Role of Mitochondrial Deacetylase Sirt3 in Skeletal Homeostasis

线粒体脱乙酰酶 Sirt3 在骨骼稳态中的作用

• 批准号: 10268962
• 负责人: Ha-Neui Kim
• 金额: $ 30.06万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-16 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268962
• 关键词: Acetate-CoA Ligase; Acetylation; Affect; Age-Related Bone Loss; Aging; BCL2 gene; Bone Marrow; Bone Resorption; Calcium; Cell Death; Cell physiology; Complex; Data; Deacetylase; Disease; Elderly; Estrogens; Excision; Foundations; G1 Arrest; Generations; Genes; Goals; Homeostasis; Immunity; In Vitro; Insulin Resistance; Knowledge; Light; Link; Lysine; Maintenance; Metabolic Activation; Metabolism; Mitochondria; Mitochondrial Proteins; Multienzyme Complexes; Mus; Musculoskeletal Diseases; Nerve Degeneration; Osteoblasts; Osteoclasts; Oxidative Phosphorylation; Play; Post-Translational Protein Processing; Process; Production; Proteins; Proteome; Reactive Oxygen Species; Research; Role; Sirtuins; Skeleton; Source; Testing; Work; bone cell; bone loss; bone mass; cell type; human disease; insight; macrophage; mineralization; mitochondrial dysfunction; mouse model; novel; osteoblast differentiation; osteoclastogenesis; prevent; progenitor; skeletal

Mitochondria are the major source of cellular ATP generated through the process of oxidative phosphorylation. Recent insights have revealed that mitochondria are also involved in other tasks, such as cell death, reactive oxygen species (ROS) generation, immunity, and calcium homeostasis. Mitochondrial protein lysine acetylation plays a key role in these metabolic processes. Although still not completely understood, this post-translational modification of mitochondrial protein networks seem to be important for maintenance of healthy, properly functioning mitochondria. Unsurprisingly, in human diseases as well as mouse models, mitochondrial dysfunction has been linked to numerous aspects of aging, including neurodegeneration, cardiopathologies, insulin resistance, and bone loss. However, it is largely unknown whether the reduced bone mass with advancing age is due to a change in mitochondrial function(s) in osteoclasts or in other cell types. The primary goal of the work proposed in this application is to establish the role of mitochondrial function in bone cell physiology and disease. In order to investigate mitochondrial function, we will manipulate Sirtuin 3 (Sirt3), a molecule with critical roles for both mitochondrial function and energy homeostasis that has not clearly been studied in osteoclasts and osteoblasts. Sirt3 is an NAD+- dependent deacetylase localized primarily to the mitochondria, with a unique role in the activation of metabolic enzymes and Complex I subunits. Sirt3 deacetylates and activates Ndufa9, an important Complex I subunit, leading to enhancement of cellular ATP levels and oxidative phosphorylation. Sirt3 can also interact with the acetyl-CoA synthetase, affect G1 arrest induced by loss of Bcl-2 and, thereby, regulate cell death. Loss of all of these roles can contribute to mitochondrial dysfunction and aberrantly enhanced ROS levels, most likely by down-regulating Complex I activity. Our preliminary data shows that the anti-osteoclastogenic actions of estrogens are associated with decreased Complex I activity and ATP production in early osteoclast precursors, and that Sirt3 expression is upregulated during osteoclastogenesis. Further, global deletion of Sirt3 in mice prevents age-related bone loss, accompanied by a decrease in bone resorption. Removal of Sirt3 from osteoblast progenitors in vitro also reduces osteoblast differentiation and mineralization. These observations form the foundation for the hypothesis that Sirt3 plays an essential role in skeletal homeostasis by regulating mitochondrial function(s) in osteoclasts and osteoblasts. To test these hypotheses, we will examine whether deletion of Sirt3 in osteoclasts (Aim 1) and osteoblasts (Aim 2) prevents the loss of bone mass caused by advancing age or estrogen deficiency. Further, we will perform in vitro studies to identify Sirt3 target proteins in osteoclasts and osteoblasts that are responsible for their effects on differentiation and function using quantitative analysis of global proteome and lysine acetylome in primary bone marrow-derived macrophages with or without Sirt3 genes (Aim 3). Successful completion of this work will shed light on novel mechanisms that contribute to ost oporosis and will advance knowledge of how the skeleton responds to changes in mitochondrial function.

线粒体是通过氧化磷酸化过程产生的细胞 ATP 的主要来源。最近的研究表明，线粒体还参与其他任务，例如细胞死亡、活性氧 (ROS) 生成、免疫和钙稳态。线粒体蛋白赖氨酸乙酰化在这些代谢过程中起着关键作用。尽管尚未完全了解，但线粒体蛋白质网络的这种翻译后修饰似乎对于维持健康、正常运作的线粒体很重要。毫不奇怪，在人类疾病和小鼠模型中，线粒体功能障碍与衰老的许多方面有关，包括神经退行性变、心脏病、胰岛素抵抗和骨质流失。然而，目前尚不清楚骨量随年龄增长而减少是否是由于破骨细胞或其他细胞类型中线粒体功能的变化所致。本申请提出的工作的主要目标是确定线粒体功能在骨细胞生理学和疾病中的作用。为了研究线粒体功能，我们将操纵 Sirtuin 3 (Sirt3)，这是一种对线粒体功能和能量稳态具有关键作用的分子，尚未在破骨细胞和成骨细胞中进行明确研究。 Sirt3 是一种 NAD+ 依赖性脱乙酰酶，主要定位于线粒体，在代谢酶和复合物 I 亚基的激活中具有独特的作用。 Sirt3 脱乙酰并激活 Ndufa9（一种重要的复合物 I 亚基），导致细胞 ATP 水平和氧化磷酸化增强。 Sirt3 还可以与乙酰辅酶 A 合成酶相互作用，影响 Bcl-2 缺失引起的 G1 停滞，从而调节细胞死亡。所有这些作用的丧失可能导致线粒体功能障碍和 ROS 水平异常升高，很可能是通过下调复合物 I 活性来实现的。我们的初步数据表明，雌激素的抗破骨作用与早期破骨细胞前体中复合物 I 活性和 ATP 生成的降低有关，并且 Sirt3 表达在破骨细胞生成过程中上调。此外，小鼠体内 Sirt3 的整体缺失可以防止与年龄相关的骨质流失，并伴有骨吸收的减少。体外从成骨细胞祖细胞中去除 Sirt3 也会降低成骨细胞分化和矿化。这些观察结果为以下假设奠定了基础：Sirt3 通过调节破骨细胞和成骨细胞中的线粒体功能，在骨骼稳态中发挥重要作用。为了检验这些假设，我们将检查破骨细胞（目标 1）和成骨细胞（目标 2）中 Sirt3 的缺失是否可以防止因年龄增长或雌激素缺乏而导致的骨量损失。此外，我们将进行体外研究，通过对有或没有 Sirt3 基因的原代骨髓源性巨噬细胞中的整体蛋白质组和赖氨酸乙酰组进行定量分析，确定破骨细胞和成骨细胞中的 Sirt3 靶蛋白，这些靶蛋白对分化和功能产生影响（目标 3）。这项工作的成功完成将揭示导致骨质疏松的新机制，并将增进对骨骼如何反应的了解。 线粒体功能的变化。