Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding  

作　　者：Liliana Pérez 

机构地区：[1]Department of Physiology and Biophysics,Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey

出　　处：《World Journal of Biological Chemistry》2011年第11期246-251,共6页世界生物化学杂志（英文版）（电子版）

基　　金：Supported by Grants from the National Institutes of Health,No.AG029859; the National Center of the American Heart Association,No.0330335N;the New Jersey Commission on Cancer Research(NJCCR703010)to Fan H

摘　　要：AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.AIM: To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding. METHODS: Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS: Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE. CONCLUSION: The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.

关 键 词：ADAM17 ECTODOMAIN SHEDDING L-SELECTIN Tumor NECROSIS factor-α converting enzyme 

分 类 号：R363[医药卫生—病理学]