ILA-ECM_LeClaire_2023_Lawrence LeClaire.pptx

ILA: Extracellular Matrix Lawrence LeClaire, PhD November 20, 2023 Suggested Reading: Marks Chapter 47 Objectives • List the functions of the extracellular matrix • Describe the structure of the major ECM proteins: collagens, elastin, laminin • Describe the steps in the production of collagen • De...

ILA: Extracellular Matrix Lawrence LeClaire, PhD November 20, 2023 Suggested Reading: Marks Chapter 47 Objectives • List the functions of the extracellular matrix • Describe the structure of the major ECM proteins: collagens, elastin, laminin • Describe the steps in the production of collagen • Describe the effects of defects in production of ECM proteins: scurvy, OI, JEB • Describe glycosaminoglycans and proteoglycans including their structure, synthesis, degradation, and defects therein • Discuss the roles of MMPs and TIMPs in ECM Extracellular Matrix • Many different components in matrix • Forms bulk of connective tissue, low percentage is cells • Basal lamina is a special form of ECM • Hydration is important to density and deformability of matrix ECM Functions • Provides structure and support, holds cells and tissues together • Limits movement and migration • Barrier to microorganisms and large molecules • Damage allows movement of materials not normally mobile in matrix • Infectious agents, metastatic cancer cells, proteins lost in urine in kidney disease Collagen • One of the most abundant proteins in the body, especially Collagen Type I • Found in loose connective tissue, bone, tendons, skin, blood vessels, cornea • Triple helical structure with repetitive nature • About 33% glycine, 21 % proline and hydroxyproline • Many sections are Gly-X-Y, glycine-anything-(proline or hydroxyproline) to give proper helix and place glycine side chain in center Hydroxylated Amino Acids • Proline and lysine can be hydroxylated, requires α-ketoglutarate and ascorbate (Vitamin C) • Hydroxyproline needed for hydrogen bonding to stabilize triple helix, hydroxylysine for cross-linking helices into larger structures or carbohydrate addition Importance of Vitamin C • Lack of vitamin C means less stability of collagen and tissues where it is important • Skin less stable, blood vessels easily injured, gums less stable so teeth are loose • Symptoms of scurvy • Sailors on long voyages got scurvy (“ye scurvy dogs”) on standard diet • Vitamin C in citrus fruits, British navy started carrying limes on ships to prevent it, source of slang term “limeys” Scurvy Song by PINK (from Spongebob) Lysyl Oxidase • Besides carbohydrate addition, two crosslinking reactions • Requires conversion to allysine • With a lysine, form a Schiff base • Covalent link between different helices, allows fibers to be made that are bound fibrils • Two allysine can react by aldol condensation to make lysinonorleucine Collagen IV • Makes a mesh rather than fibrils, important in basement membranes and basal lamina • Globular domains at carboxy terminus interact to make dimer • Amino termini aggregate from 4 molecules to make 7S domain • Meshwork formed Many Collagen Types • There are 28 known collagens • Fall into various categories • Found in different tissues • Defects may cause specific or general problems Osteogenesis Imperfecta • Defect in collagen synthesis, varies a lot in severity • Can cause “brittle bone” disease • Some forms give blue sclera • Some mild, some lethal Basis of Various OI Forms • Sometimes collagen itself • Maybe a mutation at lysine • C-terminal = greatest problem as fibril starts winding at C-terminus, blocked early • Collagen mutations generally show a type of dominant-negative effect • Processing defects due to enzyme deficiencies more likely to be recessive Elastin • Abundant in elastic fibers, found in smooth muscle, endothelium, near chondrocytes and fibroblasts • Allows blood vessels to deform, lungs to expand and contract with breathing without using energy • Alternating hydrophobic and hydrophilic domains in protein • Stretching exposes hydrophobic regions, water organizes into cages • On relaxation, water can reorganize, provides impetus for full relaxation of molecule Laminin Structure • Three polypeptides in a cross configuration • Globular domains N-terminal region, then rigid coils • Disulfide bonds link coils • Only α-chain has C-terminal extension, binds to ECM • Collagen, sulfated lipids, proteoglycans bound • Various combinations of α, β and γ make different forms • 5 α, 3 β, 3 γ known, 45 possible combinations, only 18 identified Laminin Functions • Most abundant protein in basal laminae after Type IV Collagen • Binds it to ECM molecules and cell integrins, provides stability • Defects in Laminin 5 or 6 cause junctional epidermolysis bullosa (JEB), dermis and epidermis not held together properly http://emedicine.medscape.com/article/909549-overview Glycosaminoglycans • Repeating disaccharide units, most one acid form and one with nitrogen and acetyl and/or sulfate • Sulfate added from PAPS • Iduronic acid made from glucuronic acid by an epimerase • Generally are side chains on proteoglycans • Hyaluronic acid can exist separately Proteoglycan Linkage • Added on serine or threonine • Linker of xylose and galactose to start • Repeat unit added to linker • Many repeat units added Order of Synthesis • Addition may be of normal or modified sugar • Some modifications made to growing chain • Sulfate added to existing chain sugars Proteoglycan Structure • The bottle brush proteins • Many