Identification of novel multicomponent protein complexes of the critical transcription factors Steroidogenic factor 1 and Liver Receptor Homolog 1 essential to embryonic stem cells and induced pluripotent stem cells

细胞基因表达的转录调控在疾病和健康中都起着重要的作用。大多数控制基因表达和细胞编程的分子事件都需要瞬时多蛋白复合体，这些复合体对不同的生理信号做出反应，以解释DNA中发现的增强子和启动子。在这些机器的单个部件的结构和功能分析方面取得了很大进展，但对多部件组装的详细机制知之甚少。我建议解决这个问题，但限制研究范围，专注于基本转录因子类固醇生成因子1(SF-1)和肝脏受体同源基因1(LRH-1)及其共同调节因子，它们对胚胎干细胞(ES)和诱导多能干细胞(IPS)至关重要。这些转录因子将评估在适当的DNA分子存在下或通过掺入翻译后修饰(PTM)在体外获得稳定转录蛋白质组件的可能性。在存在优化的DNA片段和共同调节蛋白质的情况下，全长SF-1和LRH-1的稳定性有望提高，从而获得结构研究的机会。PTMS的引入可以进一步提高蛋白质的稳定性，并可能有助于寻找新的蛋白质结合伙伴。所选择的将PTMS整合到蛋白质中的半合成策略的显著优势在于产生了能够进行结晶学研究的均一修饰蛋白质。对稳定蛋白组装的研究将为理解SF-1和LRH-1的结构-功能关系以及如何设计和使用特定的调节剂来调节内分泌代谢疾病和癌症的异常活动提供必要的信息。

Transcriptional regulation of the cellular gene expression plays an important role in both sickness and health. Most molecular events in controlling gene expression and cell programming require transient multi protein complexes that act in response to different physiological signals to interpret enhancers and promoters found in the DNA. Much progress is made in structure and function analyses of individual components of theses machines, but few detailed mechanisms are known for multi-component assemblies. I propose to work on this problem but limit the scope of research by focusing on the essential transcription factors Steroidogenic factor 1 (SF-1) and Liver Receptor Homolog 1 (LRH-1) and their co-regulators, which are critical to embryonic stem (ES) cells and to induced pluripotent stem (iPS) cells. The possibility to achieve stable transcriptional protein assemblies in vitro in the presence of appropriate DNA molecules or by the incorporation of posttranslational modifications (PTMs) will be evaluated with these transcription factors. The stability of full-length SF-1 and LRH-1 in the presence of optimized DNA fragments and co-regulating proteins is expected to improve and thus to gain access to structural studies. The introduction of PTMs can further increase protein stability and might help to identify novel protein binding partners. The remarkable advantage of the chosen semi-synthetic strategy to incorporate PTMs into proteins lies in the generation of homogeneously modified proteins that will allow crystallographic studies. The investigation of the resulting stabilized protein assemblies will provide necessary information for understanding the SF-1 and LRH-1 structure-function relationship and how one could design and use specific modulators to tune aberrant activities which underlie endocrine metabolic diseases and cancer.