Defining degradation pathways of RGS2 using high-throughput siRNA screening
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Benita Sjögren, Ph.D. Department of Pharmacology & Toxicology Michigan State University Drug Discovery Interdisciplinary Event May 14, 2015
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Defining degradation pathways of RGS2 using high-throughput siRNA screening
- 1. Defining degradation pathways of RGS2 using high-throughput siRNA screening Benita Sjögren, Ph.D. Department of Pharmacology & Toxicology
- 2. Regulator of G protein signaling 2 (RGS2) as a drug target GTP α-GDP γ β GDP α-GTP γ β Pi RGS RGS protein active α-GDP γ β GDP α-GTP γ β 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 37⁰C 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