Murphy Genome Biology 2011, 12:233 http://genomebiology.com/2011/12/11/233 PROTEIN FAMILY REVIEW Tissue inhibitors of metalloproteinases Gillian Murphy* now been identified in species as widely separated as Abstract Trichoplax, Hydra, molluscs, worms and insects, as well Orchestration of the growth and remodeling of as in vertebrates such as fish and birds. Plants do have tissues and responses of cells to their extracellular metzincins, but no plant TIMP ortholog has been environment is mediated by metalloproteinases of the identified. Metzincin clan. This group of proteins comprises several TIMP1 was originally cloned in 1985 when it was found families of endopeptidases in which a zinc atom is to have an erythroid potentiating activity [1] and to be an liganded at the catalytic site to three histidine residues inhibitor of metalloproteinases [2]. TIMP2 was cloned in and an invariant methionine residue. Tissue inhibitors 1990 by Stetler-Stevenson et al. [3], TIMP3 by Pavloff and of metalloproteinases (TIMPs) are endogenous protein colleagues in 1992 [4], and TIMP4 in 1996 [5]. ese regulators of the matrix metalloproteinase (MMPs) proteins act as significant regulators of the activities of family, and also of families such as the disintegrin MMPs and, in some instances, of other metalloendo- metalloproteinases (ADAM and ADAMTS). TIMPs peptidases of the metzincin clan, namely the disintegrin therefore have a pivotal role in determining the metalloproteinases (ADAM) and the disintegrin metallo- inuence of the extracellular matrix, of cell adhesion proteinases with thrombospondin motifs (ADAMTS). molecules, and of many cytokines, chemokines and TIMPs inhibit with a 1:1 molar stoichiometry. eir growth factors on cell phenotype. The TIMP family is importance in modulating the ability of a cell to control an ancient one, with a single representative in lower its extracellular environment, from the remodeling of the eukaryotes and four members in mammals. Although extracellular matrix to the interaction of cells via adhesion much is known about their mechanism of action and signaling molecules such as growth factors has long in proteinase regulation in mammalian cells, less is been appreciated [6], but the significance of TIMPs as known about their functions in lower organisms. both proteinase inhibitors and signaling molecules in their Recently, non-inhibitory functions of TIMPs have been own right is only just beginning to be documented [7]. identied in mammalian cells, including signaling roles e four mammalian TIMPs are thought to be products downstream of specic receptors. There are clearly still of gene duplication because there is a single gene in questions to be answered with regard to their overall insects, but orthologs of all four proteins are not found in roles in biology. all vertebrates. e TIMP proteins share a similar domain structure, composed of an amino-terminal domain and a carboxy-terminal sub-domain. TIMP1 and TIMP3 seem Gene organization and evolutionary history to have originated earlier than TIMP2 and TIMP4, with e naturally occurring inhibitory activities of the matrix TIMP1 having undergone the least evolutionary change metalloproteinases (MMPs) were initially identified in (Figure 1). e amino-terminal domain of human TIMP3 many cell and tissue culture studies, carried out over is more closely related to that of the Drosophila melano- several decades. Between 1985 and 1996, however, four gaster TIMP in terms of sequence, isoelectric point, and members of the tissue inhibitor of metalloproteinases functional properties, such as the inhibition of MT1- (TIMP) family were definitively identified at the gene MMP and ADAM17. Hence, it has been suggested that level in mammals. In fact, orthologs of the TIMPs are TIMP3 might have more of the preserved functions of widely distributed across the animal kingdom and have the ancestral protein than do the other human TIMPs. Invertebrate TIMPs differ more markedly in sequence than vertebrate TIMPs, having differing disulfide bond *Correspondence: [email protected] Department of Oncology, University of Cambridge, Cancer Research UK arrangements [6], but they maintain the N- and C- Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge domain structure of vertebrate TIMPs. Nematode TIMPs, CB2 0RE, UK exceptionally, are single-domain proteins with similarity to the (inhibitory) N-domain of mammalian TIMPs. e © 2010 BioMed Central Ltd © 2011 BioMed Central Ltd human genes TIMP1 (chromosome Xp11.3-11.23), Murphy Genome Biology 2011, 12:233 Page 2 of 7 http://genomebiology.com/2011/12/11/233 T1_mo T1_dog T1_bov T1_pig T1_rab T1_hu T3_medaka T3_fugu T3_tetraodon T3_Xenl T3_mo T3_hu T3_shark T3_chic T2_bream T2_trout T2_zebraf T2_chick T2_xenl T2_bov T2_rab T2_mo T2_hu T2_dog T4_hu T4_bov T4_rat T4_mo Drosophila 0.7 Figure 1. A phylogenetic tree showing the inferred evolutionary relationships