The molecular mechanisms of Pdx1 destabilization by SPOP

项目总结：《SPOP研究Pdx1失稳的分子机制》 少年派：格蕾丝·A·亚瑟 胰腺和十二指肠同源框1(Pdx1)是内分泌胰腺所必需的转录因子 B细胞特性的发展、维持和胰岛素表达的调节。因此，或的突变 Pdx1水平不足与胰腺发育和胰岛素反应受损有关。特定的 氨基酸替换与特定的糖尿病表型相关，包括2型糖尿病和 成熟起病的青年糖尿病(MODY4)和PDX1的过度表达与胰腺导管有关 腺癌。鉴于它在糖尿病和癌症中的重要性，Pdx1的功能和稳定性的研究是 必须对疾病的分子基础有一个完整的了解。PDX1 分子间相互作用不仅对胰岛素基因表达的激活至关重要，而且通过与 共激活剂，但也对其葡萄糖调节的稳定性。Pdx1(Pdx1-C)的C末端与 泛素连接酶适配器SPOP以葡萄糖依赖的方式促进Pdx1的蛋白酶体降解。它 我的中心假设是Pdx1翻译后修饰调节其分子间相互作用 因此，稳定性和转录因子活性。这项提案将调查两国之间的互动 PDX1-C和SPOP，以表征围绕其关联的环境以及 细胞内Pdx1稳定性的相关性。目的1阐明Pdx1-SPOP的分子机制 用核磁共振、X射线结晶学和荧光研究体外相互作用 偏振结合分析。AIM 2将在Pdx1的磷酸化和其SPOP之间建立联系- 使用体外激酶分析以及随后的质谱学和核磁共振表征相关联的稳定性。 此外，将通过荧光来探测磷酸化Pdx1和SPOP的结合，这是稳定性的代理 极化分析，就像目标1一样。最后，目标3将把我的体外发现转化为细胞，在那里我将使用co. 荧光显微镜监测Pdx1和SPOP的定位以了解它们在细胞内的相互作用 并评估葡萄糖依赖的Pdx1活性和稳定性。该项目将使用基础科学方法来 研究Pdx1-SPOP相互作用，从分子水平了解糖尿病的表型。在……里面 除了它的科学价值，这项提案还提供了一个强有力的培训计划，其中策划了 专业知识、对科学方法的理解和专业发展技能是优先考虑的。这个 建议的多管齐下的Pdx1-SPOP表征方法确保接触到广泛的 将在未来的生物医学研究中为我服务的实验技术。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了解 与真核基因调控中心的项目和合作以及机构培训赠款 为学员提供独特的科学和专业发展环境。最后，赞助商 表示完全支持这项建议和我的持续培训。

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.