CSB327 Lecture 13 Notes

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Cell and Systems Biology
Maurice Ringuette

CSB327 Lecture 13 Notes (November 5, 2012) – MMPs and ADAMs 2 – Outline • MMPs breakdown stuff. • This lecture is about the dynamic interplay between MMPs, FA, and matrix turnover. • We do not make activated MMPs in our tissues. It is under extremely tight regulation. We do not make a lot in our normal tissue. You make MMPs in response to tissue injury, tumour progression and invasion, and embryogenesis. 3 – MMP substrates identified in complex biological samples by proteomic analysis: “Degradomics” • MMPs target molecules. • MMPs may be more fundamental to us for regulating the biological activity of bioactive molecules like growth factors for their cleavage, activation, silencing, shedding of cell surface receptors, etc. o They are intimately linked to growth factor signalling transduction and simultaneously remodelling the matrix. • The remodelling of the classical ECM molecules is probably the lesser of their important functions. Without regulating this symphony of signaling with these MMPs, this just falls apart. 4 – Pro-MMP-2 and MMP-9 (Secreted) • What is universal about the secreted form of MMPs? o There is no transmembrane domain. • Pre-peptide is a signal peptide. • Pro-domain is going to come off somewhere. • Cysteine switch • Catalytic domain with a zinc ion. • Hemopexin domain with a hinge region. o Not all MMPs have hemopexin domain. • Depending on the MMP, the type II FN repeat in the catalytic domain gives substrate specificity. The hemopexin domain and the FN domain help you target a specific molecule in the ECM or ECM component. • The Cys in the pro-domain will interact with Zn2+ in the catalytic domain. This folds the pro-domain. There is an intimate contact with the Cys and Zn2+. The MMP is inactive. • FN type II domain is essential for gelatinases to cleave gelatin. o Gelatin is broken down collagen. • Collagenases can only target intact collagen fibrils. It will cleave intact collagen. Once the intact collagen is cleaved, it becomes gelatin. o Without the hemopexin domain, collagenase cannot target the triple helices to start the cleavage. 5 – In inactive proMMPs, the SH group of a cysteine residue in the prodomain interacts with the catalytic zinc ion in the catalytic domain • This is inactive. • There is a tremendous number of materials, ions, and reactive oxygen species that can inactivate MMPs. In most cases, you will cleave the pro-domain. You no longer have a closed complex. This is called a MMP that is an active molecule. • MMPs are universally switched off by the cysteine switch between the cysteine residue in the pro-domain and the zinc ion in the catalytic domain. • It is called a metal-dependent endopeptidase. o They do not chew things from the end. They cleave from within. 6 • MT1-MMP is a membrane-type MMP. o This is embedded in the plasma membrane. • MMP-1 is the secreted form. o This is a soluble MMP. • Both of them can cleave collagens I, II and III and release a ¾ fragment and a ¼ fragment. They are modified collagenases because they can cleave the intact molecule. Very few MMPs can do this. Only the collagenases can target a sequence like this. o Either MMP-1 or MT1-MMP will be able to remodel a fibrillar type collagen. • MT1-MMP will be the focus of this lecture. o They are critical for migrating cancer cells as they begin to penetrate the matrix. They are at the leading edge of a migrating cell. They are remodelling the matrix as they go on. The remodelled matrix that these contractile cells sit on gets remodelled like a rail road track for the following cells to go into. • There is a soluble form of MMP and a membrane type MMP. o I will emphasize MT-MMP. 7 – Inhibition of MMPs by tissue inhibitors of metalloproteinases (TIMPs) • Cells will make MMPs, but the make natural inhibitors of MMPs called TIMPs. • TIMP-1 is a good inhibitor of soluble MMPs, but a poor inhibitor of MT-MMPs. o It will bind to MMP and inactivate the MMP. • Nature regulates the levels of TIMP to MMP to regulate the rate of matrix remodelling and activation of growth factors, etc. • TIMP-2 is an effective inhibitor of MT-MMPs. o TIMP-2 and MT1-MMP have a special relationship with respect to the invasion of cancer cells into a matrix. 8 – Dermal wound healing • In wound repair, during wound closure, first you form a provisional matrix which is clots. • In the repair process, the basal keratinocytes begin to migrate and lay down another basement membrane. You eventually remove the scar. You get repair. o The repair process is dependent on remodelling by MMPs and the activation of fibroblast cells into myofibroblast cells. They are highly contractile. They make matrix. They also make MMPs.  The MMPs will help remodel the matrix. One of them is turning over the matrix. The problem with wound repair is that depending on how large the wound is and how large you are, you get to a certain stage where the remodelling leaves a scar.  Tissue repair and tissue regeneration are two different things. The gold standard that we would like to try to do is not to have scarring.  We do not have the ability for tissue regeneration.  Newts can regenerate an entire limb, which also involves MMPs. 9 – Membrane-type MMPs (MT-MMP) “pericellular proteolysis” • We will talk about the role of MT1-MMP in cancer progression. o A cancer cell going through a matrix, as it invades interstitial matrices or by- passes the basal lamina, they need to breakdown the BM and remodel the matrix. • At the leading edge, MT1-MMP is a modified collagenase that can breakdown fibrillar collagens into ¾ and ¼ fragments. This creates an opportunity for other MMPs to breakdown the cleaved collagen. • There are several members of the MT-MMP family. o It has the same molecular design as the soluble MMPs with one major difference. There are furin cleave sites associated with them. • There are two ways of anchoring them into the membrane. o You can create a hydrophobic region that is embedded in the plasma membrane, or you can have a GPI link. • A huge number of cell types can make MT-MMPs. • What is special about MT1-MMP? 10 – Possible mechanism for localization and turnover of MT1-MMP on the cell surface • MT1-MMP with CD44 receptor involves the actin cytoskeleton. o It brings it to the leading edge of cells. o You can shed the CD44 as a result of the active MMP. o It can target other sequences such as fibrillar collagen. • MT1-MMP comes to the surface as a dimer because of the hemopexin domain that promotes dimerization. • The TIMP-2 can bind to MT1-MMP. It can silence the biological activity of MT1-MMP on the cell surface. o TIMP-2 is a good inhibitor of MT1-MMP. o You bring in actin and you can modulate its biological activity by interaction with TIMP-2. The molecular details of where the molecular interactions are occurring are not important for this course. o TIMP-2 can bind and silence MT1-MMP. • You have ecto shedding. • Even though we are focusing on one aspect of MT1-MMP, there is a huge number of non-collagenous targets that MT1-MMP can target as long as it sees the right sequence. o The MT1-MMP binding sites can be found on different types of molecules that are not necessarily related. o Do not memorize the list of targets. 11 – Schematic representation of MT-MMPs • The furin cleavage site is in the pro-domain sequence. The furin cleavage site results the removal of the pro-domain within a secretory vesicle inside the cell. • The MT1-MMP is brought to the surface in an a
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