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BC chap 49 Intro Basic components of extracellular matrix – fibrous structural proteins o Collagens o Proteoglycans – contain long GAG chains attached to protein backbone covalently GAG chains contain repeating disaccharide units that usually contain a hexosamine and a uronic acid (frequently sulfated) Synthesis starts with attachment of sugar to serine, threonine, or asparagine reside of protein – additional sugars donated by UDP-sugar precursors and add sequentially to nonreducing end of molecule Synthesized in ER and Golgi complex GAG chains are degraded by lysosomal enzymes that cleave one sugar at a time from nonreducing end of chain Inability to degrade leads to mucopolysaccharidoses o Adhesion proteins – link components of matrix to each other and to cells Fibronectin and laminin are examples Extracellular glycoproteins that contain separate distinct binding domains for proteoglycans, collagen, and fibrin Contain specific binding domains for integrins, which bind to fibronectin on external surface, span PM of cells, and adhere to proteins, which bind to intracellular actin filaments of cytoskeleton Integrins provide a mechanism for signaling between cells via both internal signals and signals generated via ECM Cell movement within ECM requires remodeling of various components of ECM – accomplished by matrix metalloproteinases (MMPs) and regulators of MMPs (tissue inhibitors of matrix metalloproteinases (TIMPs)) o Dysregulation of balance of regulators of cell movement allows cancer cells to metastasize Composition of Extracellular Matrix Fibrous proteins – collagen, elastin, and laminin o Collagen – produced principally by fibroblasts, muscle cells, and epithelial cells Type I collagen is most abundant protein in mammals – about 33% glycine and 21% proline and hydroxyproline Procollagen(I) – precursor of collagen(I) – triple helix of 3 pro-α chains twisted around each other Polymerization of collagen(I) molecules forms collagen fibrils, which provide great tensile strength to connective tissues 3 polypeptide chains of triple helix linked by interchain hydrogen bonds Each turn of the triple helix contains 3 amino acid residues so that every 3rd amino acid (glycine) is in close contact with other two strands in center of structure (this must be a glycine, which is the only AA without a side chain) – amino acid before glycine is usually proline or hydroxyproline Procollagen(I) requires extensive posttranslational modification Hydroxylation of proline and lysine requires vitamin C as cofactor of enzymes (prolyl hydroxylase and lysyl hydroxylase) Hydroxyproline residues involved in hydrogen bond formation that helps stabilize triple helix Hydroxylysine residues are sites of attachment of disaccharide moieties (galactoseglucose) Side chains of lysine residues may be oxidized to form aldehyde (allysine) – aldehyde residues produce covalent cross-links between collagen molecules – allysine residue on one collagen molecule reacts with amino group of lysine residue on another molecule, forming covalent Schiff base that is converted to more stable covalent cross-links – aldol condensation may also occur, forming lysinonorleucine o In absence of vitamin C (scurvy), melting temperature of collagen drops dramatically because of lack of interstrand H bond formation, which is caused by lack of hydroxyproline residues 28 types of collagen and can be fibril-forming, network-forming, those that associate with fibril surfaces, those that are transmembrane proteins, endostatin-forming, and those that form periodic beaded filaments Can be composed of non-triple helical domains between triple helices Fibril-associated collagens with interrupted triple helices (FACITs) associate with fibrillary collagens without themselves forming fibers Endostatin-forming collagens are cleaved at their C-terminus to form endostatin (inhibitor of angiogenesis by inhibiting endothelial cell migration) – tumor growth dependent on blood supply, so this could prevent tumor proliferation Network-forming collagens form a mesh-like structure because of large noncollagenous domains at carboxy terminus Type XXV collagen has been associated with neuronal plaques that develop during Alzheimer’s disease Type I, II, and III collagens form fibrils that assemble into large insoluble fibers – fibrils strengthened through covalent cross-links between lysine residues on adjacent fibrils Types of collagen that do not form fibrils perform a series of distinct roles Fibril-associated collagens bind to surface of collagen fibrils and link them to other matrix-forming components Transmembrane collagens form anchoring fibrils that link components of extracellular matrix to underlying connective tissue Network-forming collagens form flexible collagen that is part of basement membrane and basal lamina that surround many cells Collagen synthesized in ER as preprocollagen – presequence acts as signal sequence for protein