کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532691 | 961696 | 2013 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Quantifiable analysis of cellular pathway inhibition of a Nedd8-activating enzyme inhibitor, MLN4924, using AlphaScreen
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Cellular effects of a Nedd8-activating enzyme (NAE) inhibitor, MLN4924, using the AlphaScreen format were explored. MLN4924 acts as a substrate-assisted inhibitor of NAE by forming a tight binding Nedd8-MLN4924 adduct. The inhibited enzyme can no longer transfer Nedd8 downstream to modify and activate the E3 cullin-RING ligases. This results in the stabilization of proteins regulated by the proteasome, leading to cell death. These studies monitored the endogenous cellular changes to NAEâ¼Nedd8 thioester, the formation of the Nedd8-MLN4924 adduct, and the reduction in the Cul1-Nedd8. Lysates derived from MLN4924-treated HCT116 cells showed that whereas the β-subunit of NAE remained constant, reductions of both NAEâ¼Nedd8 thioester and Cul1-Nedd8 levels occurred with a concomitant rise of the adduct. Moreover, the formation of the Nedd8-MLN4924 adduct was approximately stoichiometric with the concentration of NAEβ. Higher density 384-well cell-based assays illustrated the kinetics of enzyme inactivation across a wider range of MLN4924 concentrations, showing a rapid loss of NAEâ¼Nedd8 thioester and Cul1-Nedd8. The reduction of NAEâ¼Nedd8 thioester precedes the loss of Cul1-Nedd8 at twice the rate. Finally, these results clearly demonstrate the utility of the homogeneous assay for quantitative assessment of these endogenous cellular components in a 384-well plate in response to inhibition of NAE by MLN4924.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 439, Issue 2, 15 August 2013, Pages 109-115
Journal: Analytical Biochemistry - Volume 439, Issue 2, 15 August 2013, Pages 109-115
نویسندگان
Zhong-Hua Yan, Anne Burkhardt, Huay-Keng Loke, Jesse Chen, Qing Xu, Pam Brauer, Jingya Ma, Yafang Lin, Khris Garcia, Lawrence R. Dick, Michael E. Bembenek,