A Proteomic Screen for Muscle E3 LigaseSubstrates in Cachexia.

描述（申请人提供）：癌症相关肌肉萎缩的治疗是癌症患者临床护理和管理的一大挑战。在药理学上，癌症的这方面已被证明是谜，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，在包括癌症在内的许多疾病中，肌肉萎缩主要是由于泛素蛋白酶体系统激活引起的蛋白质水解增加。我们利用基因组转录因子结合（ChIP-on-chip）分析，在成肌细胞和成肌细胞向肌管转变过程中，发现了一系列受MyoD1（肌发生的主要调节因子）转录调控的E3连接酶。我们假设这些E3连接酶通常在肌肉稳态中起作用，而癌症中的肌肉萎缩可能部分归因于肌肉特异性E3连接酶的不适当、持续激活。因此，这些E3连接酶（及其调节因子）可以作为治疗癌症和其他疾病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设。首先，我们将对Rnf11的底物进行蛋白质组学筛选，Rnf11是先前在MyoD1基因组筛选中发现的E3连接酶组的成员，在使用地塞米松治疗的完全分化的C2C12肌管中作为肌肉萎缩的组织培养模型。其次，我们将尝试通过Rnf11 E3连接酶在体外和我们的肌肉萎缩组织培养模型中验证这些假定底物的泛素化。本提案旨在开发创新和以前未探索的方法来对抗与癌症相关的肌肉萎缩，因此为其临床管理提供了新的可能性。

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.