Transcript of Defining degradation pathways of RGS2 using high-throughput siRNA screening
- 1. Defining degradation pathways of RGS2 using high-throughput
siRNA screening Benita Sjgren, Ph.D. Department of Pharmacology
& Toxicology
- 2. Regulator of G protein signaling 2 (RGS2) as a drug target
GTP -GDP GDP -GTP Pi RGS RGS protein active -GDP GDP -GTP Pi RGS
SIGNAL RGS protein enhanced SIGNAL GTP RGS2 KO mice are
hypertensive, show prolonged response to vasoconstrictors (e.g.
Angiotensin II). Heximer et al. J Clin Invest. 2003 and have
worsened heart failure in TAC models. Takimoto et al , J Clin
Invest. 2009 Enhancing RGS2 function could be a novel avenue in
cardiovascular therapeutics But how to achieve it? Blocking the
enzymes involved in RGS2 protein degradation should increase RGS2
protein function RGS2 is rapidly degraded through the ubiquitin
proteasomal pathway >600
- 3. siRNA screening approach Gene involved in RGS2 protein
degradation RGS2 protein chemiluminescence 48h at 37C siGENOME
SMART POOL siRNA (Dharmacon) Druggable genome Subset ~3,200 genes
Lipofectamine RNAiMAX + SMART POOL (triplicate) 384-well plateAdd
cells 384-well plate Celltiter Fluor viability assay PathHunterTM
ProLabel assay Controls: Non-targeting siRNA RGS2 Viability PLK-1
siTOX Kif-11 DAVID david.abcc.ncifcrf.gov GeneGo portal. genego.com
PathHunter ProLabel assay, DiscoveRx Stable HEK-293 cell line Top
hit genes involved in ubiquitin-proteasomal degradation was F-box
protein 44 (FBXO44) and Cullin 4B (CUL4B)
- 4. RGS2 is degraded by a novel CUL4B/DDB1/FBXO44 E3 ligase
RGS2chemiluminescence (%change) R G S 2 F B X O 4 4 C U L 4 A C U L
4 B -7 5 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 *** *** *** s iR
N A RGS2-HAproteinhalf-life(min) N T F B X O 4 4 C U L 4 A C U L 4
B 0 1 0 2 0 3 0 4 0 5 0 *** * s iR N A
RGS2proteinlevelsRGS2proteinhalf-life X RGS2 protein levels
Funding: NIGMS (R01 GM110195), American Heart Association
(15SDG21630002) MSU Richard Neubig Center for Chemical Genomics
Martha Larsen Steven Swaney University of Iowa Kevin Glenn