Regulation of Mitochondrial Metabolism by Post-Translational Modifications

描述(申请人提供)：线粒体是新陈代谢和信号传递的中心，其功能对除少数真核细胞类型之外的所有细胞类型都是必不可少的。这些细胞器的普遍功能障碍与广泛的先天代谢错误有关，并与越来越多的常见人类疾病有关，包括2型糖尿病(T2D)、癌症和心脏病。然而，在这些疾病中，线粒体功能障碍的具体变化通常是不明确的，并且几乎总是不受治疗干预的影响。因此，明确界定导致代谢紊乱的致病线粒体改变并设计新的治疗策略来治疗这些疾病是线粒体医学的主要挑战。新出现的证据，包括我们最近的蛋白质组学数据，已经揭示了线粒体蛋白质充满了在健康和疾病状态之间动态变化的磷酸化和乙酰化位点，并且这些修饰经常在相同的蛋白质上联合发现。这些数据表明，这些翻译后修饰(PTM)在调节线粒体代谢方面非常重要，这些修饰的异常水平是线粒体病理生理学的相关改变之一。如果这是真的，这可能会推动开发一种新的治疗策略：通过操纵细胞信号过程来控制线粒体新陈代谢。然而，尽管有这些关于线粒体PTM的早期研究，我们对它们在线粒体生理学中的作用的了解仍处于起步阶段，原因有很多。首先，目前还不清楚这些修饰中的哪些实际上对调节蛋白质功能是重要的。其次，关于执行这些修饰的酶(例如，激酶)的信息明显缺乏。这些实质性的知识差距阻碍了我们理解细胞如何使用PTM来操纵线粒体功能，也阻碍了我们利用这些信息来获得潜在的治疗益处。这项建议采取了一种彻底和创新的方法，通过将重点生物化学与一系列最先进的质谱学工具相结合来解决这些知识差距。特别是，这项建议的总体目标是：1)利用体外生物化学和基于细胞的代谢组学和生物能量学，阐明磷酸化如何影响参与酮类生成和β-氧化的部分肝脏线粒体蛋白的活性；2)通过基于活性和靶向的质谱学技术，确定线粒体中执行这些事件的激酶；以及3)建立伴随肥胖和T2D发病的动态线粒体蛋白乙酰化事件的补充、定量图谱。这些目标的完成将为线粒体关键代谢途径的调控提供新的见解，将为未来对线粒体翻译后修饰之间相互关系的机制研究提供基础，并将有助于揭示线粒体功能障碍治疗的新靶点。

DESCRIPTION (provided by applicant): Mitochondria are centers of metabolism and signaling whose function is essential to all but a few eukaryotic cell types. General dysfunction of these organelles is implicated in a wide range of inborn errors of metabolism, and in an increasing number of common human diseases, including type 2 diabetes (T2D), cancer and heart disease. However, the specific alterations that underlie mitochondrial dysfunction in these disorders are most often poorly defined, and are nearly always impervious to therapeutic intervention. As such, clearly defining the pathogenic mitochondrial alterations that underlie metabolic disorders and devising new therapeutic strategies to treat these conditions represent principal challenges in mitochondrial medicine. Emerging evidence, including our own recent proteomic data, have revealed that mitochondrial proteins are replete with phosphorylation and acetylation sites that change dynamically between healthy and diseased states, and that these modifications are frequently found jointly on the same proteins. These data suggest that these post- translational modifications (PTMs) are widely important in regulating mitochondrial metabolism, and that aberrant levels of these modifications are among the relevant alterations underlying mitochondrial pathophysiology. If true, this could motivate the development of a novel therapeutic strategy: the control of mitochondrial metabolism via manipulation of cellular signaling processes. However, despite these promising early studies on mitochondrial PTMs, our understanding of their role in mitochondrial physiology remains in its infancy for a number of reasons. First, it remains unclear which of these modifications are actually important for regulating protein function. Second, there is a striking lack of information regarding the enzymes (e.g., kinases) that perform these modifications. These substantial knowledge gaps prevent us from understanding how cells use PTMs to manipulate mitochondrial function, and from exploiting this information for potential therapeutic benefit. This proposal takes a thorough and innovative approach to addressing these knowledge gaps by blending focused biochemistry with a range of state-of-the-art mass spectrometry tools. In particular, the overarching goals of this proposal are to 1) elucidate how phosphorylation affects the activities of select liver mitochondrial proteins involved in ketogenesis and beta-oxidation using in vitro biochemistry and cell-based metabolomics and bioenergetics, 2) to identify kinases in mitochondria that execute these events through activity-based and targeted mass spectrometry techniques, and 3) to establish a complementary, quantitative map of the dynamic mitochondrial protein acetylation events that accompany the onset of obesity and T2D. Completion of these aims will provide new insight into the regulation of key mitochondrial metabolic pathways, will provide a foundation for future mechanistic studies into the interrelationship between mitochondrial post-translational modifications, and will help reveal new therapeutic targets for the treatment of mitochondrial dysfunction.