Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting -: Fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-α-converting enzyme
Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting -: Fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-α-converting enzyme
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DOI：
10.1074/jbc.m500897200
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发表时间：
2005-04-22
影响因子：
4.8
通讯作者：
Murphy, G
中科院分区：
文献类型：
--
作者：
Lee, MH;Rapti, M;Murphy, G
金属蛋白酶组织抑制剂（Tissue inhibitors of metalloproteinases，TIMPs）是基质金属蛋白酶（matrix metalloproteinases，亚当斯）和血小板反应蛋白重复序列（thrombospondin repeats，ADAM-TS）的内源性抑制剂。有四种哺乳动物TIMP（TIMP-1至TIMP-4），并且每种TIMP具有其自身的金属蛋白酶抑制谱。TIMP-4是最新被克隆的TIMP成员，并且从未报道其对肿瘤坏死因子-α-转化酶（TACE，ADAM-17）具有活性。在这里，我们用TACE检查了TIMP-4的全长和N-末端结构域形式（N-TIMP-4）的抑制特性，并显示N-TIMP-4是比其全长对应物更优越的上级抑制剂。尽管全长TIMP-4显示出对TACE的可忽略的活性，但N-TIMP-4是具有低纳摩尔结合亲和力的缓慢紧密结合抑制剂。我们的研究结果表明，C-末端亚结构域的TIMPs有显着的影响，他们的活动与亚当斯。为了进一步阐明支持TIMP/TACE相互作用的分子基础，我们用TIMP-3的表面残基雕刻N-TIMP-4，TIMP-3是该酶唯一的天然TIMP抑制剂。仅将三个残基Pro-Phe-Gly移植到N-TIMP-4的AB环上导致结合亲和力增强10倍;所得突变体的Ki值几乎与TIMP-3的Ki值相当。EF环的进一步突变支持了我们早期关于TACE在该位点对亮氨酸的偏好的发现。结合我们以前使用TIMP-1和TIMP-2支架进行TACE靶向突变的经验，我们最终解决了TACE对TIMP-3选择性敏感性的奥秘。
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs ( a disintegrin and metalloproteinase) and the ADAM-TS ( ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme ( TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 ( N-TIMP-4) with TACE and showed that N- TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N- TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N- TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K-i values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.