HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Caterina, N. C. M. Articles by Engler, J. A. Search for Related Content PubMed PubMed Citation Articles by Caterina, N. C. M. Articles by Engler, J. A. Social Bookmarking                      What's this?

Volume 272, Number 51, Issue of December 19, 1997 pp. 32141-32149

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

(Received for publication, June 12, 1997, and in revised form, September 30, 1997)

Nancy C. M. Caterina , L. Jack Windsor ^§¶ , Audra E. Yermovsky ^¶ , M. Kirby Bodden , Kenneth B. Taylor ^¶ , Henning Birkedal-Hansen and Jeffrey A. Engler ^§¶

From the Departments of ^¶ Biochemistry and Molecular Genetics, ^§ Oral Biology, and  Restorative Dentistry and the Research Center for Oral Biology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the  National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

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  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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S O Wurtz, A-S Schrohl, N M. Sorensen, U Lademann, I J Christensen, H Mouridsen, and N Brunner Tissue inhibitor of metalloproteinases-1 in breast cancer Endocr. Relat. Cancer, June 1, 2005; 12(2): 215 - 227. [Abstract] [Full Text] [PDF]

				L. Perez-Martinez and D. M. Jaworski Tissue Inhibitor of Metalloproteinase-2 Promotes Neuronal Differentiation by Acting as an Anti-Mitogenic Signal J. Neurosci., May 18, 2005; 25(20): 4917 - 4929. [Abstract] [Full Text] [PDF]

				D. L. Steele, O. El-Kabbani, P. Dunten, L.J. Windsor, R.U. Kammlott, R.L. Crowther, C. Michoud, J. A. Engler, and J.J. Birktoft Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1 Protein Eng. Des. Sel., June 1, 2000; 13(6): 397 - 405. [Abstract] [Full Text] [PDF]

				J. D. Mott, C. L. Thomas, M. T. Rosenbach, K. Takahara, D. S. Greenspan, and M. J. Banda Post-translational Proteolytic Processing of Procollagen C-terminal Proteinase Enhancer Releases a Metalloproteinase Inhibitor J. Biol. Chem., January 14, 2000; 275(2): 1384 - 1390. [Abstract] [Full Text] [PDF]

				A. E. Yermovsky-Kammerer and S. L. Hajduk In Vitro Import of a Nuclearly Encoded tRNA into the Mitochondrion of Trypanosoma brucei Mol. Cell. Biol., September 1, 1999; 19(9): 6253 - 6259. [Abstract] [Full Text] [PDF]

				J. J. Caterina, S. Yamada, N. C. M. Caterina, G. Longenecker, K. Holmback, J. Shi, A. E. Yermovsky, J. A. Engler, and H. Birkedal-Hansen Inactivating Mutation of the Mouse Tissue Inhibitor of Metalloproteinases-2(Timp-2) Gene Alters ProMMP-2 Activation J. Biol. Chem., August 18, 2000; 275(34): 26416 - 26422. [Abstract] [Full Text] [PDF]

				M. Bond, G. Murphy, M. R Bennett, A. Amour, V. Knauper, A. C. Newby, and A. H. Baker Localization of the Death Domain of Tissue Inhibitor of Metalloproteinase-3 to the N Terminus. METALLOPROTEINASE INHIBITION IS ASSOCIATED WITH PROAPOPTOTIC ACTIVITY J. Biol. Chem., December 22, 2000; 275(52): 41358 - 41363. [Abstract] [Full Text] [PDF]