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Structural and functional comparisons of MMP-9 forms
Matrix metalloproteinases (MMPs) constitute a family of soluble or membrane bound Zn2+-dependent endopeptidases. In the human species, this group contains more than 20 multidomain enzymes, including collagenases, gelatinases, metalloelastases, matrilysins, stromelysins and membrane-typeMMPs. These proteases are associated with physiological processes such as reproduction, organogenesis, bone remodeling, vasculogenesis and wound healing. Besides, they are also involved in a variety of pathologies, such as acute and chronic inflammation (e.g. rheumatoid arthritis and multiple sclerosis), cancer cell invasion and metastasis as well as cardiovascular, brain and lung diseases.
MMPs are multi-domain enzymes which contain a propeptide, a catalytic domain, a Zn2+-binding domain and, in most instances, also a hemopexin-like domain. The site of active proteolysis is a combination of the catalytic domain and the Zn2+-bindingdomain which holds the active zinc ion. The hemopexin-like domain is involved in the binding of a range of proteins, including substrates, cellular receptors and TIMPs. A most studied and structurally complex memberof the MMP family is MMP-9 or gelatinase B. Although all members of theMMP family possess similar characteristics, MMP-9 is exceptional. Firstly, since MMP-9 is a gelatinase, it has an additional fibronectin-like domain which improves the ability of the molecule to bind and digest large substrates (e.g. gelatins). Secondly, MMP-9 contains a prominent O-glycosylated domain. This domain is highly glycosylated and is referred to as a flexible linker since it lends the molecule the high degree of flexibility necessary for moving along collagen fibrils. Finally, besides monomers, MMP-9 has the remarkable ability to form multimers.
Literature on MMP-9 is increasing exponentially each year which makes it challenging to gain an overview on the current status of MMP-9 research. We extracted and summarized the literature highlights from the past decade. Despite the significance of MMP-9, many of its structural and functional features remain elusive. To date, the exact MMP-9 structural characterization is limited to its N-terminal and C-terminal domains and no in-depth information is available on MMP-9 multimerization. In order to clarify the structure and function of homomultimeric MMP-9, a stable full-length MMP-9 mutant was generated and produced at high yield. A protocol for separating MMP-9 monomers from multimers was optimized. A detailed analysis based on biochemical and biophysical methods yielded informationon the trimeric structure of MMP-9. Atomic force microscopic images de visu elucidated this new trimeric structure of MMP-9 multimers. Comparative analysis of structure and function revealed that TIMP-1 binds more tightly to MMP-9 homomultimers than to monomers. In view of TIMP-1-free MMP-9 secretion by neutrophils this may indicate a different functional role for MMP-9 trimers. This new insight might be critical to understand basic biological processes including inflammation, cancer cell invasion,metastasis and angiogenesis.
Several MMP inhibitors (MMPIs) weredeveloped during the past 20 years. Against all odds, most clinical trials with MMPIs had poor outcomes and severe side-effects were observed. Many explanations have been postulated, but the main problem was low selectivity of the used MMPIs, which results in a disturbance of the proteolytic network and in an inhibition of antitargets. New directions for MMP targeting strategies include (i) the development of more specific MMPIs that target a single function of the MMP and (ii) the alternative use of MMPs. For the discovery of MMPIs which target distal domains, a suitable screening method has to be developed. The use of fluorogenic naturalsubstrates rather than small peptides allowed the detection of inhibitors of the fibronectin repeats, compared to small peptides. Alternative strategies for the use of MMP-9 include exploitation of the presence of active MMP-9 in diseased tissues to activate a specific drug system, as exemplified with MMP-9 substrate-loaded nanoparticles.
MMPs are multi-domain enzymes which contain a propeptide, a catalytic domain, a Zn2+-binding domain and, in most instances, also a hemopexin-like domain. The site of active proteolysis is a combination of the catalytic domain and the Zn2+-bindingdomain which holds the active zinc ion. The hemopexin-like domain is involved in the binding of a range of proteins, including substrates, cellular receptors and TIMPs. A most studied and structurally complex memberof the MMP family is MMP-9 or gelatinase B. Although all members of theMMP family possess similar characteristics, MMP-9 is exceptional. Firstly, since MMP-9 is a gelatinase, it has an additional fibronectin-like domain which improves the ability of the molecule to bind and digest large substrates (e.g. gelatins). Secondly, MMP-9 contains a prominent O-glycosylated domain. This domain is highly glycosylated and is referred to as a flexible linker since it lends the molecule the high degree of flexibility necessary for moving along collagen fibrils. Finally, besides monomers, MMP-9 has the remarkable ability to form multimers.
Literature on MMP-9 is increasing exponentially each year which makes it challenging to gain an overview on the current status of MMP-9 research. We extracted and summarized the literature highlights from the past decade. Despite the significance of MMP-9, many of its structural and functional features remain elusive. To date, the exact MMP-9 structural characterization is limited to its N-terminal and C-terminal domains and no in-depth information is available on MMP-9 multimerization. In order to clarify the structure and function of homomultimeric MMP-9, a stable full-length MMP-9 mutant was generated and produced at high yield. A protocol for separating MMP-9 monomers from multimers was optimized. A detailed analysis based on biochemical and biophysical methods yielded informationon the trimeric structure of MMP-9. Atomic force microscopic images de visu elucidated this new trimeric structure of MMP-9 multimers. Comparative analysis of structure and function revealed that TIMP-1 binds more tightly to MMP-9 homomultimers than to monomers. In view of TIMP-1-free MMP-9 secretion by neutrophils this may indicate a different functional role for MMP-9 trimers. This new insight might be critical to understand basic biological processes including inflammation, cancer cell invasion,metastasis and angiogenesis.
Several MMP inhibitors (MMPIs) weredeveloped during the past 20 years. Against all odds, most clinical trials with MMPIs had poor outcomes and severe side-effects were observed. Many explanations have been postulated, but the main problem was low selectivity of the used MMPIs, which results in a disturbance of the proteolytic network and in an inhibition of antitargets. New directions for MMP targeting strategies include (i) the development of more specific MMPIs that target a single function of the MMP and (ii) the alternative use of MMPs. For the discovery of MMPIs which target distal domains, a suitable screening method has to be developed. The use of fluorogenic naturalsubstrates rather than small peptides allowed the detection of inhibitors of the fibronectin repeats, compared to small peptides. Alternative strategies for the use of MMP-9 include exploitation of the presence of active MMP-9 in diseased tissues to activate a specific drug system, as exemplified with MMP-9 substrate-loaded nanoparticles.
Date:1 Oct 2009 → 21 Nov 2013
Keywords:matrix metalloproteinase-9, posttranslational modification, recombinant expression
Disciplines:Immunology
Project type:PhD project