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Distinct Functions for the Catalytic and Hemopexin Domains of a Drosophila Matrix Metalloproteinase

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Distinct Functions for the Catalytic and Hemopexin Domains of a Drosophila Matrix Metalloproteinase Distinct functions for the catalytic and hemopexin domains of a Drosophila matrix metalloproteinase Bernadette M. Glasheen, Aashish T. Kabra1, and Andrea Page-McCaw2 Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180 Edited by Terry L. Orr-Weaver, Massachusetts Institute of Technology, Cambridge, MA, and approved December 29, 2008 (received for review April 29, 2008) Human matrix metalloproteinases (MMPs) are believed to contrib- Mmp1 allele Mmp1 predicted protein ute to tumor progression. Therapies based on inhibiting the cat- alytic domain of MMPs have been unsuccessful, but these studies wild-type ss pro catalytic hemopexin f1 raise the question of whether other MMP domains might be f2 appropriate targets. The genetic dissection of domain function has ss pro catalytic hemopexin been stymied in mouse because there are 24 related and partially ss pro catalytic hemopexin PC redundant MMP genes in the mouse genome. Here, we present a genetic dissection of the functions of the hemopexin and catalytic 2 (excision) ss pro domains of a canonical MMP in Drosophila melanogaster,an organism with only 2 MMPs that function nonredundantly. We Q112* (genetic null) ss pro catalytic compare the phenotypes of Mmp1 null alleles with alleles that have specific hemopexin domain lesions, and we also examine phenotypes of dominant-negative mutants. We find that, although Q273* (hemopexin deleted) ss pro catalytic the catalytic domain appears to be required for all MMP functions including extracellular matrix remodeling of the tracheal system, W439* ss pro catalytic hemopexin the hemopexin domain is required specifically for tissue invasion (hemopexin truncated) events later in metamorphosis but not for tracheal remodeling. Thus, we find that this MMP hemopexin domain has an apparent Mmp1 transgene Mmp1 predicted protein specialization for tissue invasion events, a finding with potential implications for inhibitor therapies. DN pro-pex ss pro hemopexin (dominant negative) cancer ͉ genetics ͉ tissue remodeling A DN E225A ss pro catalytic hemopexin (dominant negative) atrix metalloproteinases (MMPs) are a family of enzymes BIOLOGY Fig. 1. Predicted protein products of Mmp1 alleles and transgenes. (Upper) Mrequired for tissue remodeling, and their expression is Predicted products from alleles at the Mmp1 genomic locus. The wild-type DEVELOPMENTAL up-regulated in tumors and inflamed tissue. As proteases that protein has the typical domain structure of a secreted MMP, including a signal cleave extracellular matrix, MMPs have the potential to break sequence (ss), an inhibitory pro domain (pro), a catalytic domain (cat) (the down tissues, remove physical barriers, and liberate signaling catalytic core is shown as a v-shaped indentation), a flexible hinge domain molecules. All of these processes occur during tissue remodeling (shown as a black wavy line), and a hemopexin domain (pex) containing 4 and during tumor progression. MMP mutant phenotypes in mice hemopexin loops (each light gray). We have identified cDNAs for 2 splice forms and flies demonstrate that MMPs are required for tissue remod- of Mmp1 as shown, form 1 (f1, known as PD in Flybase) and form 2 (f2). Flybase eling (reviewed in ref. 1), and many lines of evidence implicate predicts an additional splice form PC, shown for completeness. The dashed line MMPs in promoting tumor progression including clinical data, over the catalytic domain denotes the recombinant fragment used for gen- erating anti-catalytic monoclonal antibodies. Allele 2, generated by P- mouse tumor studies, cell culture studies, and substrate analysis element excision (18), deletes most of the coding sequence. Q112*, Q273*, (reviewed in refs. 2 and 3). Thus, MMPs are considered potential and W439*, all recovered in an EMS mutagenesis screen (18), each contain a pharmaceutical targets for cancer therapies, although some nonsense mutation causing premature termination as shown. (Lower) Mmp1 MMPs may be important for inhibiting tumor progression transgenes with dominant-negative activity, used under UAS transcriptional (reviewed in ref. 3). control. DN Pro-pex is a deletion construct lacking the entire catalytic domain. In the 1990s, the pharmaceutical industry performed clinical DN E225A is a point mutant that ablates the conserved E225 at the catalytic trials to test several MMP inhibitors that had been effective in core, rendering the catalytic domain nonfunctional. preventing tumor progression in mouse (4–7). These compounds were designed to inhibit MMP catalysis at the active site (8). teinases) can reversibly occupy the active site of the catalytic Unfortunately, in patients MMP inhibitors caused musculoskel- domain and thus regulate its activity (10). The pro domain acts etal pain and inflammation, which decreased the tolerated dose as a negative regulator of catalytic activity by occupying the possibly below effective levels (9). From these studies, it can be concluded that the broad-spectrum inhibition of MMP catalysis is not a workable strategy for patient therapies as MMP catalysis Author contributions: A.P.