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The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3
A Amour1, C G Knight, A Webster
1School of Biological Sciences, University of East Anglia, Norwich, UK.
Abstract:
A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.
Insights
Researchers developed a catalytically active ADAM-10 enzyme. This enzyme
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- ADAM-10 (a disintegrin and metalloproteinase domain) is a key metalloproteinase involved in various biological processes.
- Understanding ADAM-10's catalytic activity and inhibition is crucial for therapeutic development.
- Previous studies have characterized metallosheddase cleavage sites but detailed inhibition profiles are needed.
Purpose of the Study:
- To express and characterize a catalytically active recombinant soluble form of human ADAM-10.
- To investigate the inhibitory effects of matrix metalloproteinase (MMP) inhibitors on ADAM-10 activity.
- To compare the inhibitory profiles of TIMP-1 and TIMP-3 against ADAM-10 and TACE.
Main Methods:
- Expression of human ADAM-10 catalytic domain as an Fc fusion protein in myeloma cells.
- Assessing catalytic activity using myelin basic protein degradation and quenched fluorescent substrate assays.
- Testing inhibition by hydroxamate-based MMP inhibitors, TIMP-1, TIMP-2, TIMP-3, and TIMP-4.
Main Results:
- Recombinant ADAM-10 demonstrated catalytic activity, cleaving substrates like myelin basic protein and peptides from TNF-alpha, CD40L, and APP.
- Hydroxamate MMP inhibitors also inhibited ADAM-10.
- TIMP-1 and TIMP-3 inhibited ADAM-10 with low nanomolar apparent inhibition constants (0.1 nM and 0.9 nM, respectively), while TIMP-2 and TIMP-4 did not.
- TIMP-1 inhibition was specific to ADAM-10, unlike TIMP-3 which also inhibits TACE.
Conclusions:
- The study successfully produced an active ADAM-10 enzyme for further investigation.
- ADAM-10 activity can be inhibited by hydroxamate-based MMP inhibitors and specifically by TIMP-1 and TIMP-3.
- TIMP-1 serves as a valuable tool for distinguishing ADAM-10 activity from TACE in cellular assays.