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作用。控制基因表达和细胞编程的大多数分子事件都需要瞬态多蛋白复合物来响应不同的生理信号来解释DNA中的增强子和启动子。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。我建议研究这个问题，但限制研究范围，将重点放在必要的转录因子甾体生成因子1 （SF-1）和肝脏受体同源物1 （LRH-1）及其共同调节因子上，它们对胚胎干细胞（ES）细胞和诱导多能干细胞（iPS）细胞至关重要。在合适的DNA分子存在或通过结合翻译后修饰（PTMs）在体外实现稳定转录蛋白组装的可能性将用这些转录因子进行评估。在优化的DNA片段和共调节蛋白存在下，全长SF-1和LRH-1的稳定性有望得到改善，从而获得结构研究的机会。ptm的引入可以进一步提高蛋白质的稳定性，并可能有助于识别新的蛋白质结合伙伴。所选择的半合成策略将PTMs纳入蛋白质的显著优势在于产生均匀修饰的蛋白质，这将允许晶体学研究。对稳定蛋白组合的研究将为理解SF-1和LRH-1的结构-功能关系以及如何设计和使用特异性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.