A Proteomic Screen for Muscle E3 LigaseSubstrates in Cachexia.

描述(申请人提供)：治疗与癌症相关的肌肉萎缩在癌症患者的临床护理和管理中是一个巨大的挑战。在药理学上，癌症的这一方面已被证明是谜，因为恶病质背后的蛋白质降解程序的分子细节尚未阐明。最近的研究表明，包括癌症在内的许多疾病中的肌肉萎缩，主要是由于泛素蛋白酶体系统的激活导致蛋白分解增加。我们利用基因组转录因子结合(芯片上的)分析，在成肌细胞和成肌细胞向肌管的转变过程中，鉴定了一组E3连接酶，这些连接酶受肌肉发生的主要调节因子MyoD1的转录调控。我们假设这些E3连接酶在肌肉内稳态中正常发挥作用，癌症中的肌肉萎缩可能部分是由于肌肉特异性E3连接酶不适当的、持续的激活所致。因此，这些E3连接酶(和它们的调节器)可能是治疗癌症和其他疾病中肌肉萎缩的可行药物靶点。在这里，我们将从两个具体目标来探讨这一假说。首先，我们将使用地塞米松治疗作为肌肉萎缩的组织培养模型，在完全分化的C2C12肌管中进行Rnf11底物的蛋白质组筛选，Rnf11是我们之前在MyoD1基因组筛选中确定的E3连接酶组的成员。其次，我们将尝试在体外和我们的肌肉萎缩组织培养模型中验证Rnf11 E3连接酶对这些假定底物的泛素化作用。这项建议旨在开发创新的和以前从未探索过的方法来对抗与癌症相关的肌肉萎缩，因此为其临床治疗提供了新的可能性。

DESCRIPTION (provided by applicant): Treatment of muscle wasting associated with cancer poses a great challenge in the clinical care and management of cancer patients. Pharmacologically, this aspect of cancer has proven enigmatic, as the molecular details of the protein degradation programs underlying cachexia have not been elucidated. Recent studies have shown that muscle wasting in a number of diseases, including cancer, is principally due to an increase in proteolysis caused by activation of the ubiquitin proteasome system. We identified a cadre of E3 ligases that are transcriptionally regulated by MyoD1, a master regulator of myogenesis, in myoblasts and during the myoblast to myotube transition using genomic transcription factor binding (ChIP-on-chip) analyses. We hypothesize that these E3 ligases normally function in muscle homeostasis and that muscle wasting in cancer may be due in part to inappropriate, sustained activation of muscle-specific E3 ligases. Thus, these E3 ligases (and their regulators) could represent viable drug targets in the treatment of muscle wasting in cancer and other diseases. Here, we will explore this hypothesis in two specific aims. First, we will conduct a proteomic screen for substrates of Rnf11, a member of the group of E3 ligases previously identified in our MyoD1 genomic screen, in fully differentiated C2C12 myotubes using dexamethasone treatment as a tissue culture model of muscle atrophy. Second, we will attempt to validate ubiquitylation of these putative substrates by the Rnf11 E3 ligase in vitro and in our tissue culture model of muscle atrophy. This Proposal is designed to develop innovative and previously unexplored approaches to combat muscle wasting associated with cancer and therefore presents novel possibilities for its clinical management.