Abstract
Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.
Highlights
Collagen catabolism is normally a well regulated physiological process critical to tissue and organ development, morphogenesis, and wound healing [1]
The ␣1(V)Gly⌿{PO2H-CH2}Val THPI, whose sequence is based on ␣1(V)436 – 450, is a low nanomolar inhibitor of matrix metalloproteinases (MMPs)-2 and MMP-9 but not MMP-1, MMP-3, MMP-8, MMP-13, and MT1-MMP [26]
Identification of MMP-9 Domains Participating in Triple-helical Peptidase Activity—We examined the role of the HPX-like domain and fibronectin type II (FN II) inserts in MMP-9 catalysis using deletion mutants
Summary
Collagen catabolism (collagenolysis) is normally a well regulated physiological process critical to tissue and organ development, morphogenesis, and wound healing [1]. JULY 18, 2008 VOLUME 283 NUMBER 29 described as exhibiting collagenolytic behavior, requiring that they catalyze the hydrolysis of the collagen triple helix [1]. Collagenolysis may be divided into several steps as follows: (a) binding of the triple helix; (b) proper orientation of the MMP domains; (c) unwinding of the triple helix and/or trapping of relaxed triplehelical backbones; (d) binding of individual substrate strands to the active site; and (e) rapid sequential processing of all three strands [20]. One approach to identify MMP residues and regions that participate in the individual collagenolytic steps is to use triple-helical substrates in combination with site-specific or domain mutagenesis (14, 20 –25). The synthesis of the protected Fmoc-Gly⌿{PO2HCH2}Leu building block proceeded as described previously [26], with the exception of iodobutane being used instead of iodopropane to prepare the
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
