Abstract
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families. There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets. TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-alpha-converting enzyme, ADAM-17). The molecular basis for such inactivity is unknown. Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L). Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP. Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE. The binding affinity of the mutant (V4S/TIMP-3-AB-loop/V69L/T98L) (K (app)(i) 0.14 nm) surpassed that of TIMP-3 (K (app)(i) 0.22 nm), the only natural TIMP inhibitor of the enzyme. The requirement for leucine is absolute for the transformation in inhibitory pattern. On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1. Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity.
Highlights
The mammalian matrix metalloproteinases (MMPs,1 matrixins) and their close associates, ADAM proteinases, are important modulators of cell-cell and cell-matrix interactions
In our previous study with membrane-type 1 (MT1)-MMP, we created a series of N-terminal domain tissue inhibitors of metalloproteinases (TIMPs)-1 mutants that displayed partial to full inhibitory functions against the enzyme [18]
At the outset of the current project, we subjected tumor necrosis factor-␣-converting enzyme (TACE) to affinity measurements with three of these mutants (T98L/V4A/P6V/ TIMP-2-AB-loop, threonine 98 residue with leucine (T98L)/V4A/P6V, and V4A/P6V/TIMP-2-ABloop), and the results are summarized in Table I. (The composition of the TIMP-2-AB-loop mutant is explained in Fig. 1.) Interestingly, we found that N-TIMP-1 mutants originally designed against an MT1-MMP backdrop were active TACE inhibitors
Summary
Materials—All kinetic assays were performed at 27 °C in fluorescence assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 0.05% Brij-35, 1% Me2SO, 0.02% NaN3) with a PerkinElmer Life Sciences LS-50B spectrofluorometer equipped with thermostatic cuvette holders. Inhibition Constant Measurement (Kiapp)—With the exception of MMP-19, MPs (0.15 nM) were preincubated with increasing concentrations of N-TIMP-1 mutants (up to 800 nM, depending on the potency of the inhibitors) at room temperature. Due to the low kcat/Km rate of MMP-19 with its fluorescent substrate, significantly more MMP-19 enzyme (5 nM) was used for the inhibition constant assays. Quenched fluorescent peptides were added to a final concentration of 1 M (2 M for MMP-19) to initiate the assays. Association Rate Constant Measurement (kon)—kon measurements were performed by adding N-TIMP-1 mutants (up to 400 nM) to 0.05 nM. Where P is the product concentration, V0 is the initial velocity, Vs is the steady state velocity, and k is the apparent first order rate constant of equilibrium between enzyme and TIMP complex. Where P is the product concentration, V0 is the initial velocity, Vs is the steady state velocity, and k is the apparent first order rate constant of equilibrium between enzyme and TIMP complex. kon values were calculated by linear regression of k on TIMP concentrations
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.
