Regulation of Peroxisomal Metabolism by Lysine Acylation

长链脂肪酸氧化紊乱(LC-FAOD)是一组异质性疾病，其特征是 无法分解线粒体中的长链脂肪酸以获取能量。过氧化物型脂肪酸 氧化(FAO)是通往线粒体FAO的一条平行途径，可以用来减轻脂肪酸 LC-FAOD患者体内蓄积情况。然而，目前还没有刺激的药物手段。 人类体内的过氧酶体粮农组织。开发新的过氧化物酶刺激疗法的能力受到以下限制 关于调节粮农组织过氧化物酶活性的因素的知识差距。在这里，建议 Sirtuin-5(SIRT5)和赖氨酸琥珀酸化-被SIRT5逆转的翻译后修饰-代表着一种 操控过氧化体功能的新机制。当给小鼠喂食一种叫做 二羧酸(DCA)，赖氨酸琥珀酸化累积在过氧化体蛋白上。进一步的初步数据 提示赖氨酸琥珀酸化可增强过氧化体功能。SIRT5逆转这些影响的能力 目前仍不清楚。这项拨款的中心假设是，喂养DCA可以改善动物的疾病病理 通过驱动蛋白琥珀酸化和过氧化体激活建立小鼠线粒体LC-FAOD模型。这是 由初步数据支持，在7天的DCA喂养中，LC-Fod的肌肉功能得到改善 老鼠模型。核心假设将在三个具体目标上得到充分探讨。1)目标1将量化 DCA喂养和SIRT5消融对过氧化体酰基组的影响。SIRT5部分本地化到 过氧化物体，但其活性还没有表征。一个定量的、部位水平的赖氨酸“酰基组”±DCA 将汇编肝脏、肌肉和心脏--LC-FAOD中受影响的关键组织--以及所有SIRT5的喂养情况 已确定目标地点。2)目的2描述DCA供血±SIRT5消融对心脏功能的影响 过氧化物体酶和途径通量。这将使用纯化的重组蛋白，培养的细胞 在过氧化酶体和SIRT5缺陷小鼠中操纵SIRT5水平。代谢组学，14C底物通量 研究和酶稳定性/功能测试将被用来确定可逆赖氨酸PTM如何影响 过氧化物酶。3)目标3将在LC-FAOD小鼠模型上测试DCA喂养作为一种治疗策略的作用。它是 提出DCA将从肝脏分布到外周器官，作为能量来源通过 部分链缩短和过氧化体功能增益。轻、重度LC-FAOD小鼠模型±Long-Fod 将评估长期DCA喂养的肝脏、心脏和肌肉功能以及对禁食的反应 压力。在这种情况下，SIRT5的消融可能会进一步增强过氧化体功能。为了测试这一点，LC-FAOD 鼠标模型将被交叉到SIRT5-/-背景上。这些具体目标的完成将共同形成 操控过氧化体功能治疗LC-FAOD的关键新知识。这些机制也是 与衰老、肾损伤、糖尿病、癌症和许多其他疾病有关，特征是 过氧酶体功能和脂代谢紊乱。

Long-chain fatty acid oxidation disorders (LC-FAODs) are a heterogenous group of disorders characterized by the inability to break down long-chain fatty acids in the mitochondria for energy. Peroxisomal fatty acid oxidation (FAO) is a parallel pathway to mitochondrial FAO that could be leveraged to alleviate fatty acid accumulation in patients with LC-FAODs. However, there is currently no pharmacological means of stimulating peroxisomal FAO in humans. The ability to develop new peroxisome-stimulating therapies is limited by knowledge gaps regarding the factors that regulate activity of peroxisomal FAO enzymes. Here, it is proposed that sirtuin-5 (Sirt5) and lysine succinylation—a post-translational modification reversed by Sirt5—represent a new mechanism for manipulating peroxisomal function. When mice are fed a class of fatty acids called dicarboxylic acids (DCAs), lysine succinylation accumulates on peroxisomal proteins. Further preliminary data suggest that lysine succinylation increases peroxisomal function. The capacity of Sirt5 to reverse these effects remains unclear. The central hypothesis of this grant is that feeding DCAs can improve disease pathology in mouse models of mitochondrial LC-FAOD by driving protein succinylation and peroxisomal activation. This is supported by preliminary data in which seven days of DCA feeding improved muscle function in an LC-FAOD mouse model. The central hypothesis will be fully explored in three Specific Aims. 1) Aim 1 will quantify the effects of DCA feeding and Sirt5 ablation on the peroxisomal acylome. Sirt5 partially localizes to the peroxisome but its activity there has not been characterized. A quantitative, site-level lysine “acylome” ± DCA feeding will be compiled for liver, muscle, and heart—the key tissues affected in LC-FAODs—and all Sirt5 target sites identified. 2) Aim 2 is to delineate the effects of DCA feeding ± Sirt5 ablation on the function of peroxisomal enzymes and pathway fluxes. This will be done using purified recombinant proteins, cultured cells with manipulated Sirt5 levels in the peroxisome, and Sirt5-deficient mice. Metabolomics, 14C-substrate flux studies, and enzyme stability/function testing will be used to determine how reversible lysine PTMs affect the peroxisome. 3) Aim 3 will be to test DCA feeding as a therapeutic strategy in LC-FAOD mouse models. It is proposed that DCAs will distribute beyond the liver to the peripheral organs, serving as a source of energy via partial chain shortening and peroxisomal gain-of-function. Mild and severe LC-FAOD mouse models ± long- term DCA feeding will be evaluated for liver, heart, and muscle functioning as well as the response to fasting stress. Ablation of Sirt5 in this context may further enhance peroxisomal function. To test this, the LC-FAOD mouse models will be crossed onto a Sirt5-/- background. Together, completion of these Specific Aims will form critical new knowledge for manipulating peroxisomal function to treat LC-FAODs. These mechanisms are also relevant to aging, kidney injury, diabetes, cancer, and many other diseases characterized by impaired peroxisomal function and dysfunctional lipid metabolism.