Structure and Mechanism of Transcription Factors in Pancreatic Beta Cells

项目总结 胰腺和十二指肠同源框1(Pdx1)是一种转录因子，协调动态组装 对胰腺β细胞的功能至关重要的复合体。这些复合体的破坏导致了 2型或青年成熟发作型糖尿病的分子基础；与其在发育中的作用有关， Pdx1基因异常与胰腺癌有关。对这种重要蛋白质的机制研究已经 没有跟上对其在胰腺健康中的核心作用的了解的进展。这是一个中心假设 这一建议的结论是，Pdx1反式激活结构域中结合基序的独特结构特征，以及 它们的翻译后修饰，在调节Pdx1‘S的力量中起着至关重要的作用 蛋白质之间的相互作用。这个项目提供了一个独特的机会，可以在这方面产生持续的影响 具有挑战性的领域，因为它利用创新的13C直接检测核磁共振策略-在PI中定制设计 实验室探索转录因子及其形成的复合体的生物物理。在这方面，第一个 该项目的具体目的是表征Pdx1 C-末端结构域在将Pdx1稳定性与 通过与泛素连接酶适配器蛋白SPOP的相互作用，进行葡萄糖传感。这个目标是对工作的检验 假设是多价Pdx1-SPOP相互作用提供了促进Pdx1周转所需的亲和力， 而由单个SPOP结合基序介导的弱相互作用将失败。这预示着损失 通过致病突变或临床干预的个体相互作用足以防止Pdx1- SPOP交互。核磁共振和结晶学将为SPOP的招募提供结构洞察 机制，而结合分析进一步检验了多价模型。这些生物物理研究将是 连接到培养细胞的功能分析，以确定分子细节揭示了驱动 表型。第二个具体目标是评估Pdx1招募共激活子Set7的机制 到蛋白质复合体。这个目标的工作假设是Pdx1-Set7的相互作用是由N- Pdx1的终端结构域和Set7的N-set结构域。结合分析和核磁共振研究将开发结构- 将在单元格中验证的函数关系。第三个具体目的是研究Pdx1的调节 通过翻译后修饰发挥作用。磷酸化和甲基化建立时间控制 超过Pdx1相互作用的特异性和半衰期。这一目标的工作假说是，磷酸化修饰了 Pdx1与SPOP和Set7的相互作用强度。另外，由Set7甲基化的Pdx1刺激Pdx1 活动。因此，这个目的的工作假说也是Pdx1的甲基化，而不是直接的组蛋白甲基化， 负责β-细胞中SET7的基因调控。蛋白质Pdx1、SPOP和Set7都是已知的 糖尿病的致病因素，以及具有广泛原发组织分布的腺癌的病因。 这个项目的发现有望推广到所有涉及SPOP家族的蛋白质-蛋白质相互作用 以及在一系列重要的生物医学背景下与之相互作用的转录因子。

PROJECT SUMMARY The pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that coordinates the dynamic assembly of complexes essential for the function of pancreatic β-cells. Disruption of these complexes contributes to the molecular basis for type 2 or maturity onset diabetes of the young (MODY); related to its role in development, dysregulation of Pdx1 is associated with pancreatic cancer. Mechanistic studies of this essential protein have not kept up with advances in understanding of its central role in pancreatic health. It is the central hypothesis of this proposal that the unique structural features of binding motifs in the Pdx1 transactivation domains, and their modulation by post-translational modification, play an essential role in regulating the strength of Pdx1's protein-protein interactions. This project presents a unique opportunity to have a sustained impact in this challenging area because it leverages an innovative 13C direct-detect NMR strategy custom-designed in the PI’s laboratory to probe the biophysics of transcription factors and the complexes they form. In this context, the first specific aim of this project is to characterize the role of the Pdx1 C-terminal domain in linking Pdx1 stability to glucose sensing, through interactions with the ubiquitin ligase adaptor protein SPOP. This aim tests the working hypothesis is that multivalent Pdx1-SPOP interactions provide the avidity required to promote Pdx1 turnover, whereas the weak interactions mediated by individual SPOP binding motifs would fail. This predicts that loss of individual interactions through disease-driving mutations, or clinical intervention, is sufficient to prevent Pdx1- SPOP interactions. NMR and crystallography will provide structural insights into the SPOP recruitment mechanism, while binding assays further test the multivalence model. These biophysical studies will be connected to functional assays in cultured cells to establish that the molecular details uncovered drive phenotypes. The second specific aim is to evaluate the mechanism whereby Pdx1 recruits the co-activator Set7 to protein complexes. This aim’s working hypothesis is that Pdx1-Set7 interactions are mediated by the N- terminal domain of Pdx1 and the Set7 N-Set domain. Binding assays and NMR studies will develop structure- function relationships that will be validated in cells. The third specific aim is to investigate the regulation of Pdx1 function through post-translational modifications. Phosphorylation and methylation establish temporal control over Pdx1 interaction specificity and half-life. This aim’s working hypothesis is that phosphorylation modifies the strength of Pdx1 interactions with both SPOP and Set7. Separately, methylation of Pdx1 by Set7 stimulates Pdx1 activity. Thus, it is also the working hypothesis of this aim that methylation of Pdx1, not direct histone methylation, is responsible for gene regulation by Set7 in β-cells. The proteins Pdx1, SPOP, and Set7 are all known contributors to diabetes, as well as to the etiology of adenocarcinomas with broad primary tissue distributions. Findings from this project are expected to generalize to all protein-protein interactions involving SPOP, the family of SET paralogs, and the transcription factors that interact with them in a range of significant biomedical contexts.