Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding.
Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding.
复制标题
DOI：
10.4331/wjbc.v2.i11.246
复制
发表时间：
2011-11-26
期刊：
影响因子：
--
通讯作者：
Fan, Huizhou
中科院分区：
文献类型：
--
作者：
Li, Xiaojin;Perez, Liliana;Fan, Huizhou
目的：确定主要脱落酶肿瘤坏死因子- α转换酶（TACE; ADAM17）的细胞尾是否控制蛋白外结构域脱落。方法：采用定点诱变法获得TACE变异体。将在细胞外、富含半胱氨酸的解体素结构域（CRD）和/或缺失的细胞尾中替换氨基酸的TACE表达质粒，以及增强的绿色荧光蛋白的表达载体，转染到稳定表达跨膜l -选择素或转化生长因子(TGF)- α的脱落缺陷M1突变体中。流式细胞术检测TACE底物在细胞表面的表达水平。结果：与已发表的数据一致，CRD中的单点突变（C600Y）导致脱落缺陷。然而，从C600Y TACE变异中去除细胞尾部分恢复了tgf - α和l -选择素的外域切割。细胞尾缺失突变体与C600Y TACE相比，C600Y TACE具有与C600Y TACE相似的功能。结论：细胞尾具有抑制作用，当它从影响酶功能的另一突变的酶中去除时，这种抑制作用变得明显。
AIM: To determine if the cytotail of the principal sheddase tumor necrosis factor-alpha 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)-alpha. 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-alpha 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.