Mechanisms of metabolic rate depression: following nature's lead

我的研究目的是发现自然界中代谢停滞现象背后的调控分子原理和机制。为了在严酷的环境挑战中生存，如零下温度、缺氧或干旱条件，许多动物利用代谢率抑制(MRD)进入麻木或休眠状态。我的实验室分析了其中涉及的生物化学。利用分子工具和脊椎动物冬眠(地面松鼠)、耐冻性(林蛙)、耐缺氧(海龟)和夏眠(非洲爪蛙)(以及部分无脊椎动物)的模型，我们探索了酶和蛋白质的适应，抑制和重新排序ATP昂贵的细胞功能的机制，以及控制MRD和控制低代谢状态下长期细胞保存和寿命延长的细胞信号和基因表达的变化。今后五年的持续工作和拟议工作的目标涉及调节低代谢的新概念。(A)转录抑制：我们最近关于冬眠和耐缺氧的工作表明，表观遗传机制参与了低代谢过程中基因转录的全局沉默；这些机制包括DNA甲基化和组蛋白的翻译后修饰(PTM)(改变的乙酰化、磷酸化)。这一新的洞察力将通过对跨低代谢系统的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(例如，组蛋白乙酰酶和脱乙酰酶、DNA甲基转移酶)的活性和调节的变化的量化来发展。研究还将分析SUMO化(一种在冬眠地鼠细胞核中突出的PTM)在抑制转录因子(TF)的作用中的作用，从而帮助抑制MRD期间的整体基因表达。(B)微型RNA：我的实验室提供了第一个证据，证明微型RNA作为转录后控制mRNA转录本的关键机制，有助于抑制ATP昂贵的翻译，并在MRD期间提供转录本的存储。我们继续的研究将考察microRNA作为动物系统低代谢原理的作用，并重点研究选定的microRNA在控制特定转录因子和其他蛋白质合成方面的作用。(C)信号和基因/蛋白质表达：我们最近的工作表明，在MRD期间，选定的信号通路和TF在抑制ATP昂贵的功能(例如蛋白质合成、细胞周期)或上调细胞保护(例如抗氧化剂、伴侣)方面具有靶向性作用。拟议的研究分析了在低代谢过程中其他代谢功能的重组(例如，抗细胞凋亡和自噬)以及参与应激反应的转录因子(例如，P53、STATS、Smads)。(D)酶调控：研究将探索我们工作中确定的酶调控的新概念：例如，脱氢酶酶的磷酸化调控，作为影响酶活性和调控的PTM的乙酰化。动力学、生物信息学、建模和稳定性(使用我们新的差示扫描荧光方法)研究将被整合，以了解酶/蛋白质在低代谢系统中的适应。总体而言，这项研究将对理解自然界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具有关键的潜在应用，例如将诱导性麻木作为一种医疗干预策略的开发。

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.