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Fibrous Proteins DrVivek Joshi, MD Fibrous Proteins Cylindrical shape: long rod like Low solubility in aqueous solutions With a structural rather than a dynamic role in the cell or organism Characteristically contain larger amounts of regular secondary structure Examples: Collagen, elastin, keratin 2 Collagen Most abundant protein in the human body Comprises about 25 % of total mammalian protein Present in all tissues and organs Provides the framework that gives the tissues their form and structural strength Types and organization are dictated by the structural role played in a particular organ 3 Collagen Types and organization are dictated by the structural role played in a particular organ Organ/Tissue Collagen characteristics Tendons Bundled in tight parallel fibers that provide great strength Bone Fibers arranged at an angle to each other so as to resist mechanical shear from any direction Skin Loosely woven flexible fibers Cornea Ordered so as to be nearly crystalline, transparent 4 Collagen More then 20 distinct types in humans Made up of about 30 distinct polypeptide chains (from 30 genes) Type Tissue I COL1A1, COL1A2 Most connective tissues, including bone II COL2A1 Cartilage, vitreous humor III COL3A1 Extensible connective tissues such as skin, lung, and the vascular system IV COL4A1, COL4A6 Basement membranes Minor component in tissues, containing collagen I V COL5A1, COL5A6 VI COL6A1, COL6A3 Most connective tissues VII VIII COL7A1 Anchoring fibrils COL8A1, COL8A2 Endothelium, other tissues IX COL9A1, COL9A3 COL10A1 Tissues containing collagen II XI COL11A1, COL11A2 COL2A1 Tissues containing collagen II XII COL12A1 Tissues containing collagen I XIII XIV COL13A1 Many tissues COL14A1 Tissues containing collagen I XV COL15A1 Many tissues XVI COL16A1 Many tissues XVII COL17A1 Skin hemidesmosones XVIII XIX COL18A1 Many tissues (e.g., liver, kidney) COL19A1 Rhabdomyosarcoma cells X 5 Genes Hypertrophic cartilage Collagen Subdivided into classes based primarily on the structures they form C l a s s T y p e F i b r i l - f o r m i nI g, I I , Ne t wo r k - l i k e I V, VI I I , FACI T s XI I , Be a d e d I X, Ot h e r s XI V, f i bVrI iI l s T r a n s m e mdbormaani eX nI I I , XV, XVI I XVI I I V, and XI X f i l a m eVnI t s An c h o r i n g 6 I I I , XVI , XI X A. Collagen All types have a triple helical structure Composed of 3 left-handed polypeptide helices twisted around each other to form a right-handed superhelix * Stabilized by H-bonds between individual polypeptide chains * Helices with approximately 3 residues per turn 7 Collagen Amino acid sequence primarily consists of large numbers of repeating triplets with the sequence of Gly-X-Y 8 X = frequently proline Y = often hydroxyproline or hydroxylysine repeating structure is an absolute requirement for the formation of the triple helix glycine – the only residue with an R-group small enough to fit within the central core of the superhelix proline and hydroxyproline – confer rigidity because conformationally inflexible hydroxyproline- involved in H-bond formation that helps to stabilize the triple helix hydroxylysine – site of attachment of carbohydrate moieties (most commonly glucose and galactose) Biosynthesis of Collagen 1. Synthesis of pro- chain Ribosomes start the synthesis of prepro--chain with a signal sequence Signal sequence directs growing polypeptide chain into the cisternae of the rough ER The signal sequence facilitates the binding of ribosomes to the RER and directs the passage of pre pro chain in thr RER. 9 Signal sequence is rapidly cleaved in the ER to yield pro--chain. Biosynthesis of Collagen 2. Modification of pro chain A. Post-translational hydroxylation of proline and lysine in RER Catalyzed by prolyl hydroxylase and lysyl hydroxylase Requires molecular oxygen and Vitamin C Hydroxylated collagen can be crosslinked to triple helix collagen Vitamin C deficiency-Weak collagenBleeding of gums B. Hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose 10 Modification of pro chain and Ascorbic acid • Ascorbic acid is required as a cofactor • • • • for prolyl hydroxylase and lysyl hydroxylase-Hydroxylation of the proline and lysine amino acids in collagen. Hydroxyproline and hydroxylysine are important for stabilizing collagen by cross-linking the propeptides in collagen. Defective collagen impairs