Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924

Hua Liao,Eric S. Lightcap,Khristofer Garcia,Hugues Bernard,Xiaozhen J. Liu,David C. Bouck,Jonathan L. Blank

doi:10.1074/mcp.m111.009183

Hua Liao, Eric S. Lightcap + Show 5 more

Open Access

https://doi.org/10.1074/mcp.m111.009183

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Abstract

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.

Highlights

MLN4924 is an investigational small molecule inhibitor of the NEDD8-activating enzyme (NAE)1 [1] that is currently be
Because NAE inhibition blocks the ubiquitination of a minor subset of proculture; DMEM, Dulbecco’s modified Eagle’s medium; dimethyl sulfoxide (DMSO), dimethylsulfoxide; RIPA, radioimmunoprecipitation assay
SILAC Analysis of MLN4924-Induced Protein Stabilization—As an inhibitor of the NAE, the primary effect of MLN4924 is the stabilization of proteins rapidly turned over by the Cullin-RING ubiquitin ligases (CRLs)

Summary

EXPERIMENTAL PROCEDURES

SILAC Sample Preparation—A375 cells were grown for 11 days with three passages in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and containing either 0.5 mM each of L-Lysine-2HCl and L-Arginine-HCl or 13C615N2 L-Lysine2HCl and 13C615N4 L-Arginine-HCl. Following treatment with 650 nM MLN4924 or vehicle (0.065% DMSO) for the times indicated, A375 cells were harvested by trypsinization and centrifugation and stored in 350 ␮l RLT (Qiagen #79216) and 2-mercaptoethanol at Ϫ80 °C. Following treatment with 650 nM MLN4924 or vehicle (0.065% DMSO) for the times indicated, cells were rinsed once with phosphate-buffered saline (PBS) and harvested by scraping and centrifugation. Cell pellets were lysed in RIPA buffer containing 1 ϫ protease inhibitor mixture (Calbiochem, #539131), 50 mM sodium fluoride, 50 mM sodium orthovanadate, 25 mM ␤-glycerophosphate, and benzonase, followed by freezing at Ϫ80 °C overnight and thawing at 4 °C with periodic vortexing. Following five washes in TBST and one wash in TBS, membranes were dried for 1 h at room temperature and proteins were detected and quantitated by LI-COR/Odyssey infrared image system (LI-COR Biosciences).

RESULTS

Protein quantitation

Aphidicolin regulated

Synthetic lethal

DISCUSSION