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CSB327H1 Midterm: CSB327TT1.docx

14 Pages

Cell and Systems Biology
Course Code
Maurice Ringuette

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CSB327H1 TT1 Notes Made by Man Lai Ho
Extracellular Matrix
Two Major Types of ECMs
Basal Lamina (BL) or Basement membranes: Thin, Sheet-like ECM upon which epithelial cells
oSurround Muscle and Adipose cells, Peripheral nerves
Connective Tissue ECM: Most abundant
oE.g. Dermis underlying epidermis, bone, tendon, cartilage
Matrisome: ≈300 ECM coding genes
Diverse Structural and Regulatory functions of ECM molecules (crit. for multicellular organism
Endow tissues with their distinct and complex biomechanical and physiochemical properties
Serving as Adhesive 3D substratum/scaffold, for cell attachment and migration
oCollagens primarily responsible for shape of tissues
Potent regulator of all cellular activities: Proliferation, Growth, Differentiation, Adhesion,
Migration, Secretion, Communication, Apoptosis, Pattern formation, Tissue remodeling,
Dynamic reciprocity (Cross-talk): what cells secrete, in turn affects their biological activities and
lead to further changes in the activity of surrounding cells
Transition from unicellular to multicellular organisms over a billion years ago required
appearance of gene families that coded for broad spectrum of novel molecules (e.g. cell-cell
adhesive receptors, ECM molecules, Companion cell-matrix receptors)
Secretory pathway of ECM molecules
Nucleus transcription -> mRNA
Peptide/protein translation begins in the cytosol
Signal peptide exposed (no longer masked by ribosome) when ~70 AA have been polymerized
oSignal peptide
Common features: One or more basic AA residues (+) followed by continuous
stretch of 6-30 small, hydrophobic, neutral AAs
Direct protein to ER membrane
Start-transfer signal that opens pore of protein translocator
SRP (Signal recognition particle) binds to the signal peptide causes translation arrest
oMammalian SRP: Six proteins and one RNA
P54 (signal-sequence-binding-domain)
Crystal structure contains large, exposed binding pocket lined with large
number of hydrophobic Methionine residues
Met residues (forming a flexible, unbranched chain) bind to hydrophobic
domain of signal peptides
Only need 1 SRP to recognize all signal peptides
SRP-bound ribosome attaches to SRP receptor found on ER membrane
SRP and SRP receptor displaced (via GTP hydrolysis) and recycled
o3GTP -> 3GDP + 3Pi (inorganic phosphate) hydrolysis, releases SRP and SRP receptor
One GTP for SRP and one GTP for each SRP receptor (SRα and SRβ = dimer)
Translation continues and Translocation begins (Co-translation translocation)
oProtein translocator (Sec61 complex) found in ER membrane, transfer protein into ER
Signal peptidase cleaves off signal peptide on luminal side of ER membrane, releasing mature
protein into ER lumen
CSB327H1 TT1 Notes Made by Man Lai Ho
Hydrophobic signal peptide diffuse into bilayer, rapidly degraded by Signal Peptide Peptidases
Protein From ER -> Golgi apparatus -> Secretory vesicles -> Cell surface -> ECM
Fibroblast cells: Cells of connective tissue that produce and secrete ECM molecules
Occurs in various shapes (e.g. Fusiform/spindle-shaped, Stellate/star-like)
Morphologically heterogeneous
Collagens: 3 Left-handed Polyproline α-chains in a Right-handed coiled coil
Long, stiff helical structure composed of 3 left-handed collagen polypeptide chains (α-chains)
oα-chain AA sequence repeat motif: (Gly-X-Y)n; X and Y can be any AA, often Pro and
Most common exon length (Gly-X-Y)6 = 18 AA, 54 base pairs per exon
Ancestral unit that was duplicated
D banding morphology, crimped with alternating dark and light transverse bonds
Per α-chain: Proline and Hydroxyproline constitutes approx. 22% of AAs
oNo cysteine or tryptophan residues in triple helical domain of fibrillar collagens
3 α-chains Intertwined with each other into a rope-like right-handed helix (Coiled-coil; super-
oGlycine on interior of coiled coil
Molecular organization: α-chain -> Collagen molecules -> Fibrils -> Fibers (Bundle of fibrils)
oFibrils: Principal source of tensile strength in tissues; function as prime determinant of
tissue shape in which they occur
Collagen fibrillogenesis (no need to worry too much about this part)
