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Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9
Janelle L Lauer-Fields1, Thilaka Sritharan, M Sharon Stack
1Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431-0991, USA.
Abstract:
The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.
Insights
Matrix metalloproteinases (MMPs) are crucial for tumor invasion. This study developed selective triple-helical peptide (THP) substrates to accurately measure active MMPs in cancer cells, revealing non-MMP-2/-9 activity upon integrin stimulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Proteases, particularly matrix metalloproteinases (MMPs), play a complex role in tumor cell invasion and extracellular matrix (ECM) degradation.
- Distinguishing between MMP production and active MMP presence is critical for understanding tumor progression.
- Collagen hydrolysis is a key step in ECM turnover, making MMPs essential targets for study.
Purpose of the Study:
- To develop selective triple-helical peptide (THP) substrates to evaluate active matrix metalloproteinase (MMP) activity.
- To investigate the hydrolysis of specific collagen models by different MMPs.
- To screen for active MMP production in melanoma cells stimulated via alpha(2)beta(1) integrin.
Main Methods:
- Synthesized two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen.
- Performed kinetic analyses of MMP-2 and MMP-9 hydrolysis on these THP models.
- Screened for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells using THP substrates.
Main Results:
- MMP-2 and MMP-9 selectively hydrolyzed specific THP models at rates comparable to native collagen.
- Neither THP was hydrolyzed by other tested MMPs (MMP-1, -3, -13, -14), indicating high selectivity.
- Alpha(2)beta(1) integrin stimulation in melanoma cells induced significant triple-helical peptidase activity, predominantly (>95%) from non-MMP-2/-9 sources.
Conclusions:
- Triple-helical peptides (THPs) serve as highly selective substrates for specific matrix metalloproteinases (MMPs).
- THPs enable accurate evaluation of active MMP production within cellular systems.
- Melanoma cell invasion involves significant protease activity beyond MMP-2 and MMP-9, particularly upon alpha(2)beta(1) integrin stimulation.