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Updated: Feb 20, 2026
Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells
Published on: August 7, 2021
Quantitative proteomic analysis of cellular protein modulation upon inhibition of the NEDD8-activating enzyme by
Hua Liao1, Xiaozhen J Liu, Jonathan L Blank
1Discovery, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
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.
Insights
MLN4924, a Nedd8-activating enzyme inhibitor, inactivates Cullin-RING ubiquitin ligases (CRLs) to identify novel cancer-relevant substrates. This study reveals key proteins involved in cell cycle and DNA repair crucial for MLN4924
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cullin-RING ubiquitin ligases (CRLs) mediate ubiquitination for protein degradation, with many substrates linked to cancer.
- Identifying CRL substrates is crucial for understanding cancer biology and developing targeted therapies.
- MLN4924 is an investigational Nedd8-activating enzyme (NAE) inhibitor targeting CRL function.
Purpose of the Study:
- To identify novel substrates of Cullin-RING ubiquitin ligases (CRLs) using MLN4924.
- To elucidate the mechanism of action of MLN4924 in cancer cells.
- To discover potential biomarkers for MLN4924 efficacy.
Main Methods:
- Stable isotope labeling with amino acids in cell culture (SILAC) was employed to quantify protein changes.
- A375 melanoma cells were treated with MLN4924 to induce CRL inhibition.
- RNA interference (siRNA) was used to validate the role of identified proteins in MLN4924-induced cytotoxicity.
Main Results:
- Over 5000 proteins were identified and quantified per time point in MLN4924-treated cells.
- Several novel CRL substrates, including MLX, KLF5, and ORC6L, were identified.
- Thirty-eight proteins involved in cell cycle, DNA damage repair, and ubiquitin transfer were critical for MLN4924's cell-killing effects.
Conclusions:
- The combination of SILAC and RNAi is a powerful approach to study ubiquitin proteasome system modulators.
- This study identified novel CRL substrates and elucidated mechanisms underlying MLN4924's anti-cancer activity.
- The findings provide a foundation for identifying biomarkers to predict patient response to MLN4924.