wound healing. Collagen is also an important part of bone, so bone formation is also affected. Defective connective tissue also leads to fragile capillaries, resulting in abnormal bleeding Biosynthesis of Collagen 3. Assembly of pro chains to form procollagen Carboxy-terminal globular domains fold and disulfide bonds are formed. Interaction of these domains initiates winding of the triple helix Winding of the triple helix occurs from the carboxyl towards the amino terminus 12 Summary of steps 1 to 3 for modifications of the pro chains 13 Biosynthesis of Collagen 4. Secretion of procollagen The completed triple helix, with globular domains at each end, moves to the Golgi apparatus. Completed procollagen is released from the cell via secretory vesicles 5. Extracellular cleavage of procollagen molecules N- and C- procollagen peptidases remove the terminal propeptidesReleases the triple helix tropocollagen molecule 14 Biosynthesis of Collagen 6. Formation of collagen fibrils Individual tropocollagen molecules spontaneously associate to form collagen fibrils. Form an ordered, overlapping, parallel array. Adjacent molecules arranged in a staggered pattern, each overlaps its neighbor by a length approximately ¾ of a molecule 15 Biosynthesis of Collagen 7. Assembly of collagen fibrils into mature collagen fiber Cu Fibrillar array of collagen molecules serves as substrate for lysyl oxidase. An extracellular enzyme Oxidatively deaminates some of the lysyl and hydroxylysyl residues forming reactive aldehydes Stabilized by extensive cross-linking-Essential for achieving the tensile strength necessary for the proper functioning of connective tissue. 16 Summary of collagen processing 17 18 Collagen is highly stable Half-life may be as long as several months Any mutation that interferes with the ability of collagen to form cross-linked fibrils almost certainly affects the stability of collagen. Degradation of collagen-Collagenase Cleave intact collagen fibers into smaller fragments that can be further degraded by lysosomal enzymes into constituent amino acids Important during connective tissue remodeling in response to growth or injury of tissue 19 Selected disorders in collagen biosynthesis and structure Disorder Collagen defect Clinical Manifestations Osteogenesis imperfecta 1 Osteogenesis imperfecta 2 Decreased synthesis of type I Long bone fractures prior to puberty Point mutations and exon rearrangements in triple helical regions Poor secretion, premature degradation of type III Decreased hydroxylysine in types I and III Type I procollagen accumulation: N-terminal propeptide not cleaved Decreased hydroxylysine due to poor Cu distribution Hydroxyproline decrease due to ascorbic acid deficiency Perinatal lethality; malformed and soft, fragile bones Ehlers-Danlos IV Ehlers-Danlos VI Ehlers-Danlos VII Cutis laxa (occipital horn syndrome) Scurvy Translucent skin, easy bruising, arterial and colon rupture Hyperextensive skin, joint hypermobility Joint hypermobility and dislocation Lax, soft skin; occipital horn formation in adolescents Poor bone growth, poor wound healing, severe bruising Some Ehlers-Danlos IV and some Osteogenesis imperfecta results form point mutations that substitute another amino acid for glycine. Collagen Diseases 1. Ehlers Danlos Syndrome 21 Heterogeneous group of generalized connective tissue disorders Characterized by stretchy skin and loose joints Delayed motor development Ehlers-Danlos Syndrome (EDS) Inheritable defect in the metabolism of fibrillar collagen molecule Ehlers Danlos mostly affects Type III collagen (blood vessels) EDS can result from: Deficiency of collagen processing enzyme(Lysyl hydroxylase, Procollagen peptidase)-In most cases, the step of collagen synthesis which is disturbed in Ehlers-Danlos is the formation of cross-links Mutation in the amino acid sequence of Collagen I,III or V The replacement of glycine with another amino acid may lead to EDS Glycine is used because it has the smallest side chain (-H) This allows for the three alpha helices to come in close contact when they form the triple helix If glycine is replaced by any other AA, the side chain is bigger This makes it difficult for the alpha helices to come close together Thus, this forms a conformational change in collagen triple helix Collagen Diseases 2. Osteogenesis imperfecta(OI) A heterogeneous group of inherited disorders-Also k/a brittle