A therapeutic approach to muscle wasting by limiting protein breakdown

描述(申请人提供)：肌肉萎缩与停用、肌营养不良、糖皮质激素治疗(如库欣综合征)以及许多全身性疾病(如癌症恶病质、糖尿病、禁食)有关，当总的蛋白质降解率超过蛋白质合成时。大多数类型的萎缩主要通过泛素(Ub)-蛋白酶体途径增加细胞内蛋白的降解，并共享一系列共同的转录变化。目前，还没有有效的药物治疗萎缩。在蛋白酶体抑制剂和Ub-蛋白酶体途径的转录调控方面的工作表明，蛋白酶体是一个很好的靶点。然而，我最近对细胞培养的研究表明，溶酶体(自噬)过程也会导致蛋白质丢失。研究由不同原因造成的肌肉萎缩的小鼠模型(去神经支配、糖皮质激素应用和MDX小鼠--一种肌肉营养不良)，我有三个目标。1)为了确定溶酶和蛋白酶体途径在多大程度上导致失神经和糖皮质激素诱导的萎缩，我将使用这些途径的特定抑制剂，同时测量孵化肌肉中的总蛋白质降解率。2)为了了解被批准用于癌症治疗的特定蛋白酶体抑制剂VELCADE如何影响成年小鼠失神经萎缩期间的蛋白质分解，我将定量测定肌肉蛋白质组分、孵化肌肉的等长收缩能力，并通过使用实时荧光聚合酶链式反应和蛋白质印迹法检测已知转录因子或泛素连接酶的已知转录因子或泛素连接酶，确定激活这些通路的分子。(失神经萎缩有望通过FOXO转录因子或NFkB激活。)3)为了探讨VELCADE给药对mdx小鼠的疗效，我将测试能够增加dystrophin含量的VELCADE是否可以改善离体肌营养不良肌肉的收缩功能，以及突变型dystrophin含量的增加是由于对这种短暂蛋白质的特定作用，还是通过影响整体蛋白质降解(通过Western blotting和特异性蛋白质降解分析)。总而言之，这些研究应该会推进我们开发可行的肌肉萎缩疗法的长期目标。关联性。肌肉萎缩是肌肉停用、禁食和许多系统性疾病(如癌症、艾滋病或肌肉营养不良)的一种经常使人虚弱的并发症。由于这些肌肉萎缩有许多共同特征，我们对减缓蛋白质分解的疗法和机制的研究可能会为这些人带来一种可行的治疗方法。

DESCRIPTION (provided by applicant): Muscle atrophy occurs with disuse, muscular dystrophy, glucocorticoid treatment (as in Cushing's syndrome), and with many systemic diseases (e.g. cancer cachexia, diabetes, fasting), when overall rates of protein degradation exceed protein synthesis. Most types of atrophy have a general increase in degradation of intracellular proteins mainly through the ubiquitin(Ub)-proteasome pathway and share a common set of transcriptional changes. Currently, no effective pharmaceutical treatments for atrophy are available. Work on proteasome inhibitors and the transcriptional regulation of the Ub- proteasome pathway has suggested that the proteasome is an excellent target. However, my recent studies with cell culture suggest that the lysosomal (autophagic) process also contributes to protein loss. Studying mouse models of muscle wasting with very different causes (denervation, glucocorticoid administration, and mdx mice - a muscular dystrophy), I have three aims. 1) To determine to what extent the lysosmal and proteasomal pathways contribute to denervation and glucocorticoid-induced atrophy, I will use specific inhibitors of these pathways while measuring overall protein degradation rates in incubated muscles. 2) To learn how Velcade, a specific proteasome inhibitor approved for cancer therapy, can influence protein breakdown during denervation atrophy in adult mice, I will quantify muscle protein fractions, isometric contractile ability in incubated muscle and determine the molecules that activate these pathways by using real-time PCR and Western blotting for known transcription factors or ubiquitin ligases. (Denervation atrophy would be expected to be activated via the FOXO transcription factors or possibly through NFkB.) 3) To explore the efficacy of Velcade administraction to mdx mice, I will test whether Velcade, which can increase dystrophin content, can improve dytrophic muscle contractile function in isolated muscles and whether the increased content of mutant dystrophin it due to specific effects on this short-lived protein or a result of effects on overall protein degradation (by Western blotting and specific protein degradation assays). Taken together, these studies should advance our long-term goal of developing viable therapies for muscle wasting. RELEVANCE. Muscle wasting is an often debilitating complication of muscle disuse, fasting and many systemic diseases such as cancer, AIDS, or muscular dystrophy. Since these muscle atrophies share many common features, our research on therapies and mechanisms of slowing protein breakdown may lead to a viable treatment for these individuals.