Structual and Calcium Regulatory Proteins in Sarcopenia

摘要 我们的长期目标是确定骨骼肌萎缩的分子机制。 随年龄增长的表现。我们假设Key中特定氨基酸残基的损伤与年龄有关。 骨骼肌蛋白质通过改变蛋白质的结构和功能来影响骨骼肌的性能。在……里面 支持这一假说，我们之前的调查清楚地表明，单位力量的下降是 部分原因是由于特定位点的翻译后修饰导致肌球蛋白结构的变化。 然而，肌球蛋白的变化不能解释与年龄相关的全部肌肉功能障碍。我们之前的发现 表明年龄引起的钙敏感性改变，与肌钙蛋白C密切相关。 肌球蛋白亚型不能解释的II型纤维缩短速度的显著降低 开关，这表明调控蛋白，肌球蛋白轻链，参与其中。这项提案有两个 目的：目的：1.确定肌钙蛋白C在年龄相关性骨骼肌功能障碍中的作用(减少 钙敏感性)。我们预测，随着年龄的增长，肌钙蛋白C中的特定氨基酸会被修饰，从而使 对钙不太敏感的蛋白质。目的2.确定肌球蛋白轻链(MLC1f和MLC3f)在增龄反应中的作用。 相关的骨骼肌功能障碍(收缩速度减慢)。为了从机械上测试 这两种蛋白质导致肌肉功能障碍，我们将使用三种方法。首先，我们将提纯肌钙蛋白 C和肌球蛋白轻链，并检测它们的生理相互作用。第二，我们将 确定体内翻译后化学修饰的类型和特定的氨基酸位置 利用蛋白质组学技术进行修饰。第三，我们将使用重组腺病毒技术来超越- 表达不同年龄大鼠肌球蛋白轻链亚型，从力学角度测试肌球蛋白轻链亚型的变化 蛋白质的表达直接有助于减缓收缩。Fischer 344大鼠将成为我们的衰老模型 集中在代表动物寿命的三个年龄组(成人、老年和老年)。这项建议 研究汇聚了一支强大的研究团队和多种技术的组合，包括生理学， 生物化学、蛋白质组学和分子生物学，以测试有关新角色的基本问题 肌钙蛋白C和肌球蛋白轻链在衰老中的作用一旦我们装备了这种化学修饰和 在特定的修饰部位，我们将能够测试这些体内修饰是否对改变负责 蛋白质结构/功能随年龄增长。叙述性 在衰老过程中，有许多机会使适当转录的多肽和蛋白质成为 结构上发生了变化。先前的研究表明，由于翻译后蛋白的积累，改变的蛋白质 修改，与功能丧失相关。因此，识别特定的更改是至关重要的 蛋白质在衰老过程中的作用，并确定它们在与年龄相关的肌肉功能障碍中的作用。目前，年龄的作用- 特定蛋白质氨基酸对蛋白质结构和功能的相关翻译后修饰 在关键的骨骼肌中，蛋白质是未知的。这项提案研究了两种关键的骨骼肌蛋白质， 肌钙蛋白C和肌球蛋白轻链，这是解释年龄相关性肌肉功能障碍的候选方法。

Abstract Our long term goal is to determine the molecular mechanisms underlying the diminished skeletal muscle performance with age. We hypothesize that age-dependent damage of specific amino acid residues in key skeletal muscle proteins impacts skeletal muscle performance by altering protein structure and function. In support of this hypothesis, our previous investigations clearly demonstrate that the decline in specific force is due, in part, to changes in the structure of myosin resulting from site-specific post-translational modifications. However, changes in myosin do not explain the total age-related muscle dysfunction. Our previous findings indicate an age-induced alteration in calcium sensitivity, strongly implicating Troponin C. There is a significant reduction in shortening velocity in type II fibers that cannot be explained by myosin isoform switching, suggesting that the regulatory proteins, myosin light chains, are involved. This proposal has two aims: AIM 1. Determine the role of Troponin C in age-related skeletal muscle dysfunction (reduction in calcium sensitivity). We predict that specific amino acids are modified in Troponin C with age, rendering the protein less sensitive to calcium. AIM 2. Determine the role of myosin light chains (MLC1f and MLC3f) in age- related skeletal muscle dysfunction (reduction in contraction velocity). In order to test mechanistically how these two proteins contribute to muscle dysfunction we will use three approaches. First, we will purify Troponin C and myosin light chains from different aged rats and test their physiological interactions. Second we will identify the in vivo type of post-translational chemical modification and the specific amino acid site of modification using proteomic technology. Third, we will use Recombinant Adenovirus technology to over- express specific myosin light chain isoforms in different aged rats to test mechanistically how alterations in protein expression contribute directly to the slowing of contraction. The Fischer 344 rat will be our aging model concentrating on three age groups (adult, old and aged) representing the lifespan of the animal. This proposed research brings together a powerful research team and a combination of techniques, including physiology, biochemistry, proteomics, and molecular biology to test fundamental questions regarding novel roles for Troponin C and myosin light chains in aging. Once we are armed with the type of chemical modification and the specific site of modification, we will be able to test if these in vivo modifications are responsible for altered protein structure/function with age. Narrative During aging there are many opportunities for appropriately transcribed peptides and proteins to become structurally altered. Previous studies show that accumulation of altered proteins, due to post-translational modifications, is correlated with a loss of function. Therefore, it is critical to identify the alterations of specific proteins during aging and to define their roles in age-related muscle dysfunction. Presently, the role of age- related post-translational modifications of specific protein amino acids on protein structure and protein function in key skeletal muscle proteins is unknown. This proposal investigates two key skeletal muscle proteins, Troponin C and myosin light chains, which are candidates to explain age-related muscle dysfunction.