glycosyl chains added to protein core • May be >100 chains, 95% carbohydrate by weight Forming the Matrix • Must connect components • Hyaluronic acid backbone • Link protein bind HA to proteoglycans with noncovalent bonds • Negative charges bind positive ions, hydrogen bonds to water, forms hydrated gel • Flexible, acts as filter • Small molecules can move, larger molecules and cells limited Matrix and Cells • Proteoglycans bind to fibronectin • Fibronectin binds to integrins in cell membranes and to collagen fibrils • Cells attached to all matrix components in some fashion Cartilage • High number of negative charges attracts cations, creates high osmotic pressure • Water drawn in, network under tension • Tension balances swelling at equilibrium • Can be compressed, will regain shape after pressure released Matrix Life Cycle • Components both synthesized and degraded • Near cell, less organized • Intercellular matrix is more organized with a longer half-life for molecules • Synovial fluid has smaller components Matrix Degradation • Components enter cells by endocytosis • Endosomes fuse with lysosomes • Lysosomal hydrolases break down into sugars and amino acids • Lack of any enzyme will prevent degradation, accumulation of partially degraded material • Lysosomal storage disorders, mucopolysaccharidoses Chemical Degradation • May be modified by glycation, reactive oxygen species, or other chemicals or enzymes • Diabetes one example, high glucose and other changes lead to modification • Alter function, not filter properly or maintain barriers, important in kidney function MPS Disease Defects Integrins • Transmembrane proteins that connect to matrix and cytoskeleton • Signals may pass in either direction • An α-β dimer, 18 α and 10 β known, 24 combinations seen in cells • Some cells have inducible integrins • White blood cells don’t normally express them, but can be induced by cytokines at injury site; integrins are made, adhere to endothelium, and begin process of diapadesis MMPs and TIMPs • Matrix metalloproteinases contain zinc; degrade all ECM proteins • MMPs are important for migration and remodeling • Destruction of ECM by MMPs can release growth factors; when growth factors are bound to ECM proteins, the growth factors are inactive • MMPs are synthesized with propeptide which contains a cysteine that blocks active site; propeptide removal is necessary for activation of MMPs • Highly regulated, many processes; one is control of TIMP (Tissue Inhibitors of Metalloproteinases) expression Summary • ECM has many different components • Collagen fibril can only be formed if triple helix forms and is processed properly • Elastin uses no energy to stretch and contract • Laminin is important in basal laminae • Everything turns over at its own speed • GAGs are carbohydrate portions of proteoglycans, important in function • Failure in degradation of GAGs lead to MPS • Fibronectin attaches matrix proteins to integrins on cells • Integrins link cytoskeleton to matrix • MMPs and TIMPs are important in remodeling matrix and preventing remodeling Reminder of Steps • Order is important for correct outcome • Different events take place in different locations Roles of GAGs Assessment Questions Individuals who develop scurvy suffer from sore and bleeding gums and loss of teeth. This is due to the synthesis of a defective collagen molecule. The step that is affected in collagen biosynthesis contributing to scurvy is which of the following? A. The formation of lysyl cross-links between collagen molecules B. The formation of disulfide bonds, which initiates tropocollagen formation C. The formation of collagen fibrils D. The hydroxylation of proline residues, which stabilizes the collagen structure E. Secretion of tropocollagen into the extracellular matrix Assessment Questions Which one of the following alterations would reduce the ability of cartilage to cushion weight-bearing activities at joints? A. Increased concentration of sulfated sugars on the proteoglycans B. Loss of positive charges on the proteoglycans C. Gain of negative charges on the proteoglycans D. Increased concentration of glucuronic acid residues E. Loss of negative charges on the proteoglycans Assessment Questions The underlying mechanism that allows elastin to exhibit elastic properties (expansion and contraction) is which of the following? A. Breaking of disulfide bonds during expansion and reformation of these bonds during contraction B. A decrease in entropy during expansion and an increase in entropy during contraction C. The breaking of salt bridges during expansion and reformation of salt bridges during contraction D. Hydroxylation of elastin during expansion and decarboxylation of elastin during contraction E. Proteolysis during expansion and re-synthesis during contraction Assessment Questions The movement of tumor cells from their site of origin to other locations within the body requires the activity of which of the following proteins? A. Elastin B. Matrix metalloproteinases C. Proteoglycans D. Laminin E. Collagen Assessment Questions Fibronectin is frequently absent in malignant fibroblast cells. One of the major functions of fibronectin is which of the following? A. To coordinate collagen deposition within the extracellular matrix B. To extend glycosaminoglycan chains using nucleotide sugars C. To regulate glycosaminoglycan production D. To inhibit the action of matrix metalloproteinases E. To fix the position of cells within the extracellular matrix