Structual and Calcium Regulatory Proteins in Sarcopenia

描述(由申请人提供)：我们的长期目标是确定骨骼肌性能随年龄增长而减弱的分子机制。我们假设，关键骨骼肌蛋白质中特定氨基酸残基的年龄依赖性损伤通过改变蛋白质的结构和功能来影响骨骼肌的性能。为了支持这一假说，我们之前的研究清楚地表明，比力的下降部分是由于特定部位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。我们以前的发现表明，年龄导致钙敏感性的改变，这与肌钙蛋白C密切相关。II型纤维的缩短速度显著降低，这不能用肌球蛋白异构体转换来解释，这表明调控蛋白，肌球蛋白轻链，参与了这一过程。这项建议有两个目的：目的1.确定肌钙蛋白C在年龄相关性骨骼肌功能障碍(钙敏感性降低)中的作用。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，使蛋白质对钙的敏感度降低。目的2.确定肌球蛋白轻链(MLC1f和MLC3f)在年龄相关性骨骼肌功能障碍(收缩速度减慢)中的作用。为了从机制上测试这两种蛋白质如何导致肌肉功能障碍，我们将使用三种方法。首先，我们将从不同年龄的大鼠中提纯肌钙蛋白C和肌球蛋白轻链，并检测它们之间的生理相互作用。其次，我们将利用蛋白质组学技术确定体内翻译后化学修饰的类型和修饰的特定氨基酸位点。第三，我们将使用重组腺病毒技术在不同年龄的大鼠中过表达特定的肌球蛋白轻链亚型，从机械上测试蛋白质表达的变化如何直接导致收缩放缓。Fischer 344大鼠将是我们的衰老模型，集中在代表动物寿命的三个年龄组(成人、老年和老年)。这项拟议的研究汇集了一支强大的研究团队和包括生理学、生物化学、蛋白质组学和分子生物学在内的多种技术组合，以测试有关肌钙蛋白C和肌球蛋白轻链在衰老中的新角色的基本问题。一旦我们掌握了化学修饰的类型和特定的修饰部位，我们就能够测试这些体内修饰是否会导致蛋白质结构/功能随着年龄的变化而改变。与公共卫生相关：在衰老过程中，适当转录的多肽和蛋白质有许多机会发生结构改变。先前的研究表明，由于翻译后修饰而导致的改变蛋白质的积累与功能丧失有关。因此，确定特定蛋白质在衰老过程中的变化并确定它们在衰老相关肌肉功能障碍中的作用是至关重要的。目前，特定蛋白质氨基酸的年龄相关翻译后修饰对关键骨骼肌蛋白质的蛋白质结构和蛋白质功能的影响尚不清楚。这项建议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，它们是解释年龄相关性肌肉功能障碍的候选对象。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.