Regulation of the Pax-5 Proto-Oncogen by Redox Mec,

描述(申请人提供)：Pax DNA结合蛋白家族包括在人类和其他高等真核生物中组织特异性基因表达的基本调节因子。Pax蛋白对于分化的细胞和组织的形成是必不可少的，然而，DNA结合活性的增加会导致肿瘤转化和肿瘤的发生。例如，导致Pax-5过度表达的染色体异常和基因重排与人类B系淋巴瘤有关。Pax-5介导的淋巴瘤发生的机制尚不清楚，但多条证据表明，在正常的B细胞中，Pax-5的剂量受到转录和翻译后机制的精细调控。作为Pax-5翻译后调控的一种机制，我们认为Pax-5的DNA结合在一定程度上是由其DNA结合区中高度保守的半胱氨酸残基的氧化还原状态来调节的。因此，Pax-5(和其他Pax家族成员)与DNA的结合可能会因氧化应激而减少。到目前为止，这一假说只在有限的体外模型系统中进行了测试，这些模型系统不能充分反映体内转录活动控制的动态平衡机制的复杂性。此外，目前还没有确定Pax-5的S硫基化(谷胱甘肽基化)是否是体内控制其活性的重要机制。在这一应用中，我们提出了一种遗传方法，绕过了以前遇到的与转基因分析和其他体外实验方案相关的缺陷。我们的实验将解决一个相对未被探索的分子生物学领域，对理解细胞如何维持关键调控因子的准确水平具有深远的影响。我们的研究最终将有助于设计治疗Pax相关癌症(淋巴瘤、星形细胞瘤和横纹肌肉瘤)的新疗法。

DESCRIPTION (provided by applicant): The Pax family of DNA-binding proteins includes essential regulators of tissue-specific gene expression in humans and other higher eukaryotes. Pax proteins are essential for the formation of differentiated cells and tissues, however, increasing levels of DNA binding activity results in neoplastic transformation and tumorigenesis. For example, chromosomal abnormalities and gene rearrangements resulting in overexpression of Pax-5 are associated with B lineage lymphomas in humans. Mechanisms contributing to Pax-5-mediated lymphomagenesis are not understood, but multiple lines of evidence suggest that the dosage of Pax-5 is exquisitely regulated in normal B cells by transcriptional and post-translational mechanisms. As one mechanism contributing to post-translational regulation of Pax-5, we propose that Pax- 5 DNA binding is regulated, in part, by the redox status of highly conserved cysteine residues in its paired DNA-binding domain. Thus, DNA binding by Pax-5 (and other Pax family members) may be reduced in response to oxidative stress. To date, this hypothesis has only been tested using limited in vitro model systems that do not adequately reflect the complexity of homeostatic mechanisms governing transcriptional activity in vivo. Moreover, it has not been determined whether S-thiolation (glutathionylation) of Pax-5 is an important mechanism for controlling its activity in vivo. In this application, we propose a genetic approach that bypasses previously encountered deficiencies associated with transfection assays and other in vitro experimental protocols. Our experiments will address a relatively unexplored area of molecular biology with profound implications for understanding how cells maintain precise levels of key regulatory factors. Our studies will eventually aid in devising new therapeutics for treating Pax-related cancers (lymphoma, astrocytoma, and rhabdomyosarcoma).