04/19/10

EXTRACELLULAR MATRIX PROTEINS AND

PROTEINASES

By,Raghu

AmbekarPhotonics Research of Bio/nano Environments

Department of Electrical & Computer EngineeringUniversity of Illinois Urbana - Champaign

BioE 506

04/19/10

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

http://kentsimmons.uwinnipeg.ca/cm1504

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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)

Collagen molecule

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Classification of collagen

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

1. Fibril-forming collagens

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Classification of collagen

Forms network in basement (Collagen IV) and Descemet’s membrane (Collagen VIII) Molecular filtration Example : Types IV, VIII, X

2. Network-forming collagens

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Classification of collagen

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

3. Fibril-associated collagens with interrupted triple helices (FACITs)

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Classification of collagen

Provides functional integrity by connecting epithelium to stroma Example : Type VII

4. Anchoring collagens

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Classification of collagen

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

5. Beaded-filament-forming collagens

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Type I Fibril assembly

Chain recognition sequence

Fibril assembly is determined by chain recognition sequence in C-propeptide

Fish scale

Bone osteon

Tendon

Skin

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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 Spread of cancer from a primary tumor to distant sites of the body 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 Major role of MMPs was to facilitate the breakdown of physical barriers, thus promoting invasion, intravasation, extravasation and migration 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

Structure and content of collagen governs the delivery of therapeutic molecules in tumors

Penetration of therapeutic molecules improved by developing agents that modify ECM and increase diffusion

Detect tumor collagen noninvasively to quantify collagen content and estimate drug delivery characteristics

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Modification of collagen for therapeuticsUses Second-harmonic generation (SHG) for imaging only collagen fibers

Conditions :

Non-centrosymmetric (collagen, microtubules)

Lasers (high intensity)

Advantages :

No staining

3D imaging

No photobleaching

SAMPLE

Red Wavelength=800 nm

SHG: BlueWavelength=400 nm

Collagen stained red and imaged by fluorescence

microscopy

Collagen imaged by SHG microscopy

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Modification of collagen for therapeutics

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

Chronic relaxin treatment degrades tumor matrix and improve macromolecular diffusion in tumors

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THANK YOU!