-M. designed research; B.M.G., A.T.K., and A.P.-M. performed is required in normal physiology. Other domains may be more research; B.M.G., A.T.K., and A.P.-M. analyzed data; and A.P.-M. wrote the paper. appropriate inhibitor targets, and this possibility raises a ques- The authors declare no conflict of interest. tion about MMP structure/function: Do the domains participate This article is a PNAS Direct Submission. equally in different biological processes, or do some domains Freely available online through the PNAS open access option. participate in some processes and not others? 1Present address: Neuroscience Institute and Department of Neurology, Albany Medical MMPs contain 3 highly conserved domains: the pro, catalytic, College, Albany, NY 12205. and hemopexin domains (see Fig. 1). The catalytic domain 2To whom correspondence should be addressed. E-mail: [email protected]. mediates proteolysis of substrates and is often expressed in This article contains supporting information online at www.pnas.org/cgi/content/full/ isolation for in vitro proteolysis assays. Endogenous MMP 0804171106/DCSupplemental. protein inhibitors called TIMPs (tissue inhibitors of metallopro- © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804171106 PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2659–2664 Downloaded by guest on September 26, 2021 active site; it is removed for enzyme activation. The hemopexin animals, Table 1), these were much lower than levels in null domain, which is connected to the catalytic domain by a flexible animals (Ϸ45%), and we did not observe the taut stretched hinge, is a beta-propeller structure comprised of 4 repeating dorsal trunks typical of the null animals. It is likely that the loops, each of which is homologous to the blood protein he- tracheal breaks in the hemopexin mutant larvae are caused by mopexin. This domain is believed to mediate protein–protein the overall reduction in Mmp1 protein (see below) rather than interactions and to contribute to substrate specificity (11–15). by the lack of the hemopexin domain. Animals mutant for either One method of assessing the functions of different domains is hemopexin allele Q273*orW439* frequently survived to meta- through genetic analysis of mutants. However, a complication of morphosis, when they died with body malformations associated MMP genetic analysis in mouse models is that there are 24 MMP with a failure of disc eversion (Table 1 and Fig. 3). Transhet- genes that exhibit partial redundancy (reviewed in ref. 1). In erozygotes of the genotype Q273*/W439* also had normal larval contrast, the fruitfly Drosophila melanogaster has only 2 MMP dorsal trunks with infrequent breaks, survived to pupariation, genes, Mmp1 and Mmp2, and each has the conserved domain and sometimes failed to evert imaginal discs during metamor- structure typical of mammalian MMPs (16–18). Each Drosophila phosis. Early in disc eversion, imaginal disc peripodial and stalk MMP is required for viability, participating in different aspects cells traverse 2 layers of basement membrane to invade the larval of postembryonic tissue remodeling. Mmp1 is required for larval body wall (23). Srivastava et al. have demonstrated that in tracheal elongation and for tissue invasion during disc eversion animals compromised for Mmp1 function, the normal invasion of during metamorphosis (18, 19); Mmp2 is required for histolysis basement membranes by disc epithelia fails, leading to failures of and epithelial fusion during metamorphosis, and it is not re- disc eversion (19). The observation that both Mmp1 hemopexin quired in the larval tracheal system (18). Interestingly, both mutants fail to evert discs indicates that the hemopexin domain Drosophila MMPs have been demonstrated by 3 groups to be is required selectively for developmental tissue invasion but not required for tumor invasion using 2 different Drosophila tumor for remodeling the tracheal tubes (Table 1). models (19–22), suggesting a conservation of pathological and To understand these alleles better, we examined Mmp1 wild- physiological MMP function in Drosophila and humans. Here, we type and mutant protein mobility and expression levels by analyze the phenotypes of Mmp1 mutants disrupted for the Western blot analysis with anti-Mmp1-catalytic-domain mono- hemopexin domain and compare them to Mmp1 null mutants. clonal antibodies
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