Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

This project is to identify direct substrates of several E3 enzymes using the novel Orthogonal Ubiquitin Transfer (OUT) technology and analyze the roles of the ubiquitination enzymes and identified substrates in regulation of mammalian cells. It will require diverse expertise from chemical engineering of the enzymatic complexes to biological validations of candidate substrates identified by biochemical purification and mass spectrometry. Dr. Hiroaki Kiyokawa, Professor of Pharmacology at Northwestern University has been collaborating closely with Dr. Jun Yin, Associate Professor of Chemistry at Georgia State University for six years, and played a key role in developing the OUT technology especially in procedures using mammalian cells such as lentiviral transduction, tandem affinity purification, LC-MS/MS and data analysis using bioinformatics. As a co-PI of this application, Dr. Kiyokawa and his staff will conduct experimental procedures for Specific Aim 3 and also part of Aims 1 & 2 together with Dr. Yin’s group, as follows: (1) Construct lentiviral vectors for mammalian expression of xUB, xE1, xE2 and xE3 (2) Package recombinant lentiviruses using 293FT cells and plasmids encoding viral components (3) Infect mammalian cells with recombinant lentiviruses and establish cell lines with stable expression of various combinations of xE1-xE2-xE3 enzymes (4) Harvest viral transduced cells and conduct tandem affinity purification using Ni2+-agarose and streptoavidin-agarose beads (5) Analyze purified proteins by immunoblotting and pull down assays for quality control (6) Prepare samples for LC-MS/MS and conduct the analysis in the Proteomics core at Northwestern University (7) Analyze MS/MS datasets and discuss with Dr. Yin to identify candidates of biologically relevant substrate proteins for each E1-E2-E3 combination (8) Construct mammalian cells with stable or transient expression of siRNAs against the select E3 enzymes (9) Quantify levels and ubiquitination of candidate substrate proteins in cells with by siRNA-mediated silencing of the E3 proteins (10) Develop cell biological systems to determine the roles of identified and verified ubiquitination substrates in human diseases such as cancer and neurological disorders. (11) Discuss about strategies, trouble shooting, data analysis and planning future projects with Dr. Yin on the monthly basis (12) Write manuscripts and progress reports, and present data in scientific meetings, together with Dr. Yin