?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.
Transcription factors play critical roles in regulated gene expression, but they are also contributors to many diseases as a result of impaired regulation or function. It is imperative that we understand the structures, functions, and interactions of transcription factors in disease phenotypes, such as diabetes, so that we may apply those findings in a broader biological context. The proposed work will positively impact public health by establishing a molecular understanding of a transcription factor and its interactions that contribute to diabetes, which may ultimately translate to new treatment options.