Lecture 2 notes.docx

6 Pages
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Department
Cell and Systems Biology
Course Code
CSB327H1
Professor
Maurice Ringuette

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Lecture 2 – Collagen Classification, nomenclature and supramolecular organization of Type 1 Collagen Fibrils and Fibres Supramolecular Assemblies and Types of Collagens - We can classify collagens based on their supramolecular organization - Fibril forming = signature = t1c o Can see collagen molecules are lined up in a staggered way, can begin to see dark like bands due to repeated staggering - Network forming – e.g. type 4 collagen signature collagen of basal lamina Type 1 Procollagen Molecule - Always a triple helix bc it is collagenous - Signature molecular composition a1(I) a2(I),2considered a heterotrimer o Genes: COL1A1 and COL1A2 for a1 and a2 chains respectively - Homotrimers = three of the same a-chains i.e. only requiring one transcriptional unit Nomenclature - a1(1)a2(II) = does not exist in nature - diff combinations of alpha chains can exist but number in brackets must be same - a1(IV)a2(IV)a3(IV)2 = no, bc four alpha chains cannot exist in a collagenous form - a3(IV)a4(IV)a5(IV) = yes, t4c has 6 genes - Genes  COL(collagen #)A(alpha chain #) e.g. COL1A2 codes for alpha 2 chain of collagen type 1 - Every type of collagen is encoded by a diff transcriptional unit - 46 diff types of transcriptional units for alpha chains Fibroblasts – morphologically heterogeneous - Fibroblasts are CT cells that produce and secrete ECM molecules (e.g. collagens) unlike epithelial cells they can be spindle shaped - Must less polarized than epithelial cell but have internal organization, can be very motile depending on how they’re stimulated - Although epithelial cells can only move as a sheet, fibroblasts can move as a cohort - If stressed enough fibroblasts can turn into myofibroblasts which can pump out a lot of matrix e.g. T1C which hardens the tissue causing fibrosis Tissue specific 3d arrays of T1C: - parallel bundles (e.g. tendon + ligaments) - orthogonal lattices (e.g. cornea), hybrid arrangement - concentric weaves (e.g. bone) - weaves (e.g. skin)  allows for stretching - 3d organization reflects direction of applied forces, Overview of Fibrillar collagen synthesis and assembly - PP enters ER to be translated  hydroxylation of alpha chain  folding and trimerization  exit via secretory vesicles to golgi  secretion out of PM  removal of N- and C- propeptides of procollagen into tropocollagen  staggered self-assembly + cross-linking to form fibril  fibril bundle together to form fiber - Lysyl oxidase = glue for collagen, if you go through fibrosis = over cross linking via lysyl oxidase activity Post-Translational Modifications of Fibrillar Collagens in ER: 1) Cis-trans isomerisation a. Isomerization of proline residues in the ER is rate limiting step b. Right-handed polyproline type 1 (PPI) helix /w cis-proline repeats is converted into left-handed poly-proline type II (PPII) helix via Peptidyl prolyl cis-trans isomerise (PPI) c. Activity of PPI is obligatory; as the nascent a-chain PP is spooled into the ER, PPI acts to convert it into left-handed trans helix form to allow for the final triple helix collagen to become a right handed conformation 2) Hydroxylation: triple helix stability and scurvy a. Main rxn: Proline (Prolyl hydroxylase + Co-factors)  4-hydroxyproline (predominant form) and 3-hydroxyproline b. Hydroxylation of Proline and Lysine residues occurs before triple helix formation, c. This hydroxylation greatly increases thermal stability of the triple helix d. Per a1(I) chain: proline and hydroxyproline constitute approx. 22% of AA e. Enzymes involved: i. Prolyl-3-hydroxylase ii. Prolyl-4-hydroxylase iii. Lysyl hydroxylase f. Co-factors required: ascorbate (Vitamin C), Fe2+, 2-oxoglutarate and O2 g. Wherever hydroxylation occurs = hydrogen bonding h. Scurvy i. Lack of Vita C = Lack of gulonolactone oxidase which is critical in the chain of biosynthesis; only primates and guinea pigs get scurvy bc they are the only mammals that cannot synthesize Vita C ii. Nutritional disease caused by ascorbic acid/vita C deficiencies iii. Symptoms = BV rupturing, blood wept from hair follicles, corkscrew hairs, hemorrhaging beneath fingers and toenails, gum reddening and blackening, bruises can cause internal bleeding, skin became dough-like iv. One of the most rapid forms of collagen remodelling is in periodontal ligaments  undergo remodelling on a more continual basis than our bones i. Interstrand H=bonding i. Collagen w/o HO-proline denatures in vitro as it is heated to near body temp. Levels (@ 37 degrees, <25% helical content) 1. The only thing keeping it together is the glycine h-bonding b/w strands of alpha chains; For each gly-x-y triplet, one h-bond forms bw amide hydrogen atom of glycine in one chain and the carbonyl oxygen atom of residue x in an adjacent chain ii. w/o h-bonds b/w alpha chains provided by HO-proline residues, the triple helix of collagen molecules is unstable @ temp > 20 C o iii. absence of vita c = no HO-pro = triple helix in ER which cannot survive @ body temp  reason for symptoms of scurvy j. a,a’-dipyridyl is an Fe2+ chelator which prevents the hydroxylation of proline residues thus inhibiting the secretion of collagen  used as a form of ER quality control i. Protein synthesis is measured by the incorporation of C14-proline ii. use of this chelator = phenocopying the absence of Vita C 3) Glycosylation and Disulfide Bond Formation a. Glycosylation i. Hydroxylysine residues can be further modified by sugar transferases that add galactose and glucose residues to triple helices ii. Glycosylation is reqd for internalization of collagen via cell surface collagen receptors during ECM remodelling b. Disulfide bond formation and molecular chaperones i. Formation of disulfide bonds by protein disulfide isomerise (PDI) b/w a-chains is reqd for registration @ c-terminal ends (PDI ensures correct initial chain association) ii. The 3 a-chains associate, align and the triple helix forms in a zipper-like fashion from c- to n-terminal iii. Molecular chaperones involved: 1. BiP – Binding immunoglobulin protein/HSP70a 2. HSP47/Serpin H1 – collagen specific ; KO of this protein = no more collagen secretion wrt to formation of stable fibrils a. Does its job and stays in ER, reqd for stable helix assembly iv. triple helix formation is biosynthetically deter
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