oCollagen molecule/Tropocollagen): In vivo nucleators (e.g. T5C for Type 1 collagen)
oStaggered molecules: Organizers at site of assembly (e.g. Fibronectin and integrins)
Fibril: In vitro self-polymerization
oFiber: Fibril pattern/orientation (e.g. proteoglycans, glycoproteins)
oFibril forming type 1 collagen has approximately 50 binding partners
Supramolecular assemblies of collagens (Types of collagens; classification)
oFibril forming (Collagen 1, 2, 3, 5, 11)
oNetwork forming (Collagen 4)
oAnchoring fibrils (Collagen 7)
oFACIT (Fibril Associated Collagen with Interrupted Triple helices) collagens (Collagens
9, 12, 14)
oBeaded filaments (ignored)
oHexagonal networking-forming (ignored)
At least 28 different types of collagens encoded by 46 distinct polypeptides; always 3 α-chains
Cell-Matrix Interactions in Fibripositors: Integrins and Actinomyosin Pulling Forces
Integrin: Transmembrane matrix receptor, Heterodimer with α and β subunits
o24 integrin heterodimers derived from combination of 18 α an 8 β subunits in mammals
Low affinity, High avidity (accumulation of multiple affinities)
oIntegrins α1β1 and α10β1 prefer Type V collagen; α2β1 and α11β1 prefer fibrillar
oNo intrinsic enzymatic activity; Signal transduction via protein adaptors, scaffolds,
kinases, Monomeric GTPases
Binds to matrix (e.g. collagen fibril) -> Adaptor/linker complex within endodomain
-> Interacts with actin cytoskeleton (F-actin filaments) -> Actinomyosin contractile
forces via Myosin II motor -> Pulling force on adhesive substrata
CSB327H1 TT1 Notes Made by Man Lai Ho
Signalling crosstalk between collagen-binding receptors
DDRs 1 and 2 (Discoidin Domainreceptors) has intrinsic tyrosine kinase
Heart Fibroblast migration on collagen and shaping of ECM by cells
oEmbryonic heart explants cultured on collagen gel for 4 days, contain mixture of
cardiomyocytes, fibroblasts and Tropocollagen
Tropocollagen makes the dense tracks of collagen
Procollagen cannot form polymers in vitro
oAligned collagen fibres serve as adhesive substratum that promote directional fibroblast
migration; formed between explants mainly due to pulling/tensional forces by fibroblast
Fibrillar collagens (Types 1, 2, 3)
Type I Procollagen molecule: α1(I)2α2(I) Heterotrimer (3 α-chains: 2 α1 chains and 1 α2 chain)
2 distinct genes: COL1A1 gene codes for α1 chains; COL1A2 genes codes for α2 chains
Non-collagenous N-term and C-term
Used as prototype for fibrillar collagens
Tissue specific 3D arrays of T1C; 3D organization reflects directions of forces applied
(Approximate 3 kg/70 kg of body weight)
oParallel bundles (e.g. tendon, ligaments)
oOrthogonal lattices (e.g. cornea)
Insure maximum optical activity
oConcentric weaves (e.g. bone)
oWeaves (e.g. skin)
Western blot analysis of T1C: Separation of pepsin-digested T1C on SDS PAGE
oCollagens fibril/fibres too large to go through SDS-page gel. Pepsin/protease digestion
release collagen molecules from each other
oTag collagen with Anti-collagen antibody (Primary) -> Tag primary with Horseradish
Peroxidase (Secondary antibody) -> Run through gel -> Check chemiluminescence
oDimer and Trimers are molecules that are not cleaved completely
Type II: α1(II)3 Homotrimer
Genetic disease: Some types of chondrodyspla sias
Type III: α1(II)3 Homotrimer
Genetic disease: EDS IV, Aortic aneurysms
Fibrillar collagen synthesis and assembly
Post-translational modifications of fibrillar collagens in ER
Cis-Trans Isomerization of Proline residues (Rate-limiting step)
oRight-handed PP1 chain –(Peptidyl Prolyl Isomerase (PPI))-> Left-handed PP2 chain
Polyproline Type 1 helix (cis-proline repeats); Polyproline Type 2 (trans-proline
oProline: Imino-acid with 5-member ring that is sterically restricted around N-Cα bond
Proline residues disrupt or induce secondary structures
Hydroxylation of Proline and Lysine residues (Occurs before formation of triple helix)
oProline –(Prolyl hydroxylase + Co-factors)-> 4-Hydroxyproline (predominant form) and
Hydroxylation of proline residues greatly increases thermal stability of triple helix
For each Gly-X-Y triplet, 1 H-bond between amine hydrogen of glycine in
one chain and carbonyl oxygen atom of residue X in adjacent chain
α-chains further H-bonded to each other by HO-Pro residues

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