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Structure and Mechanism of Transcription Factors in Pancreatic Beta Cells

胰腺β细胞转录因子的结构和机制

基本信息

批准号：
10200033
负责人：
Scott A Showalter
金额：
$ 39.16万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10200033
关键词：
Active Sites Adaptor Signaling Protein Adenocarcinoma Affect Affinity Area Automobile Driving Avidity Beta Cell Binding Binding Sites Biological Biological Assay Biological Models Biological Process Biophysics C-terminal Cells Clinical Communities Complex Coupled Crystallography Cultured Cells Custom DNA Binding Domain Data Development Diabetes Mellitus Disease Down-Regulation Drug or chemical Tissue Distribution Duodenum Etiology Family Fluorescence Microscopy Gene Expression Gene Expression Regulation Genetic Enhancer Element Genetic Transcription Genome Glucose Half-Life Health Homeobox In Vitro Individual Intervention Knowledge Laboratories Link Literature Maintenance Malignant neoplasm of pancreas Mediating Metabolic Metabolic Control Methylation Methyltransferase Modeling Modification Molecular Molecular Conformation Mutagenesis Mutation N-terminal Nuclear Pancreas Performance Phenotype Phosphorylation Play Positioning Attribute Post-Translational Protein Processing Protein Family Proteins Regulation Regulator Genes Role SET Domain Sequence Homology Serine Signal Transduction Site Specificity Structure Structure of beta Cell of islet Structure-Activity Relationship Surface Testing Transactivation Translating Ursidae Family base biophysical analysis crosslink design experience flexibility histone methylation inhibitor/antagonist innovation insight maturity onset diabetes of the young mutant novel paralogous gene prevent promoter protein complex protein protein interaction recruit scaffold transcription factor ubiquitin ligase

项目摘要

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.

项目摘要胰腺和十二指肠同源框1（Pdx 1）是协调动态组装的转录因子，对胰腺β细胞功能至关重要的复合物。这些复合物的破坏有助于 2型或青年成熟期糖尿病（MODY）的分子基础;与其在发育中的作用有关， Pdx 1的失调与胰腺癌有关。对这种必需蛋白质的机制研究没有跟上对其在胰腺健康中的核心作用的理解的进展。这是核心假设 Pdx 1反式激活结构域中结合基序的独特结构特征，它们通过翻译后修饰的调节，在调节Pdx 1的强度中起重要作用。蛋白质相互作用这个项目提供了一个独特的机会，在这方面产生持续的影响。具有挑战性的领域，因为它利用了PI中定制设计的创新13 C直接检测NMR策略该实验室旨在探索转录因子及其形成的复合物的生物物理学。在这方面，第一该项目的具体目的是表征Pdx 1 C-末端结构域在连接Pdx 1稳定性与葡萄糖传感，通过与泛素连接酶衔接蛋白SPOP相互作用。这一目标考验着假设是多价Pdx 1-SPOP相互作用提供促进Pdx 1周转所需的亲合力，而由单个SPOP结合基序介导的弱相互作用将失败。这预示着，通过疾病驱动突变或临床干预的个体相互作用足以预防Pdx 1- SPOP相互作用。NMR和晶体学将提供SPOP募集的结构见解机制，而结合试验进一步测试多价模型。这些生物物理学研究将是连接到培养细胞的功能测定，以确定未揭示的分子细节驱动表型第二个具体目的是评估Pdx 1募集辅激活因子Set 7的机制到蛋白质复合物。这个目标的工作假设是Pdx 1-Set 7相互作用是由N- Pdx 1的末端结构域和Set 7 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Pdx 1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Pdx 1相互作用特异性和半衰期。这个目标的工作假设是磷酸化修饰了 Pdx 1与SPOP和Set 7相互作用的强度。另外，Set 7对Pdx 1的甲基化刺激了Pdx 1 活动因此，这也是这一目标的工作假设，即Pdx 1的甲基化，而不是直接的组蛋白甲基化，负责β细胞中Set 7的基因调控。蛋白质Pdx 1、SPOP和Set 7都是已知的糖尿病的贡献者，以及广泛的原发组织分布的腺癌的病因。该项目的发现有望推广到所有涉及SPOP家族的蛋白质-蛋白质相互作用的SET旁系同源物，和转录因子，与他们在一系列重要的生物医学背景。