Potential Binding Partners of Tissue Inhibitor of Metalloproteinase-3

Abstract

Abstract: : Purpose: Tissue Inhibitor of Matrix Metalloproteinases-3 (TIMP-3) is a 24kDa protein synthesized by the retinal pigment epithelium and present in Bruch’s membrane. TIMP-3 can inhibit matrix metalloproteinases (MMPs) as well as ADAMs (a-disintegrin and a metalloproteinases domain) and ADAMTSs (ADAM with thrombospondin-like repeats) within the adamalysin family. Mutations of certain cysteine residues to serine result in deposition of excessive amounts of TIMP-3 in Bruch’s membrane, producing early blindness in patients with Sorsby fundus dystrophy. In this study, we proposed to identify potential binding partners of TIMP-3 in the retina. Methods: cDNA inserts of a primary human fetal RPE library were transformed into the yeast reporter strain, AH109, along with a second plasmid for in vivo recombination to generate cDNA-GAL4 activation domain fusion constructs targeted to the nucleus. The complete coding sequence, or the N- or C-terminal domain of human wild type TIMP-3 was used to transform AH109 to generate TIMP-3-GAL4 binding domain fusion constructs. Growth on nutritionally deficient plates denoted an interaction. Sequencing of the clones followed by BLAST searches allowed identification of potential binding partners. Co-transformation and mating experiments confirmed the interactions and ruled out false positives. Results: We have identified 16 clones that appear to be good candidates for putative binding partners of TIMP-3. Binding region has also been determined by using truncated TIMP-3 protein as bait. Some interactions have been confirmed by co-immunoprecipitation experiments in eukaryotic cells systems. Conclusion: Detailed analysis of the interactions of TIMP-3 and potential binding partners will contribute significantly to the understanding of the molecular function of TIMP-3.

Keywords: extracellular matrix • protein structure/function • molecular biology