and is cleaved, forming procollagen within ER Procollagen is transported to Golgi apparatus – 3 procollagen molecules associate through formation of interstrand and intrastrand disulfide bonds at carboxy terminus 3 molecules align and form triple helix from carboxy end toward amino end, forming tropocollagen, which contains a triple helical segment between 2 globular ends (amino and carboxy terminal extensions) Tropocollagen is secreted from cell, extensions are removed using extracellular proteases, and mature collagen takes its place in ECM Individual fibrils of collagen line up in highly ordered fashion to form collagen fiber Elastin – major protein in elastic fibers – elastic fibers also contain microfibrils (composed of several acidic glycoproteins, mostly fibrillin-1 and fibrillin-2 Tropoelastin – precursor of elastin, highly soluble, and synthesized in rER for eventual secretion Contains 2 types of alternating domains o Hydrophilic sequence rich in lysine and alanine residues o Hydrophobic sequence rich in valine, proline, and glycine, frequently near repeats of VPGVG or VGGVG Upon secretion from cell, tropoelastin is aligned with microfibrils, and lysyl oxidase initiates reactions that cross-link elastin molecules, using lysine residues within hydrophilic alternating domains 2-4 lysine residues are cross-linked to form stable structure – net result is generation of fibrous mesh that encircles cells Supravalvular aortic stenosis (SVAS) caused by insufficiency of elastin in vessel wall, leading to narrowing of large elastic arteries – levels of elastin in vessel walls may regulate number of smooth muscle cell rings that encircle the vessel (low levels of elastin cause smooth muscle hypertrophy, leading to narrowing and stenosis of artery) When elastic fibers are stretched, amorphous elastin structure is stretched, exposing repeating hydrophobic regions of molecule to aqueous environment, leading to a decrease in entropy of water because water molecules need to rearrange to form cages around the hydrophobic domains When stretching force is removed, elastin takes on original structure because of increase in entropy that occurs because water no longer needs to form cages about hydrophobic domains o Laminin – second most abundant protein in basal lamina (after type IV collagen) – provides additional structural support for tissues through ability to bind to type IV collagen, other molecules present in ECM, and cell surface-associated proteins Heterotrimeric protein composed of α, β, and γ-subunits Nomenclature for these is expressed as α1-5β1-3γ1-3 Major feature is the α-helix, which joins the 3 subunits together and forms a rigid rod Only α-chain has significant carboxy-terminal extension past rod-like structure Laminin extensions allow laminin to bind to other components of ECM and provide stability for the structure Laminin binds collagen, sulfated lipids, and proteoglycans in ECM Laminin synthesized with leader sequence that targets three chains to ER Chain association occurs in Golgi apparatus before secretion from the cell After laminin is secreted by cell, amino-terminal extensions promote self-association as well as binding to other ECM components Disulfide linkages formed to stabilize trimer, but much less posttranslational processing of laminin than there is of collagen and elastin Junctional epidermolysis bullosa (JEB) caused by defects in structures of laminin 5 or laminin 6 – severe spontaneous blistering of skin and mucous membranes Congenital muscular dystrophy (CMD) results from defect in laminin 2, which is component of bridge that links muscle cell cytoskeleton to ECM – lack of this bridge triggers muscle cell apoptosis ECM serves to keep cells from moving to other locations and prevent large molecules and particles, such as microorganisms, from reaching contiguous and distant cells o Infections spread, in part, because infections agent alters containing capacity of ECM o Cancer cells metastasize by altering integrity of ECM o Rheumatoid arthritis and osteoarthritis involve damage to functional capacity of ECM o Alterations in structural characteristics of matrix of renal glomerulus may allow proteins to be excreted into urine (indication of inexorable decline in renal function) o Ehlers-Dahlos syndrome – caused by mutations that affect specific collagen genes o Marfan syndrome – defect in fibrillin o Deficiencies of lysosomal enzymes involved in normal degradation of molecules of matrix result in diseases such as mucopolysaccharidoses Proteoglycans – form gels around fibrous structural proteins – consist of repeating units of disaccharides (GAGs) linked to core protein o One sugar is either N-acetylglucosamine or N-acetylgalactosamine, and the other is usually acidic (either glucuronic acid or iduronic acid) – sugars modified by addition of sulfate groups to parent sugar o Negatively charged carboxylate and sulfate groups on proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules, creating