Regulation of Proteolysis in Crustacean Muscles

骨骼肌不仅在动物的运动中很重要，而且还构成了蛋白质的主要代谢储备，当饮食摄入量不足以满足动物的营养需求时，可以迅速动员这些蛋白质。在饥饿引起的萎缩过程中，肌肉蛋白质被降解为氨基酸，然后被其他组织用来合成生命所必需的蛋白质。此外，废用、神经损伤和疾病也会导致肌肉萎缩。在一些疾病中，比如肌营养不良症，消瘦非常严重，以至于个体死亡。对任何动物的蛋白质分解的细胞内调节都知之甚少。甲壳动物的爪肌为阐明蛋白降解机制提供了良好的模型系统。在每个蜕皮周期中，爪肌经历一系列的萎缩和恢复，其中蛋白质含量在蜕皮前下降40%，并在蜕皮后重新合成。当贝壳脱落时，萎缩显然促进了一对大爪子的收缩。蛋白质降解似乎由五种酶介导：四种钙依赖蛋白水解酶(CDPs)和一种多催化蛋白水解酶(MCP)。这些酶在中性pH下具有活性，并能降解肌肉的收缩蛋白。这项建议研究了在形态和生物化学上与脊椎动物骨骼肌相似的肌肉中细胞内蛋白分解的调节。由于萎缩与类固醇蜕皮激素水平升高不谋而合，实验将研究培养中爪肌蛋白质周转(合成和降解)的激素控制。其他实验将确定控制蛋白质分解的信号通路中的主要步骤。CDP和内源性调节因子将被提纯，用于进一步鉴定和生产抗体。MOLT诱导的单核细胞趋化蛋白的表达、含量和细胞内分布的变化将被量化。除了确定这些酶在蛋白质分解中的功能外，这些研究还将产生关于所有动物细胞共同的蛋白质分解过程调节的新的和基本的信息。

Skeletal muscle is not only important in animal locomotion, but also constitutes a major metabolic store of protein that can be quickly mobilized when dietary intake is insufficient to meet an animal's nutritional needs. During starvation-induced atrophy, muscle protein is degraded to amino acids, which are then used by other tissues for the synthesis of proteins essential for life. In addition, muscle atrophy can be induced by disuse, nerve injury, and disease. In some diseases, such as muscular dystrophy, wasting is so severe that the individual dies. The intracellular regulation of protein breakdown in any animal is poorly understood. The crustacean claw muscle provides a favorable model system for elucidating proteolytic mechanisms. During each intermolt cycle, the claw muscle undergoes a sequential atrophy and restoration, in which protein content decreases 40% during the premolt period and is resynthesized following molt. Atrophy apparently facilitates withdrawal of the pair of large claws at the time the shell is shed. Protein degradation appears to be mediated by five enzymes: four calcium- dependent proteinases (CDPs) and a multicatalytic proteinase (MCP). These enzymes are active at neutral pH and degrade the contractile proteins of muscle. This proposal examines the regulation of intracellular proteolysis in a muscle that resembles, both morphologically and biochemically, vertebrate skeletal muscle. Since atrophy coincides with elevated levels of a steroid molting hormone, experiments will investigate the hormonal control of protein turnover (synthesis and degradation) in claw muscles in culture. Other experiments will identify the major steps in the signal pathway controlling proteolysis. The CDPs and endogenous regulatory factors will be purified for further characterization and production of antibodies. Molt-induced changes in the expression, content, and intracellular distribution of the MCP will be quantified. In addition to defining the function of these enzymes in protein breakdown, these studies will yield new and fundamental information on the regulation of proteolytic processes common to all animal cells.