Metalloproteinases in Extracellular Matrix Breakdown
LAB MEMBERS: Ngee Han Lim, Ph.D Postdoctoral Fellow
Keittisak Suwan, Ph.D. Postdoctoral Fellow
Linda Troeberg, Ph.D. Postdoctoral Fellow
Robert Visse, Ph.D. Postdoctoral Fellow
Kazuhiro Yamamoto, Ph.D. Postdoctoral Fellow
Alan Lyons Senior Technician
Salvatore Santamaria Ph.D. Student
Simone Scilabra Ph.D. Student
The objectives of the group are to investigate biological and pathological functions of metalloproteinases that participate in degradation of extracellular matrix and to elucidate molecular mechanisms that regulate their enzymatic activities. These enzymes play important roles in a diverse array of biological processes such as embryonic development, organ morphogenesis, cell migration, differentiation and apoptosis, and are diseases such as arthritis, cancer, atherosclerosis, tissue ulcerations, neurodegenerative disorders and fibrosis. Our current research focuses on the mechanisms that regulate activities of collagenases and aggrecanases, proteinases that play major roles in cartilage matrix breakdown during progression of various types of arthritides, and on the structural and functional studies of endogenous metalloproteinase inhibitors, tissue inhibitors of metalloproteinases (TIMPs).
Mechanisms of action of collagenases on triple helical collagens
Collagens are the major structural proteins of all connective tissues and basement membranes. Interstitial fibrilar collagen types I, II and III are most abundant, and they consist of three α chains in a triple-helical conformation. Breakdown of these fibrilar collagens results in irreversible tissue damage such as seen in arthritic cartilage and rupture of aneurysmal aortic walls. The triple-helical structure of interstitial collagens is highly resistant to most proteinases, but is cleaved by collagenases at a single site approximately ¾ away from the N-terminus of the 300-nm long molecule (triple helicase activity). Our main objective is to understand how collagenase express the triple helicase activity.
Typical vertebrate collagenases are matrix metalloproteinases (MMPs), e.g., MMP-1, MMP-8 and MMP-13. They consist of an N-terminal propeptide, a catalytic domain, a hinge region and a C-terminal hemopexin-like domain. The catalytic domain of collagenases can hydrolyse non-collagenous proteins or peptides, but if fails to cleave triple helical interstitial collagens. Using human collagenase 1 (MMP-1), we demonstrated the ability of MMP-1 to unwind triple-helical collagen using a catalytically inactive, but correctly folded, full-length MMP-1 (E200A) mutant. In the presence of this mutant, the catalytic domain of MMP-1 or MMP-3 exhibits collagenolytic activity. Similarly, leukocyte elastase, which does not have collagenolytic activity, cleaved type I collagen in the presence of MMP-1(E200A). These results indicate that biding of MMP-1(E200A) to triple helical collagen locally unwinds the triple helices and allows other non-collagenolytic proteinase to cleave this region. We are currently investigating the MMP-1-collagen interaction using techniques such as deuterium/hydrogen exchange-mass spectrometry, mutagenesis, and x-ray crystallography with synthetic triple helical peptides.
Aggrecanases in cartilage destruction
Degradation of articular cartilage seriously impairs the function of joints and it is the hallmark of various types of arthritides. The primary cause of this process is due to the elevated proteolytic enzyme activities that degrade aggrecan proteoglycan and collagens. The loss of aggrecan is an early and crucial event in the progression of arthritic diseases, and it is mainly due to elevated levels of metalloproteinase activities that cleave the core protein of aggrecan. Two major aggrecan-degrading proteinases (aggrecanases) are ADAMTS-4 and ADAMTS-5, zinc metalloproteinases belonging to the Adamalysin family with thrombospondin (TS) type I motif.
