the naturally occurring inhibitory activities of the matrix metalloproteinases ( mmps ) were initially identified in many cell and tissue culture studies , carried out over several decades . between 1985 and 1996 , however , four members of the tissue inhibitor of metalloproteinases ( timp ) family were definitively identified at the gene level in mammals . in fact , orthologs of the timps are widely distributed across the animal kingdom and have now been identified in species as widely separated as trichoplax , hydra , molluscs , worms and insects , as well as in vertebrates such as fish and birds . timp1 was originally cloned in 1985 when it was found to have an erythroid potentiating activity   and to be an inhibitor of metalloproteinases . these proteins act as significant regulators of the activities of mmps and , in some instances , of other metalloendopeptidases of the metzincin clan , namely the disintegrin metalloproteinases ( adam ) and the disintegrin metalloproteinases with thrombospondin motifs ( adamts ) . their importance in modulating the ability of a cell to control its extracellular environment , from the remodeling of the extracellular matrix to the interaction of cells via adhesion and signaling molecules such as growth factors has long been appreciated , but the significance of timps as both proteinase inhibitors and signaling molecules in their own right is only just beginning to be documented . the four mammalian timps are thought to be products of gene duplication because there is a single gene in insects , but orthologs of all four proteins are not found in all vertebrates . the timp proteins share a similar domain structure , composed of an amino - terminal domain and a carboxy - terminal sub - domain . timp1 and timp3 seem to have originated earlier than timp2 and timp4 , with timp1 having undergone the least evolutionary change ( figure 1 ) . the amino - terminal domain of human timp3 is more closely related to that of the drosophila melanogaster timp in terms of sequence , isoelectric point , and functional properties , such as the inhibition of mt1-mmp and adam17 . hence , it has been suggested that timp3 might have more of the preserved functions of the ancestral protein than do the other human timps . invertebrate timps differ more markedly in sequence than vertebrate timps , having differing disulfide bond arrangements , but they maintain the n- and c - domain structure of vertebrate timps . nematode timps , exceptionally , are single - domain proteins with similarity to the ( inhibitory ) n - domain of mammalian timps . the human genes timp1 ( chromosome xp11.3 - 11.23 ) , timp3 ( chromosome 22q12.3 ) and timp4 ( chromosome 3p25 ) are each nested within an intron of human synapsin genes , but with reverse directionality ( that is , reading 3 ' and 5 ' ) ( figure 2 ) . synapsins comprise a multigene family of phosphoproteins that are neuron specific and are the most abundant protein of the synaptic vesicle . phylogenetic analysis shows that the synapsin - timp gene nesting relationship began as far back as drosophila , suggesting a strong linkage between these two gene families , although the significance of this association is not known . by contrast , the human timp2 gene ( chromosome 17q25 ) hosts the gene differential display clone 8 ( ddc8 ) within its first intron ( figure 2 ) and there is evidence of alternative splicing between the two genes . a phylogenetic tree showing the inferred evolutionary relationships between the vertebrate timps , rooted by the timp from drosophila melanogaster . reproduced from   with permission from elsevier . 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 . ensembl ids are : syn i - ensg00000008056 ; syn ii - ensg00000157152 ; syn iii - ensg00000185666 ; and timp2-ensg00000035862 . the human timps comprise 184 to 194 amino acids that form an n - domain and a c - sub - domain that are stabilized by six disulfide bonds . the timps are about 40% identical and have overlapping abilities to inhibit individual mmps ( table 1 ) . their amnio - terminal domain is the inhibitory domain and it binds to the active site of the mmps . mmps do not form covalent bonds with timps , neither do they cleave timps , but they do form tight 1:1 complexes with timps with inhibition constants in the sub - nanomolar range . prommp2 and prommp9 zymogens of mmps are secreted latent forms of the mmp in which a cleavable propeptide is inserted into the catalytic cleft ; this propeptide must be removed proteolytically to allow activity . these pro - enzymes bind to timps in an interaction between the carboxy - terminal non - inhibitory domain of the timp and the carboxy - terminal hemopexin domain of the prommp . these complexes act as mmp inhibitors because the amino - terminal inhibitory domain of timps is not blocked . the n - domain has a characteristic ' oligonucleotide and oligosaccharide ( ob ) ' fold , composed of five beta strands arranged in a twisted  barrel and three  helices ( figure 3a ) . the ability of these proteins to inhibit the mmps is largely due to the interaction of a wedge - shaped ridge on the n - domain , which binds within the active - site cleft of the target mmp ( figure 3b ) . the c - domain has two parallel  strands that are connected by an  helix to two anti - parallel  strands . this structure provides the ability of timps to interact with the hemopexin domain of some mmps , including the pro - forms of some mmps , and could have a stabilizing role . these do not appear to play a role in the proteins ' inhibitory activities but are important for interactions with the cellular environment . notably , timp3 is incorporated into the extracellular matrix of tissues by charge interactions with sulfated glycosaminoglycans and could be classified