The molecular mechanisms of Pdx1 destabilization by SPOP

SPOP 使 Pdx1 不稳定的分子机制

基本信息

批准号：
10318108
负责人：
Emery Thomas Usher
金额：
$ 3.45万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318108
关键词：
Acquaintances Affinity Amino Acid Substitution B-Lymphocytes Basic Science Binding Binding Proteins Binding Sites Biochemical Biochemistry Biological Biological Assay Biology Biomedical Research Biophysics C-terminal Cells Charge Complement Crystallography Development Diabetes Mellitus Disease Doctor of Philosophy Duodenum Ensure Environment Event Exposure to Fluorescence Fluorescence Polarization Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Glucose Grant Health Homeobox Impairment In Vitro Insulin Investigation Link Maintenance Malignant Neoplasms Malignant neoplasm of pancreas Mass Spectrum Analysis Mediating Methods Microbiology Microscopy Molecular Molecular Biology Monitor Mutation Non-Insulin-Dependent Diabetes Mellitus Nuclear Magnetic Resonance Pancreas Pancreatic Diseases Pancreatic Ductal Adenocarcinoma Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Play Post-Translational Protein Processing Protein Binding Domain Proteins Proxy Public Health Regulation Research Role Structure Structure of beta Cell of islet System Techniques Thermodynamics Training Transactivation Transcriptional Activation Translating Ubiquitin Work X-Ray Crystallography base biophysical properties combat design diabetic disease phenotype endocrine pancreas development experimental study homeodomain innovation insight inter-alpha-inhibitor intermolecular interaction link protein novel overexpression pancreas development programs protein folding protein protein interaction response skill acquisition structural biology transcription factor ubiquitin ligase

项目摘要

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.

项目摘要：“ PDX1的分子机制不稳定了Spop” PI：Grace A. Usher 胰腺和十二指肠同型BOOX 1（PDX1）是内分泌胰需要的转录因子开发，维持B细胞身份和调节胰岛素表达。因此，或不足水平的PDX1与胰腺发育和胰岛素反应受损有关。具体的氨基酸取代与特定的糖尿病表型有关，包括2型糖尿病和年轻的成熟发作糖尿病（Mody4）和PDX1的过表达与胰腺导管有关腺癌。鉴于其在糖尿病和癌症中的重要性，PDX1功能和稳定性的研究是必须完全了解疾病的分子基础。 PDX1 分子间相互作用不仅对通过与胰岛素基因表达的激活至关重要共激活因子，但也达到其葡萄糖调节的稳定性。 PDX1（PDX1-C）的C端与泛素连接酶衔接子以葡萄糖依赖性方式促进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。该建议将调查 PDX1-C和Spop来表征其关联的环境以及细胞中PDX1稳定性的关联。 AIM 1将阐明PDX1-SPOP的分子机制通过核磁共振（NMR），X射线晶体学和荧光在体外相互作用极化结合测定。 AIM 2将建立PDX1磷酸化及其Spop-之间的联系使用体外激酶测定和随后通过质谱和NMR进行表征的稳定性。此外，将通过荧光探测磷酸化的PDX1和SPOP的结合，稳定性的代理偏振分析，如AIM 1。最后，AIM 3将我的体外发现转化为细胞，其中我将使用共同点。通过荧光显微镜监测的PDX1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的PDX1活性和稳定性。该项目将使用基础科学方法研究PDX1-SPOP相互作用，朝着对糖尿病表型的分子水平理解。在除了其科学优点外，该提案提供了一个强大的培训计划，其中实验策划专业知识，对科学方法的理解以及专业发展技能是优先事项。拟议的多管齐下方法PDX1-SPOP表征可确保暴露于广泛的范围实验技术将在未来的生物医学研究努力中为我服务。生物化学，微生物学和分子生物学博士学位课程可以保证用高度跨学科的获取与真核基因监管中心和机构培训赠款的项目和联系为学员提供科学和专业发展的独特环境。最后，赞助商表示对该提案的全部支持以及我的持续培训。