They are multidomain proteinases consisting of a relatively large propeptide domain, a catalytic domain and a series of non-catalytic domains including a disintegrin, a thrombospondin, a cysteine rich, and a spacer domains. Our structural and functional studies indicated that ADAMTS-5 is about 30-fold more active on aggrecan than ADAMTS-4, and the cysteine-rich and the spacer domains play important role in their aggrecanolytic activity. We found that calcium pentosan polysulfate (CaPPS), a chemically sulphated xylanopyranose from beechwood, binds to these domains and block the aggrecanolytic activity of ADAMTS-4 and ADAMTS-5. In addition, CaPPS increases the level of tissue inhibitors or metalloproteinases-3 (TIMP-3), an endogenous inhibitor of aggrecanases in cartilage. This is due to blocking the endocytosis of TIMP-3 mediated by low-density lipoprotein receptor-related protein (LRP-1). TIMP-3, when bound to CaPPS, increases its affinity to ADAMTS-4 and -5 more than 100-fold. We are currently investigating the mechanism by which CaPPS increases the affinity between TIMP-3 and aggrecanases, and the efficacy of the TIMP-3-CaPPS complex to prevent cartilage breakdown in animal modes of osteoarthritis (OA).
TIMP-3 and its variants as potential cartilage protective agents.
TIMPs are the major endogenous regulators of MMPs and there are four TIMPs (TIMPs -1 to -4) in mammals. Among the four human TIMPs, TIMP-3 is unique for its ability to inhibit ADAM (e.g. TACE/ADAM17) and ADAMTS (e.g. aggrecanases) metalloproteinases as well as MMPs. We showed that recombinant N-TIMP-3 (N-terminal inhibitory domain of TIMP-3) blocks IL-1-stimulated cartilage breakdown in culture. We fortuitously have generated TIMP-3 mutants [(-1Ala) N-TIMP-3 with an extra alanine at the N-terminus and N-TIMP-3(T2G)] that inhibit TACE but not MMPs. They also inhibit ADAMTS-4 and ADAMTS-5 and block aggrecan degradation in cartilage explant experiments with a similar potency as the wild-type N-TIMP-3. Addition of two alanines (at the N-terminal [(-2AA) N-TIMP-3] made it more selective for ADAMTS-5 and weaker for ADAMTS-4. The inhibitory activity of these mutants cannot be explained by the current understanding of the mechanism of TIMPs to inhibit MMPs, but our study suggests that a highly selective inhibitor for ADAMs may be designed based on these molecules.
We have generated transgenic mice that express TIMP-3 or its mutants specifically in cartilage in collaboration with Dr George Bou-Gharios and we are currently testing whether TIMP-3 and engineered TIMP-3 with selectivity will block the progressive degradation of joints in a mouse model of OA.
The major proteinases that degrade cartilages are MMPs and ADAMTSs. Thus, many inhibitors to block their activities have been developed, but they are all targeted to the active site and lacks specificity in general. Recent studies clarified that those metalloproteinases have non-catalytic ancillary domains which uniquely function to assist enzymes to act on specific extracellular matrix components such as aggrecan and collagens. Thus, elucidation of the functional sites in those ancillary domain will allow us to develop new types of inhibitors which are specific to a single metalloproteinase and such inhibitors are ideal to develop therapeutics for arthritis. We are also interested in knowing whether aggrecanase inhibition is sufficient to block OA progression or inhibition of both aggrecanases and collagenases are required. This is approached by using TIMP-3 and its aggrecanse-selective mutants in the mouse model of OA. This information is essential for the development of anti-arthritic agents.
1. Murphy G, Nagase H. Localizing matrix metalloproteinase activities in the pericellular environment. FEBS J. 2011 Jan;278(1):2-15. PubMed PMID: 21087456.
2. Lim NH, Kashiwagi M, Visse R, Jones J, Enghild JJ, Brew K, Nagase H. Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications. Biochem J. 2010 Oct 1;431(1):113-22. PubMed PMID: 20645923.
3. Han S, Makareeva E, Kuznetsova NV, DeRidder AM, Sutter MB, Losert W, Phillips CL, Visse R, Nagase H, Leikin S. Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases. J Biol Chem. 2010 Jul 16;285(29):22276-81. PubMed PMID: 20463013.
4. Brew K, Nagase H. The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta. 2010 Jan;1803(1):55-71. PubMed PMID: 20080133.
5. Troeberg L, Fushimi K, Khokha R, Emonard H, Ghosh P, Nagase H. Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases. FASEB J. 2008 Oct;22(10):3515-24. PubMed PMID: 18632849.