An iTRAQ Proteomics Screen Reveals the Effects of the MDM2 Binding Ligand Nutlin-3 on Cellular Proteostasis
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- 作者：Judith Nicholson ; Kalainanghi Neelagandan ; Anne-Sophie Huart ; Kathryn Ball ; Mark P. Molloy ; Ted Hupp
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：November 2, 2012
- 年：2012
- 卷：11
- 期：11
- 页码：5464-5478
- 全文大小：585K
- 年卷期：v.11,no.11(November 2, 2012)
- ISSN：1535-3907
文摘
Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC鈥揗S/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein鈥損rotein interactions.