bone syndrome Distinguished by bones that easily bend and fracture Other common features: Retarded wound healing Rotated and distorted spine leading to a hump-back appearance Type I OI Osteogenesis Imperfecta Tarda Decresed production of α1 and 2 chains Type II OI More severe clinical presentation Death in utero or neonatal period Mutation in gene for pro- α1 and pro -2 chains of Type I collagen Most common mutation-Glycine replaced by bulky side chain amino acid 23 Ehlers-Danlos v/s Osteogenesis Imperfecta Ehlers-Danlos Defect in processing of collagen Leads to hyperelasticity and hypermobility Mostly affects collagen type III Incidence: 1:5,000 Osteogenesis Imperfecta Defect in amount of collagen processed Symptoms of brittle bones and bruising Hearing loss and dental imperfections Mostly affects collagen type I Incidence: 1:10,000 The 12-year-old son of a circus contortionist says that his skin scars and bruises easily. Physical examination reveals distensible skin and hypermobile joints.You suspect an inherited defect in which of the following molecules? A. Fibrillin B. Laminin C. Fibronectin D. Collagen E. Keratin F. Elastin A 64-year-old woman presents to your office complaining of bleeding gums while brushing her teeth. Physical examination reveals gingival bleeding and perifollicular hemorrhages. On careful history, the patient states that she lives alone and that her diet primarily consists of tea and toast. This patient’s symptoms are most likely caused by enzyme hypoactivity in which of the following compartments? Nucleus Cytoplasm Golgo apparatus Rough endoplasmic reticulum Lysosomes Extracellular space Mitochondria Elastin Synthesized as a soluble monomer called “tropoelastin” Composed primarily of small, non-polar amino acids Examples: glycine, alanine, valine Also rich in proline and lysine Some proline are hydroxylated to hydroxyproline A few lysine residues are oxidatively deaminated to allysine by lysyl 27 oxidase Occurs after secretion of tropoelastin from the cell Connective tissue protein with rubber-like properties Gives tissues and organs the capacity to stretch without tearing Elastin fibers can be stretched to several times their normal length, but recoil to their original shape when the stretching force is relaxed Found in lungs and walls of large blood vessels, elastic ligaments, and skin Elastin Desmosine A tetrafunctional cross-link unique to elastin Formed from 3 allysines and 1 lysine Formation results in an extensively interconnected, rubbery network • Can stretch and bend in any direction when stressed • Gives elasticity to connective tissue 28 Elastin Characteristics of mature, extracellular form: Highly insoluble Extremely stable Has a very low turnover rate Exhibits a variety of random coil conformations - permit the protein to stretch and subsequently recoil during the performance of its physiologic functions Elastin degradation Catalyzed by Elastase- Neutrophil elastase A powerful protease Released into the extracellular space Degrades elastin of alveolar walls as well as other structural proteins in a variety of tissues Can destroy alveolar wall if unopposed by 1antitrypsin 29 1- antitrypsin /1-AT/AAT/1-antiproteinases Inhibits proteolytic enzymes that hydrolyze and destroy proteins Present in blood and other body fluids Most of the 1-antitrypsin in plasma is synthesized and secreted by the liver The rest is synthesized by several tissues including monocytes and alveolar macrophages. 1- antitrypsin deficiency Due to different gene mutations Inheritance of 2 abnormal alleles leads to an increased risk for emphysema 1-AT methionine-Required for binding AAT to target proteases. Smoking causes oxidation and inactivation of methionine residuesmokers with AAT-Poor survival Treatment: weekly intravenous administration of 1- antitrypsin 30 Collagen v/s Elastin Collagen 1.- Many differentgentic types 2.- Triple helix Elastin One genetic type No triple helix; random coil conform ations allowing stretching 3.- (Gly-X-Y)n repeating structure No (Gly-X-Y)n repeating structure 4.- Presence ofhydroxylysine No hydroxylysine 5.- Carbohydrate containing No carbohydrate 6.- Intram olecular aldol crosslinks Intram olecular desm osine crosslinks 7.- Presense of extension peptides No extension peptidesduring biosynthesis during biosynthesis 31 Concept map for fibrous proteins, collagen, and elastin 32 Thank You