a hydrated gel, which provides flexible mechanical support for ECM and acts as a filter that allows diffusion of ions, water, and other small molecules but slows diffusion of proteins and movement of cells o Gel also acts as lubricant o Hyaluronan is only GAG that occurs as single long polysaccharide chain and is only GAG not sulfated o Proteoglycans found in interstitial connective tissues (synovial joints, vitreous humor of eye, arterial walls, bone, cartilage, and cornea) – major components of ECM in these tissues – interact with collagen, elastin, fibronectin, and laminin o Because long, negatively charged GAG chains repel each other, proteoglycans occupy a very large space and act as molecular sieves, determining which substances enter or leave cells o o o o Properties give resilience and a degree of flexibility to substances such as cartilage, permitting compression and re-expansion of molecule to occur Protein component of proteoglycans synthesized on ER, where it enters the ER lumen for initial glycosylations UDP-sugars serve as precursors that add sugar units one at a time, first to protein and then to nonreducing end of growing carbohydrate chain Glycosylation occurs initially in lumen of ER and subsequently in Golgi complex Glycosyltransferases (enzymes that add sugars to chain) are specific for sugar being added, type of linkage formed, and sugars already present in chain Once initial sugars attached to protein, alternating action of 2 glycosyltransferases adds sugars of repeating disaccharide to growing GAG chain Sulfation occurs after addition of the sugar 2’-phosphoadenosine 5’-phosphosulfate (PAPS) – also called active sulfate – provides sulfate groups Epimerase converts glucuronic acid residues to iduronic acid residues After synthesis, proteoglycans are secreted from cell, where they can form large aggregates, noncovalently attached by a “link” protein to hyaluronic acid Long polysaccharide chains of proteoglycans in cartilage contain many anionic groups, attracting cations that create a high osmotic pressure within cartilage, drawing water into its connective tissue and placing the collagen network under tension Resulting tension balances swelling pressure caused by proteoglycans Lysosomes fuse with endocytotic vesicles, and lysosomal proteases digest protein component – carbohydrate component degraded by lysosomal glycosidases Lysosomes contain both endoglycosidases and exoglycosidases Endoglycosidases cleave chains into shorter oligosaccharides Exoglycosidases – specific for each type of linkage – remove sugar residues one at a time from nonreducing ends Deficiencies of lysosomal glycosidases cause partially degraded carbs from proteoglycans, glycoproteins, and glycolipids to accumulate within membrane-enclosed vesicles inside cells (residual bodies) Mucopolysaccharidoses caused by accumulation of partially degraded GAGs – may cause deformities of skeleton and mental retardation IOntegrins Integrins Major cellular receptors for ECM proteins and provide link between internal cytoskeleton of cells (primarily actin microfilament system) and extracellular proteins, such as fibronectin, collagen, and laminin Consist of an α and β-subunit – 24 different combinations discovered Integrins involved in wide variety of cell signaling options Certain integrins, such as those associated with WBCs, normally inactive because white cell must circulate freely in bloodstream o If infection occurs, cells located in area of infection release cytokines, which activate integrins on WBCs, allowing them to bind to vascular endothelial cells (leukocyte adhesion) at site of infection o Leukocyte adhesion deficiency (LAD) – genetic disorder that results from mutations in β2-integrin such that leukocytes cannot be recruited to sites of infection o Drugs can block either β2-integrin or α4-integrins (on lymphocytes) to treat inflammatory and autoimmune disorders by interfering with WBC response to cytokines Can be activated by intracellular signaling events activating the molecule or by a binding event with extracellular portion of molecule that initiates intracellular signaling events Integrins that bind cells to ECM components – activation of specific integrins can result in migration of affected cell through ECM – operative during growth, during cellular differentiation, and in metastasis of malignant cells to neighboring tissues Movement of Tumor Cells Metastasis through blood or lymph system and colonization of target tissue requires degradation of ECM to allow for cell movement – accomplished by matrix metalloproteinases (MMPs) MMPs degrade specific ECM components (such as collagen or elastin) allowing cells access through this compartment Gelatin zymography assay – assay for determining if MMPs are present in biological sample o Polyacrylamide gels are prepared and enzyme samples are run through gel in presence of SDS o After gel is run, enzyme activity is reconstituted by substituting Triton X-100 for SDS o Assay