Department of Biomedical Sciences and Biotech Research and Innovation Centre, University of Copenhagen, Denmark.
Biochem J. 2010 Aug 15;430(1):79-86. doi: 10.1042/BJ20100649.
The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.
去整合素金属蛋白酶 ADAM12 在正常生理和疾病（如癌症）中具有重要功能。虽然人们对 ADAM12 如何发挥其促肿瘤作用知之甚少，但它的蛋白水解能力可能是一个关键组成部分。因此，选择性抑制 ADAM12 的活性可能具有很大的治疗价值，并可作为研究工具来阐明其作用机制。我们之前报道了 TIMP（金属蛋白酶组织抑制剂）对 ADAM12 的抑制谱，除了 TIMP-3 之外，TIMP-2 还具有独特的 ADAM 抑制活性。这些发现强烈表明，设计一种选择性抑制 ADAM12 的 TIMP 突变体是可行的。为此，我们对 ADAM12-TIMP 复合物形成的分子决定因素进行了特征分析，与已知的 TIMP 介导 ADAM17/TACE（肿瘤坏死因子 α 转换酶）抑制的分子要求进行了比较。使用荧光肽底物进行的动力学分析表明，N-TIMP（TIMP 的 N 端结构域）与 ADAM12 和 TACE 的分子相互作用在很大程度上是可比的，但却显示出 TIMP 介导的 ADAM12 抑制的独特特征。有趣的是，我们发现，去除 N-TIMP-2 中的 AB 环（已知会损害其与 TACE 的相互作用）会导致与 ADAM12 的亲和力增加。重要的是，我们首次使用基于细胞的表皮生长因子脱落测定法证明了 TIMP 对跨膜 ADAM12-L（全长 ADAM12）的抑制活性，验证了 N-TIMP-2 和工程化 N-TIMP-2 突变体在细胞环境中的独特抑制能力。总之，我们的研究结果支持这样一种观点，即可以设计出具有未来治疗潜力的独特 ADAM12 抑制剂。
