The molecular mechanisms of Pdx1 destabilization by SPOP

项目摘要：“Pdx 1通过SPOP去稳定化的分子机制” PI：Grace A.迎来 胰腺和十二指肠同源框1（Pdx 1）是胰腺内分泌所需的转录因子， 发育、维持B细胞特性和调节胰岛素表达。因此，突变或 Pdx 1水平不足与胰腺发育和胰岛素反应受损有关。具体 氨基酸取代与特定的糖尿病表型相关，包括2型糖尿病和 年轻人的成熟型糖尿病（MODY 4）和pdx 1的过度表达与胰腺导管 腺癌鉴于其在糖尿病和癌症中的重要性，Pdx 1功能和稳定性的研究是必要的。 这对于完全理解疾病的分子基础至关重要。Pdx1 分子间相互作用不仅对通过与胰岛素结合激活胰岛素基因表达是关键的， 辅活化剂，而且其葡萄糖调节的稳定性。Pdx 1的C末端（Pdx 1-C）与 泛素连接酶接头SPOP以葡萄糖依赖性方式促进Pdx 1的蛋白酶体降解。它 我的中心假设是Pdx 1的翻译后修饰调节了它的分子间相互作用 并因此影响稳定性和转录因子活性。本提案将研究以下因素之间的相互作用： Pdx 1-C和SPOP，以表征其关联的环境以及 Pdx 1在细胞中的稳定性。目的1阐明Pdx 1-SPOP的分子机制 通过核磁共振（NMR）、X射线晶体学和荧光进行体外相互作用 极化结合测定。目的2将建立Pdx 1磷酸化与其SPOP之间的联系。 使用体外激酶测定和随后通过质谱法和NMR表征连接的稳定性。 此外，将通过荧光探测磷酸化Pdx 1和SPOP的结合，即稳定性的代表 极化测定，如在目标1中。最后，Aim 3将把我的体外发现转化为细胞，在细胞中，我将使用co- 通过荧光显微镜监测Pdx 1和SPOP的定位，以了解它们在细胞中的相互作用 并评估葡萄糖依赖性Pdx 1活性和稳定性。该项目将使用基础科学方法， 研究Pdx 1-SPOP相互作用，以在分子水平上了解糖尿病表型。在 除了其科学价值外，该建议还提供了一个强大的培训计划，其中对实验的管理 专业知识、对科学方法的理解和专业发展技能是优先事项。的 所提出的Pdx 1-SPOP表征的多管齐下的方法确保暴露于广泛的 这些实验技术将在未来的生物医学研究中为我服务。生物化学， 微生物学和分子生物学博士课程保证了对高度跨学科的认识 项目和协会与真核基因调控中心和机构培训赠款 提供一个独特的环境，学员的科学和专业发展。最后，赞助商 表示完全支持这一建议和我继续接受培训。

Project Summary: “The molecular mechanisms of Pdx1 destabilization by SPOP” PI: Grace A. Usher Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that is required for endocrine pancreas development, maintenance of b cell identity, and regulated insulin expression. Therefore, mutations in or deficient levels of Pdx1 are associated with impaired pancreas development and insulin response. Specific amino acids substitutions are associated with particular diabetic phenotypes, including Type 2 Diabetes and Mature Onset Diabetes of the Young (MODY4), and overexpression of pdx1 is linked to pancreatic ductal adenocarcinoma. Given its significance in diabetes and cancer, the study of Pdx1 function and stability is imperative to achieve a complete understanding of the molecular underpinnings of disease. Pdx1 intermolecular interactions are critical not only to activation of insulin gene expression through association with co-activators, but also to its glucose-modulated stability. The C-terminus of Pdx1 (Pdx1-C) associates with the ubiquitin ligase adaptor SPOP in a glucose-dependent manner to facilitate proteasomal degradation of Pdx1. It is my central hypothesis that Pdx1 post-translational modifications modulate its intermolecular interactions and, therefore, stability and transcription factor activity. This proposal will investigate the interactions between Pdx1-C and SPOP to characterize the circumstances surrounding their association and the influence of association on Pdx1 stability in cells. Aim 1 will elucidate the molecular mechanisms of Pdx1-SPOP interactions in vitro by nuclear magnetic resonance (NMR), X-ray crystallography, and fluorescence polarization binding assays. Aim 2 will establish a connection between phosphorylation of Pdx1 and its SPOP- linked stability using in vitro kinase assays and subsequent characterization by mass spectrometry and NMR. Further, binding, a proxy for stability, of phosphorylated Pdx1 and SPOP will be probed through fluorescence polarization assays, as in Aim 1. Finally, Aim 3 will translate my in vitro findings into cells, wherein I will use co- localization of Pdx1 and SPOP monitored by fluorescent microscopy to understand their interactions in cells and assess glucose-dependent Pdx1 activity and stability. This project will use basic science approaches to investigate Pdx1-SPOP interactions toward a molecular-level understanding of diabetes phenotypes. In addition to its scientific merit, this proposal affords a robust training plan, wherein curation of experimental expertise, understanding of the scientific method, and professional development skills are priorities. The proposed multi-pronged approach to Pdx1-SPOP characterization ensures exposure to a broad range of experimental techniques that will serve me in future biomedical research endeavors. The Biochemistry, Microbiology, and Molecular Biology PhD program guarantees acquaintance with highly interdisciplinary projects and association with the Center for Eukaryotic Gene Regulation and an institutional training grant provides a unique environment to trainees for scientific and professional development. Finally, the Sponsor indicates full support of this proposal and my continued training.