Abstract
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 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 α-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 knownto 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.
Original language | English |
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Pages (from-to) | 79-86 |
Number of pages | 8 |
Journal | Biochemical Journal |
Volume | 430 |
Issue number | 1 |
DOIs | |
Publication status | Published - 15 Aug 2010 |
Externally published | Yes |
Keywords
- A disintegrin and metalloprotease 12 (ADAM12)
- Ectodomain shedding
- Therapeutic target
- Tissue inhibitor of metalloproteinase (TIMP)
- Tumour necrosis factor α-converting enzyme (TACE)