Replacement of Conserved Cysteines in Human Tissue Inhibitor of Metalloproteinases-1

Abstract

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP-1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/ C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity against M r 72,000 gelatinase (M r 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC 3 CQQC retained their ability to inhibit FIB-CL in a manner similar to wild-type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site "parent" mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.

Localization of the Death Domain of Tissue Inhibitor of Metalloproteinase-3 to the N Terminus

“…We constructed a recombinant TIMP-3 protein (TIMP-3 Cys1-Ser ) that is devoid of metalloproteinaseinhibitory activity by mutating the cysteine1 residue to a serine. An analogous mutation in TIMP-1 completely abrogates metalloproteinase-inhibitory activity without affecting protein conformation (36). We used a recombinant adenovirus to overexpress the TIMP-3 Cys1-Ser mutant in rat SMC and HeLa and compared its proapoptotic activity with a previously characterized recombinant adenovirus carrying the wild type TIMP-3 gene (RAd:wtTIMP-3) or a control adenovirus (RAd:66) (19,20).…”

“…In wild type TIMP-3 the N-terminal cysteine residue is a key to the TIMP-3-inhibitory mechanism, because it bidentally coordinates the Zn 2ϩ ion in the active site of metalloproteinases through the ␣-amino group and the peptide carbonyl group (41). An analogous TIMP-1 mutant completely lacks inhibitory activity toward MMP-1 and MMP-2, while maintaining structural integrity (36). Likewise, we demonstrated that the Cys 1 3 Ser mutation of TIMP-3 results in loss of inhibitory activity toward MMP-2.…”

Tissue Inhibitor of Metalloproteinase-2 Promotes Neuronal Differentiation by Acting as an Anti-Mitogenic Signal

“…To determine whether this function requires MMP inhibitory activity, a TIMP-2 mutant unable to inhibit MMPs was generated. Substitution of cysteine 72 to serine (TIMP-2 C72S ) completely abrogates MMP inhibitory activity without affecting protein conformation and MMP-independent functions (Caterina et al, 1997;Bond et al, 2000). Similar to TIMP-2, expression of the mutant TIMP-2 C72S resulted in a pronounced reduction in the number of BrdU-positive cells (vector, 52 Ϯ 6.4%; TIMP-2 C72S , 9 Ϯ 2%).…”

Inactivating Mutation of the Mouse Tissue Inhibitor of Metalloproteinases-2(Timp-2) Gene Alters ProMMP-2 Activation

“…(i) The deleted amino acid sequence (residues18 -89) contains two of the major sites of interaction of TIMPs with active MMPs (6,23) (Fig. 6A), and (ii) manipulation of the corresponding region of TIMP-1 around and including Cys-70 (Cys-72 in TIMP-2) either by mutagenesis of Cys-70 (24,25) or by proteolytic cleavage at Val-68 (26) results in ablation of inhibitory activity. The construct potentially leaves intact the first 18 residues including the NH 2 -terminal sequence, which fits into active site cleft of MMPs.…”