Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases

Fields, Gregg B.

doi:10.1385/1-59259-046-2:495

Cited by 13 publications

(19 citation statements)

References 50 publications

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“…It should be noted that the 3-3.5-fold difference seen for MMP-14 hydrolysis of fTHP-4 versus fTHP-9 is considerably less than the 38-fold difference reported for MMP-14 hydrolysis of dansyl-Pro-Leu-AlaCys(Mob)-Trp-Ala-Arg-NH 2 versus dansyl-Pro-Leu-AlaLeu-Trp-Ala-Arg-NH 2 (36). Different effects of the P 1 ′ subsite substitution on MMP-14 activity, based on different templates, further support the notion that subsite substitutions are not independent with respect to interaction with MMPs (10,(43)(44)(45). In addition, the conformation of the triple helix offers a unique three-dimensional presentation of residues to the enzyme.…”

Section: Discussionmentioning

confidence: 66%

Matrix Metalloproteinase Triple-Helical Peptidase Activities Are Differentially Regulated by Substrate Stability

et al. 2004

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Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological destruction of tissues. The turnover of the collagen triple-helical structure has been ascribed to several members of the MMP family, but the determinants for collagenolytic specificity have not been identified. The present study has compared the triple-helical peptidase activities of MMP-1 and MMP-14 (membrane-type 1 MMP; MT1-MMP). The ability of each enzyme to efficiently hydrolyze the triple helix was quantified using chemically synthesized fluorogenic triple-helical substrates that, via addition of N-terminal alkyl chains, differ in their thermal stabilities. One series of substrates was modeled after a collagenolytic MMP consensus cleavage site from types I-III collagen, while the other series had a single substitution in the P(1)' subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P(1)' subsite was greatly favored by MMP-14 but disfavored by MMP-1. An increase in substrate triple-helical thermal stability led to the decreased ability of the enzyme to cleave such substrates, but with a much more pronounced effect for MMP-1. Increased thermal stability was detrimental to enzyme turnover of substrate (k(cat)), but not binding (K(M)). Activation energies were considerably lower for MMP-14 hydrolysis of triple-helical substrates compared with MMP-1. Overall, MMP-1 was found to be less efficient at processing triple-helical structures than MMP-14. These results demonstrate that collagenolytic MMPs have subtle differences in their abilities to hydrolyze triple helices and may explain the relative collagen specificity of MMP-1.

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Section: Discussionmentioning

confidence: 66%

Matrix Metalloproteinase Triple-Helical Peptidase Activities Are Differentially Regulated by Substrate Stability

et al. 2004

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“…The Knight SSP is an excellent substrate for a wide variety of MMPs [19; 20]. It is also readily hydrolyzed by ADAM10 and ADAM17 [21] and cathepsins D and E [22].…”

Section: Resultsmentioning

confidence: 99%

“…One possible solution would be incorporating alternative fluorophores into the existing FRET substrates. Such fluorophores could have higher quantum yields [such as 5-carboxyfluorescein (Fam), with Φ F = 0.92, compared with Φ F = 0.49 for Mca] or be less susceptible to fluorescence quenching, as exemplified by the prior labeling of NFF-3 with a CyDye pair, Cy3/Cy5Q [10; 44]. …”

Section: Discussionmentioning

confidence: 99%

“…Protease activity assays are typically based on fluorescence resonance energy transfer (FRET) and utilize synthetic peptides [8; 9]. Fluorescence can be detected with outstanding sensitivity, and these continuous assays allow for rapid, convenient kinetic evaluation of proteases and thus greatly aid mechanistic studies of these enzymes [8; 10]. For screening compounds that might potentially modulate selective protease activities, there is a need for the study of substrates, which correspond, as closely as possible, to the native substrates of proteases.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of metalloproteinase protein and activity profiling

Giricz

Lauer

Fields

2011

Analytical Biochemistry

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Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.

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“…The QGIW peptide is a commonly used, collagen I-derived sequence [12]. LACW is an artificial, commercially available peptide that has been optimized for MMP-14 detection [13].…”

Section: Methodsmentioning

confidence: 99%

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels

2018

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Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

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Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases

Cited by 13 publications

References 50 publications

Matrix Metalloproteinase Triple-Helical Peptidase Activities Are Differentially Regulated by Substrate Stability

Matrix Metalloproteinase Triple-Helical Peptidase Activities Are Differentially Regulated by Substrate Stability

Comparison of metalloproteinase protein and activity profiling

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels

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