between the vertebrate TIMPs, rooted by the TIMP from Drosophila melanogaster. Reproduced from [6] with permission from Elsevier. TIMP, tissue inhibitors of metalloproteinases. TIMP3 (chromosome 22q12.3) and TIMP4 (chromosome that the Synapsin-TIMP gene nesting relationship began 3p25) are each nested within an intron of human synap­ as far back as Drosophila, suggesting a strong linkage sin genes, but with reverse directionality (that is, reading between these two gene families, although the signi­ 3’ and 5’) (Figure 2). Synapsins comprise a multigene ficance of this association is not known. By contrast, the family of phosphoproteins that are neuron specific and human TIMP2 gene (chromosome 17q25) hosts the gene are the most abundant protein of the synaptic vesicle. Differential display clone 8 (DDC8) within its first intron They are proposed to tether synaptic vesicles and regulate (Figure 2) and there is evidence of alternative splicing neurotransmitter release. Phylogenetic analysis shows between the two genes. Murphy Genome Biology 2011, 12:233 Page 3 of 7 http://genomebiology.com/2011/12/11/233 SYN1 1 2 3 4 5 6 7 8 9 10 11 12 13 5´ 502 58 92 157 90 63 143 75 103 147 88 589 1096 3´ 269 125 127 80 129 162 TIMP1 3´ 5´ 6 5 4 3 2 1 SYN2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 5´ 202 135 58 92 157 90 63 143 75 103 150 61 244 918 3´ 3´ 662 125 115 98 650 5´ TIMP4 5 4 3 2 1 SYN3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 5´ 81 473 58 92 160 90 63 143 75 103 135 88 292 1273 3´ 3´ 3864 122 112 83 422 3´ 5 4 3 2 1 TIMP3 1 2 3 4 5 TIMP2 5´ 429 101 109 125 2888 3´ 5´ 68 142 1880 3´ 1 2 3 DDC8 Figure 2. Genomic structures of human TIMP genes. The TIMP1, TIMP4 and TIMP3 genes are embedded in reverse orientation (3’ to 5’) within the synapsin genes SYN1, SYN2 and SYN3, respectively. The TIMP2 gene has the Differential display clone 8 (DDC8) gene nested in its first intron. Exon sizes in number of bases are as indicated [15,22]. Ensembl IDs are: SYN I-ENSG00000008056; SYN II-ENSG00000157152; SYN III-ENSG00000185666; and TIMP2-ENSG00000035862. TIMP, tissue inhibitors of metalloproteinases. Characteristic structural features and mechanism could have a stabilizing role. Human TIMP1 and TIMP3 The human TIMPs comprise 184 to 194 amino acids that are glycoproteins that have complex sugar chains. These form an N-domain and a C-sub-domain that are stabilized do not appear to play a role in the proteins’ inhibitory by six disulfide bonds. The TIMPs are about 40% identical activities but are important for interactions with the and have overlapping abilities to inhibit individual MMPs cellular environment. Notably, TIMP3 is incorporated (Table 1). Their amnio-terminal domain is the inhibitory into the extracellular matrix of tissues by charge inter- domain and it binds to the active site of the MMPs. actions with sulfated glycosaminoglycans and could be MMPs do not form covalent bonds with TIMPs, neither classified as a ‘matricellular’ protein. do they cleave TIMPs, but they do form tight 1:1 The α-amino and carbonyl groups of the amino- complexes with TIMPs with inhibition constants in the terminal Cys1 of the human TIMPs have been shown in sub-nanomolar range. ProMMP2 and proMMP9 zymo- crystallographic analyses to chelate the Zn2+ of the gens of MMPs are secreted latent forms of the MMP in enzyme active site; the OH group of Ser/Thr2 interacts which a cleavable propeptide is inserted into the catalytic with the nucleophilic Glu of the MMP catalytic cleft cleft; this propeptide must be removed proteolytically to (Figure 3b). The latter interaction displaces a water allow activity. These pro-enzymes bind to TIMPs in an molecule that is essential for peptide hydrolysis from the interaction between the carboxy-terminal non-inhibitory active site. TIMP1, TIMP2, TIMP3 and TIMP4 all inhibit domain of the TIMP and the carboxy-terminal hemo- all of the MMPs tested, but TIMP1 is a poor inhibitor of pexin domain of the proMMP. These complexes act as MT1-MMP, MT3-MMP, MT5-MMP and MMP19 [6,8] MMP inhibitors because the amino-terminal inhibitory (Table 1). TIMP2 is unique in that, in addition to inhibit- domain of TIMPs is not blocked. The N-domain has a ing MMP activity, it selectively interacts with MT1-MMP characteristic ‘oligonucleotide and oligosaccharide (OB)’ to facilitate the cell-surface activation of proMMP2. fold, composed of five beta strands arranged in a twisted Recent studies have shown that TIMPs can inhibit some β barrel and three α helices (Figure 3a). The ability of of the disintegrin metalloproteinases (ADAM and these proteins to inhibit the MMPs is largely due to the ADAMTS). TIMP1 inhibits ADAM10 [9], whereas interaction of a wedge-shaped ridge on the N-domain, TIMP2 inhibits ADAM12 [10]. TIMP3 has a much which binds within the active-site cleft of the target broader inhibition profile, including ADAM10 [9], MMP (Figure 3b).