the winter survival strategies of many Canadian animals but also have crucial potential applications for biomedicine such as to the development of inducible torpor as a medical intervention strategy.