Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

Project: Research project

Project Details

Description

This project is to examine potential roles of substrates of CHIP-mediated ubiquitination in induced-pluripotent stem cell (iPSC)-derived neurons, in order to reveal previously undefined functions of CHIP in Alzheimer’s disease. We will also conduct screens for more CHIP substrates in neurons, using our Orthogonal Ubiquitin Transfer (OUT) technology. These studies are within the scope of Specific Aim 3 of the original grant, R01-GM104498, and we are requesting for an administrative supplement in response to the NIH notice, NOT-AG-18-039.

The proposed studies will require diverse expertise. We will need to conduct in vitro biochemical analysis for ubiquitination of substrates, examine the expression, localization and activity of each substrate in cultured neurons derived from iPSCs, and perform proteomic screens using the CHIP OUT cascade of engineered enzymes we previously generated. Handling iPSC cultures and derived neurons takes special expertise, so we have recruited Dr. Gemma Carvill, Assistant Professor of Neurology at Northwestern, as a co-investigator.

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 eight 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 together with his staff and Dr. Carvill will conduct the following experimental procedures for both Specific Aims 1 and 2 of the administrative supplement. Many of the procedures will be conducted as collaborative efforts with Dr. Yin’s laboratory to ensure synergy between the two labs.
(1) Maintain several iPSC lines from healthy individuals and patients with familial Alzheimer’s disease
(2) Package recombinant lentiviruses using 293FT cells and plasmids encoding viral components (for expression of inducible anti-CHIP shRNA and CHIP cDNA)
(3) Infect undifferentiated iPSCs with recombinant lentiviruses and establish cell lines with tetracycline-inducible for expression of inducible anti-CHIP shRNA and CHIP cDNA
(4) Induce in vitro differentiation of iPSC into mature forebrain neurons
(5) Characterize neurons by flow cytometry, immunoblotting, immunoprecipitation and immunofluorescence microscopy
(6) Examine expression, ubiquitination, localization and activity of each substrate protein in neurons with tetracycline-inducible expression of anti-CHIP shRNA or CHIP cDNA
(7) Investigate drug responses of iPSC-derived neurons using Hsp90 inhibitors and RhoA inhibitors
(8) Work with the Stem Cell Core at Northwestern to generate the OUT mutations on the endogenous CHIP gene in iPSCs, using the CRISPR/Cas9 genome editing
(9) Package recombinant lentiviruses using 293FT cells and plasmids encoding viral components (for expression of xE1/Uba1 and xE2/UbcH7)
(10) Verify undifferentiated iPSCs that carry the xCHIP mutations by PCR/sequencing, and infect them with the recombinant lentiviruses for the expression of xUba1 and xUbcH7 enzymes
(11) Induce neuronal differentiation of the engineered iPSC lines
(12) Express HBT-xUB by adeno associated virus particles prepared by Stem Cell Core
(13) Harvest viral transduced cells and conduct tandem affinity purification using Ni2+-agarose and streptoavidin-agarose beads
(14) Prepare samples for LC-MS/MS and conduct the analysis in the Proteomics core at Emroy University
StatusActive
Effective start/end date7/1/196/30/21

Funding

  • Georgia State University (SP00014037-01//R01GM104498)
  • National Institute of General Medical Sciences (SP00014037-01//R01GM104498)

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