EXTRACELLULAR MATRIX PROTEINS AND PROTEINASES By, Raghu Ambekar Photonics Research of Bio/nano Environments Department of Electrical & Computer Engineering University of Illinois Urbana - Champaign 04/19/10
BioE 506
Outline Extracellular matrix proteins Collagen Classification Fibril assembly and collagen diseases Extracellular matrix proteinases Role of MMP in metastasis Modification of tumor collagen for therapeutics
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Extracellular matrix (ECM) Surrounds cell Provides mechanical support Controls the flow of nutrients and signals to the cells Consists of Fibrous: collagen, elastin, fibronectin, laminin Non-fibrous: Proteoglycans and polysaccharides
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http://kentsimmons.uwinnipeg.ca/cm1504
Collagen Collagen : most abundant protein found in the human body. About 1/3rd of the total proteins. Found abundantly in tendon, cartilage, bone and skin Functions: cell migration cell adhesion molecular filtration tissue repair
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Structure of collagen It has a triple-helix structure containing three α-polypeptide chains arranged in right-handed supercoil Glycine, proline, hydroxyproline 1.5 nm diameter At least 28 different collagens found The three α-chains could be same (collagen II) or different (collagen I)
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Collagen molecule
Classification of collagen 1. Fibril-forming collagens No interruptions in triple helix Regular arrangement results in characteristic “D” period of 67 nm Diameter : 50-500 nm Example : Types I, II, III, V, XI
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Classification of collagen 2. Network-forming collagens Forms network in basement (Collagen IV) and Descemet’s membrane (Collagen VIII) Molecular filtration Example : Types IV, VIII, X
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Classification of collagen 3. Fibril-associated collagens with interrupted triple helices (FACITs)
Short collagens with interruptions Linked to collagen II and carries a GAG chain Found at the surface of fibril-forming collagens Example : Types IX, XII, XIV
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Classification of collagen 4. Anchoring collagens ďƒ˜ Provides functional integrity by connecting epithelium to stroma ďƒ˜ Example : Type VII
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Classification of collagen 5. Beaded-filament-forming collagens Form structural links with cells Example : Type VI Collagen VI crosslink into tetramers that assemble into long molecular chains (microfibrils) and have beaded repeat of 105 nm
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Type I Fibril assembly Fibril assembly is determined by chain recognition sequence in C-propeptide Fish scale
Bone osteon
Tendon
Chain recognition sequence
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Skin
Diseases associated with collagen Diseases caused by mutations Subtypes of osteogenesis imperfecta (collagen I) Ehlers-Danlos syndrome (collagen I and V) Alport syndrome (collagen IV) Certain arterial aneurysms (collagen III) Ullrich muscular dystrophy (collagen VI) Certain chondrodysplasias (collagen IX and XI) Kniest dysplasia (collagen II)
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Role of MMP in metastasis
Metastasis Metastasis ďƒ˜ Spread of cancer from a primary tumor to distant sites of the body ďƒ˜ A defining feature of cancer
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Role of MMP in metastasis Understanding the molecular mechanisms of metastasis is crucial for the design of therapeutics Extracellular matrix metalloproteinases (MMP) associated with metastasis MMPs are capable of digesting ECM and basement membrane under physiologic conditions Collagenases degrade fibrillar collagen Stromelysins degrade proteoglycans and glycoproteins Gelatinases degrade nonfibrillar and denatured collagens At tumor sites, experiments have found Increased number of MMPs Increased levels of MMPs Reduced levels of TIMPs (Tissue inhibitors of metalloproteinases)
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Role of MMP in metastasis ďƒ˜ Major role of MMPs was to facilitate the breakdown of physical barriers, thus promoting invasion, intravasation, extravasation and migration ďƒ˜ MMPs targeted for antimetastasis therapies
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Role of MMP in metastasis Clinical trials of inhibiting MMPs to cure cancer have failed Metastasis is a complicated process MMPs contribute to every stage in tumor progression at both primary and metastatic sites Specific MMPs play a role in each stage of metastasis MMP 13, 14 – invasion MMP 9– angiogenesis Understand the role of the MMPs in each cancer setting
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Modification of collagen for therapeutics ďƒ˜ Structure and content of collagen governs the delivery of therapeutic molecules in tumors ďƒ˜ Penetration of therapeutic molecules improved by developing agents that modify ECM and increase diffusion ďƒ˜ Detect tumor collagen noninvasively to quantify collagen content and estimate drug delivery characteristics
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Modification of collagen for therapeutics Uses Second-harmonic generation (SHG) for imaging only collagen fibers Conditions :
Red Wavelength=800 nm
SHG: Blue Wavelength=400 nm
Non-centrosymmetric (collagen, microtubules) SAMPLE
Lasers (high intensity) Advantages : No staining 3D imaging No photobleaching
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Collagen stained red and imaged by fluorescence microscopy
Collagen imaged by SHG microscopy
Modification of collagen for therapeutics
ďƒ˜ SHG intensity collected from live imaging of collagen fibers provides an good estimate of diffusion coefficient in tumors
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Modification of collagen for therapeutics 0th day
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ďƒ˜ Chronic relaxin treatment degrades tumor matrix and improve macromolecular diffusion in tumors
THANK YOU! 04/19/10