as a ' matricellular ' protein . properties of human timps , summarizing their activities in inhibiting metzincin metalloproteinases and in modulating phenotypic cell behavior   angiir , angiotensin ii receptor ; efemp1 , egf - containing fibulin - like extracellular matrix protein 1 ; vegfr , vascular endothelial growth factor receptor the mode of interaction of timp1 and the catalytic domain of mmp3 . ( a ) ribbon diagram of the complex involving timp1 ( red ) and mmp3 ( blue ) . zinc and this image was prepared from the brookhaven protein data bank entry ( 1uea ) using pymol . ( b ) schematic representation of the amino - terminal ridge of timp1 , which interacts with the active site of the mmp . reproduced from   with permission from springer science+business media . the -amino and carbonyl groups of the amino - terminal cys1 of the human timps have been shown in crystallographic analyses to chelate the znof the enzyme active site ; the oh group of ser / thr2 interacts with the nucleophilic glu of the mmp catalytic cleft ( figure 3b ) . the latter interaction displaces a water molecule that is essential for peptide hydrolysis from the active site . timp1 , timp2 , timp3 and timp4 all inhibit all of the mmps tested , but timp1 is a poor inhibitor of mt1-mmp , mt3-mmp , mt5-mmp and mmp19   ( table 1 ) . timp2 is unique in that , in addition to inhibiting mmp activity , it selectively interacts with mt1-mmp to facilitate the cell - surface activation of prommp2 . recent studies have shown that timps can inhibit some of the disintegrin metalloproteinases ( adam and adamts ) . timp3 has a much broader inhibition profile , including adam10 , adam12 , adam17   and several adamtss ( adamts1 , adamts2 , adamts4 and adamts5 ) [ 12 - 14 ] . the crystal structures of a number of timp - metalloproteinase complexes have shown that the mode of inhibition described above does not vary . the timps show tissue - specific , constitutive , or inducible expression , which is regulated at the transcriptional level by various cytokines and growth factors . timp1 is widely expressed in many mammalian tissues , notably in the reproductive organs . in the central nervous system , timp1 expression is restricted to regions of persistent neuronal plasticity , such as the hippocampus , olfactory bulb , and cerebellum . timp2 is constitutively expressed in most tissues but is not inducible by common growth factors . timp3 is expressed in many tissues and is often found as a significant matrix protein in the basement membranes of the eye and kidney . the relatively restricted tissue distribution of timp4 ( in the heart , kidney , ovary , pancreas , colon , testes , brain and adipose tissue ) suggests that it plays a role in tissue - specific physiological functions , but the role of this protein is relatively understudied . the roles of timps in both normal physiology and in pathological processes have been investigated by gene ablation studies in mice , and these have indicated that there is some functional redundancy . timp1-null mice do not exhibit many inherent abnormalities , apart from alterations in the reproductive cycle and impaired learning and memory , unless the animals are challenged pathologically . timp2-null mice have neurological and motor - function abnormalities , but again they only manifest phenotypes that are associated with matrix remodeling upon challenge ( reviewed in ) . timp3 ablation causes lung emphysema - like alveolar damage   and faster apoptosis of mammary epithelial cells after weaning . furthermore , timp3 plays a key role in innate immunity by regulating the processing of tumor necrosis factor  by adam17 . the individual timp genes appear to be regulated rather differently : timp1 and timp3 are upregulated in vitro in response to phorbol myrsistate acetate and transforming growth factor  , whereas timp2 is downregulated . the timp1 , timp2 and timp3 promoters have additional common elements , such as ap1 and pea3 , that are also present in several mmp promoters . otherwise , there is little organizational conservation of the common motifs in the timp promoters . like many ' housekeeping genes ' - that is , genes that are always expressed because they encode proteins that are constantly required - timp1 , timp2 and timp4 lack obvious tata boxes . timp2 , which has a tata box and multiple transcription start sites , contains mef2 and nf - il6 binding sites in its promoter . the timp3 promoter uniquely has binding sites for the transcription factors nf-b , c - myc and p53 . timp3 has a promoter - proximal gc - rich region that mediates its basal expression , and regions further upstream that mediate serum induction . timp3 is notably regulated by gene methylation , which can lead to its downregulation in many cancer cells . the timp4 promoter has no tata box but does contain an initiator sequence and several transcription start points . timp4 also contains a myogenin - binding site , which is likely to be important for its skeletal muscle - specific expression , and an ap4 transcription factor binding site . timp - mediated inhibition of mmp activity is an important determinant of cell function , but the concept of mmp - independent timp regulation of cell behavior is now supported by the identification of both specific cell - binding partners for timp family members and specific signaling events that involve these proteins . cell signaling mechanisms mediated by timp1 and timp2 have been shown to promote cell proliferation , although the receptor - mediated events that are involved are not well understood . timp1 has anti - apoptotic effects on some cell types , possibly acting by enhancing the activities of survival and differentiation factors in a signaling network that is mediated by the tetraspanin cd63 