Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinases (TIMP)-2
Résumé
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 protumorigenic effect; however, its proteolytic capacity is likely 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 actions. We have previously reported the inhibitory profile of tissue inhibitors of metalloproteinases (TIMPs) 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. Kinetic analysis using a fluorescent-peptide substrate demonstrated that the molecular interactions of N-terminal domains of TIMPs (N-TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed striking 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, verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.
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