buffer is placed over gel and left overnight o If a lane on the gel contained MMP activity, MMP would be digesting gelatin in area of gel where MMP resided o After activity phase is complete, gel is developed with Coomassie stain, which binds to proteins in gel, including gelatin – positive result would appear as white bands on blue background Fluorescence resonance energy transfer (FRET) – more sensitive assay than gelatin zymography assay o Uses peptide substrate that contains both fluorophore and a quencher in close proximity – when excited, quencher blocks fluorescence emittance from fluorophore because of close proximity on peptide o After MMP treatment, peptide is cleaved between fluorophore and quencher, such that quencher is no longer in close proximity, resulting in strong fluorescence emittance – intensity will increase as peptide is cleaved in presence of MMP Adhesion Proteins Found in ECM and link integrins to ECM components Large multidomain proteins that allow binding to many different components simultaneously Fibronectin has binding sites for integrins, collagen, and GAGs o Alternative splicing allows many different forms of adhesion protein to be expressed, including soluble form (versus cell-associated forms) found in plasma As integrin molecule is bound to intracellular cytoskeletal proteins, adhesion proteins provide a bridge between actin cytoskeleton of cell and cells’ position within ECM Loss of adhesion protein capability can lead to physiologic or abnormal cell movement Lost when tumor cells develop (many tumor cells secrete less than normal amounts of adhesion protein material, which allows for more movement within ECM) – this increases potential for tumor cells to metastasize Matrix Metalloproteinases Zinc-containing proteases that use zinc to appropriately position water to participate in proteolytic reaction Cleave all proteins found in ECM, including collagen and laminin Important to allow cell migration and tissue remodeling during growth and differentiation Many growth factors bind to ECM components and, as bound components, do not exhibit their normal growthproducing activity o Destruction of ECM by MMPs releases these growth factors, allowing them to bind to cell surface receptors to initiate growth of tissues Cancer cells that metastasize require extensive ECM remodeling and usually use MMP activity to spread throughout body Propeptide present in newly synthesized MMPs contains critical cysteine residue which binds to zinc atom at active site of protease and prevents propeptide from exhibiting proteolytic activity o Removal of propeptide required to activate MMPs o Once activated, certain MMPs can activate other forms of MMP Regulation of MMPs include transcriptional regulation, proteolytic activation, inhibition by circulating protein α2macroglobulin, and regulation by tissue inhibitors of metalloproteinases (TIMPs) o Synthesis of TIMPs and MMPs must be coordinately regulated because dissociation of their expression can facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis Osteogenesis Imperfecta Defect in collagen production – 2 types o Reduction in synthesis of normal collagen (resulting from genetic problem) o Synthesis of mutated form of collagen – most mutations have dominant-negative effect, leading to autosomal dominant mode of transmission Many of known mutations involve substitutions of another amino acid for glycine, resulting in unstable collagen molecule because glycine is only amino acid that can fit between other 2 chains in triple helix of collagen o If mutation is near carboxy-terminal end, phenotype of disease is usually more severe than if mutation is near amino-terminal end o Mutations that replace glycine with either serine or cysteine are more stable because of hydrogen bonding capabilities of serine and ability of cysteine to form disulfide bonds, preventing the strand from unwinding Can be treated with bisphosphonates, which consist of 2 phosphates linked by carbon or nitrogen bridge (analogs of pyrophosphate where 2 phosphates are linked by oxygen) In OI, bone resorption outpaces bone formation because osteoclast activity is enhanced (reduced levels of normal collagen present to act as nucleating sites for bone formation) Bisphosphonates inhibit osteoclast action with potential to increase bone mass and its tensile strength Can occur due to mutations in genes other than collagen o Mutations in CRTAP or LEPRE1 lead to defective collagen fibers being produced o CRTAP forms complex with PH3-1 and cyclophilin to hydroxylate specific proline residue in types I and II collagen – failure to hydroxylate this proline residue leads to unstable collagen and moderate to severe forms of OI o Pattern of inheritance for these is autosomal recessive Other Diseases Lupus – alterations in cell matrix components caused by autoimmune induced trigger Type 2 diabetes – cell matrix interactions can be altered because of elevated glucose levels and nonenzymatic glycosylation