and by focal adhesion kinase , phosphoinositide 3-kinase and erk . timp2 binds to 31 integrin , leading to g1 phase growth arrest and enhanced de novo expression of the cyclin - dependent kinase inhibitor p27kip1 . , it appears to promote the development of a transformed phenotype : timp3 promotes apoptosis in several tumor cell lines and in smooth muscle cells , but this appears to involve the modulation of metalloproteinase activities . work with timp3-null mice suggests that timp3 can either promote or inhibit apoptosis depending on the model system examined . timp3 can be sequestered in the extracellular matrix in some tissues , but secreted timp3 can be rapidly endocytosed by cells such as chondrocytes through a mechanism that involves the ldl - receptor - related protein ( lrp ) . knowledge of the precise functions and spatio - temporal expression patterns of metalloproteinases are still somewhat limited . in vitro , the metalloproteinases have markedly overlapping substrate specificities and their in vivo functions will take some time to elucidate in detail ( reviewed in ) . studies of function ( for example , using transgenic mouse models ) should be coupled with studies of the metalloproteinases ' localization in tissues relative to the physiological or pathological status of those tissues . nevertheless , gene - ablation studies in mice have indicated some key roles for timps , with timp3 clearly being of some importance in both extracellular matrix remodeling and in inflammation . timp3 seems to function largely as a metalloproteinase inhibitor , but other timps directly mediate signaling pathways that lead to the regulation of growth , apoptosis and other activities . timp1 concentrations are increased in cancer patients , particularly in those with breast or colorectal carcinomas , and this increase is negatively associated with patient outcome . studies have shown the clinical utility of timp1 as a biomarker and independent prognostic factor in breast , colorectal , and several hematological cancers . there has been a growing awareness of the activity of timps as modulators of nervous system physiology and pathology , and further elucidation of their roles alongside the activities of the metalloproteinases will be of interest in this context . various polymorphisms of all the timp genes have been associated with predisposition to many different cancers . the function of timps in lower organisms is not so well elucidated and might provide fascinating insights into the roles of metalloproteases in development . although several structures of timp - proteinase complexes have been solved , there are outstanding questions as to the importance of the flexibilities of both inhibitor and enzyme during binding . this could be key to future engineering of more specific forms of timps as potential therapeutic agents . the regulation of timp function by extracellular interaction sites and by endocytosis is still to be studied fully . the role of timps in the regulation of proteinase clearance ( for example , by endocytosis ) is also not well understood . the mmp - independent , cytokine - like functions of the timps are now well recognized , and future work will involve the identification of cell surface receptors and the elucidation of downstream signaling . the pleiotropic activities of the timp family members are complex and depend upon subtle interactions with other extracellular components , as well as on direct interactions with cell - binding partners . understanding these processes and how they are modulated during disease progression will be helpful to our understanding of disease pathology . timps have rarely been considered as the basis for novel therapeutic interventions , but their use in model systems gives vital clues about the efficacy of metalloproteinase inhibitors in the abrogation of disease . adam : a disintegrin metalloproteinase ; adamts : a disintegrin metalloproteinase with thrombospondin motif ; ddc8 : differential display clone 8 ; mmp : matrix metalloproteinase ; timp : tissue inhibitor of metalloproteinases . thanks to hideaki nagase , keith brew and ian clark for invaluable advice on the manuscript , and to my ' timp ' colleagues for their hard work over the years . timp studies in the author 's laboratory have been supported by arthritis research uk , the biotechnology and biological sciences research council ( bbsrc ) , the medical research council ( mrc ) , the wellcome trust and cancer research uk .
orchestration of the growth and remodeling of tissues and responses of cells to their extracellular environment is mediated by metalloproteinases of the metzincin clan . 
 this group of proteins comprises several families of endopeptidases in which a zinc atom is liganded at the catalytic site to three histidine residues and an invariant methionine residue . 
 tissue inhibitors of metalloproteinases ( timps ) are endogenous protein regulators of the matrix metalloproteinase ( mmps ) family , and also of families such as the disintegrin metalloproteinases ( adam and adamts ) . 
 timps therefore have a pivotal role in determining the influence of the extracellular matrix , of cell adhesion molecules , and of many cytokines , chemokines and growth factors on cell phenotype . 
 the timp family is an ancient one , with a single representative in lower eukaryotes and four members in mammals . 
 although much is known about their mechanism of action in proteinase regulation in mammalian cells , less is known about their functions in lower organisms . 
 recently , non - inhibitory functions of timps have been identified in mammalian cells , including signaling roles downstream of specific receptors . 
 there are clearly still questions to be answered with regard to their overall roles in biology .