CT Flashcards
Extracellular matrix vs CT
ECM is material outside cells and epithelial/endothelial borders
CT is cells AND ECM
What tissues are CT?
Bone, cartilage, ligament, skin, teeth, tendon, basement membrane, blood vessels, adventitia, cornea, intestinal walls
Function of CT
- stabilize physical structure of tissues
- ACTIVELY regulate cell behaviour (development, migration, proliferation, cell shape, metabolic functions)
Characterization of CT based on chemical composition
- proteins
- proteoglycans
- glycoproteins
4 steps of elastin fiber synthesis
- transcriptions (tropoelastin and microfibril components)
- translation
- secretion
- fiber formation
ELN trascription regulation
ELN is gene encoding tropoelastin
Increased transcription:
IGF-1/insulin like growth factor 1 and TGF-beta/transforming growth factor beta
Decreased transcription:
TNF-alpha, bFGF (basic fibroblast growth factor), HB-EGF (heparin binding epidermal growth like factor)
Tropoelastin
- encoded by ELN
- monomer, covalently crosslinked into elastin (making very insoluble)
- secreted by ECM where it associates with microfibrils (fibrillins, MAGPs, fibulins, EMILIN-1)
Cross linking of tropoelastin
- lysyl residues covalently crosslinked by lysyl oxidase
- makes elastin very insoluble
Elastin fiber physical properties
expands and contracts (stretches). amorphous
Treatment of CT with NaOH and high temperature
- solubilizes virtually all proteins
- since elastin is so insoluble, what’s left after treatment is pure elastin
elastase
cleave insoluble elastin
elastin related disorders (6)
- cutis laxa
- supravalvular aortic stenosis
- William’s syndrome
- COPD
- Atherosclerosis
- Aortic aneurysm
Role of ELN gene in tooth movement
- After external pressure, mRNA levels of ELN increased significantly
- ELN gene could then be important in orthodontic tooth movement
Marfan’s syndrome
-mutation of fibrillin (microfibril related disorder)
Fibrilin mutation –> TGF beta –> matrix metalloproteases –> tissue degredation –>
- Cardiovascular, ocular, skeletal (bone overgrowth and joint laxity) problems
- periodontal disease
Proteoglycans
definition, location, physical properties
- form highly hydrated, gel like ground substance
- direct and indirect (via other substances being imbedded in proteoglycans) function
- intracellular, membrane associated or extracellular
- carbohydrate rich with some proteins
- bind water and cations
- polyanions
Proteoglycans function
determine visoelastic properties of joints, withstand compression, permeable, lipid metabolism, hemostasis and thrombosis, matrix assembly and cell adhesion, migration, proliferation, and differentiation.
proteoglycan associated disorders (4)
- osteoporosis
- osteoarthritis
- Ehler’s Danlos syndrome (progeroid varient, skin fragility)
- corneal diseases
Proteoglycans in the oral cavity
- postulated roles in tooth development, dentin formation/mineralization, adhesion and fusion of palatal shelves,
- roles remodeling of oral tissues –> involved in periodontal regeneration (decorin up-regulated and biglycan down-regulated)
- perlecan (heparin sulfate) may stimulate bone regeneration bu also may play role in tumorigenesis
Proteoglycan structure
- core protein
- covalently linked GAG
- proteins and GAG = proteoglycans
- different core proteins, GAG types and number of chains, and chain length of GAG makes differences in PG
GAG basic structure
- disaccharide repeating unit (long or short)
- have either glucosamine or galactosamine (amino sugar derivatives)
- one neg charged sugar
- neg charged groups either COOH or sulfate groups
- hydration and charge vary
Hyaluronic acid
heteropolymer of alternating glucuronic acid and N-acetylglucosamine
the only GAG that is not covalently linked to protein core (Free)
Major GAGs (6)
chondroitin 6-sulfate, keratin sulfate, chondroitin 4 sulfate, heparin sulfate, heparin, dermatan sulfate
Aggrecan aggregate
- large(!!!), complex, proteoglycan
- aggrecan monomers associate with hylauronic acid to make aggrecan aggregate
Glycoprotein subfamilies (3)
Fibronectins (widely distributed)
Laminins (basal laminae)
Collagens (most widely distributed protein in body)
Glycoprotein-carbohydrate
-glycoproteins covalently bound to carbohydrate:
- the amide NH2 group of asparagine: N linked
- hydroxyl OH group of serine or theonine: O linked
- hydroxyl OH group of hydroxylysine (only in collagens): O linked
Role of sugars in glycoprotein
- make more hydrophilic
- help protein folding/tertiary structure
- stabilize against hydrolysis and modulate biding of some components
Linker proteins
Some glycoproteins are linker proteins
- Fibronectin
- laminin
- chondronectin: in cartilage; links chrondocytes, type II collagen, cartilage proteoglycans
- thrombospondin: in blood plasma/ECM; links fibronectin, laminin, type V collagen, cell surface receptors, fibrinogen
- vitronectin: bone
- entactin: basal laminae
- nidogen: basal laminae
Fibronectin
location/function
- in ECM/blood plasma
- links types I, III, IV collagen, cell surface receptors, heparin, heparin sulfate, hyaluronic acid
- enable cells to interact with ECM
- determine cell morphology, embryonic cell migration, adhesion, differentiation, wound healing, structural support
Fibronectin/cancer
many cancer cells lose ability to synthesize fibronectin: may lead to migration of cancer cells in metastatic cancers
Fibronectin in oral cavity
-dental mesenchyme differentiating into odontoblasts regulated by fibronectin
Fibronectin structure
- family of glycoproteins
- 5% carbohydrate by weight
- 2 chains linked by disulfide bond near C terminal making V structure
- RGD or RGDS sequence in cell surface receptor binding domain (for binding cell surface receptors and ECM)
Laminin
location/function
- in basal laminae
- links type IV collagen (high affinity), fibrin, heparin sulfate GAGs, other laminin molecules
- enable epithelial cells to interact with ECM
- maintain polarized differentiated phenotype of epithelial cells, embryonic nerve axonal outgrowth, nerve regeneration, adhesion, motility
Laminin in oral cavity
- laminin-332 (laminin 5) contributes to growth and terminal differentiation of ameloblasts, and enamel matrix formation
- expression of alpha2 and beta2 chain in fetal oral squamous epithelium and in adult oral squamous cell carcinomas
laminin structure
- family of glycoproteins
- 13%carbohydrate by weight
- special domains bind cell surface receptors to ECM
- 3 polypeptide chains (alpha, beta, gamma); disulfide linked heterotrimer
- polypeptides in cruciform (cross) shape
- self associate to form aggregates
laminin nomenclature
- indicates chain composition of alpha, beta, gamma chain
ie. laminin 332 is (alpha3, beta3, gamma2)
Integrins function
- transmembrane glycoproteins
- cytoplasmic side binds to cytoskeletal components
- extracellular side binds to fibronectins and laminins (forms basis for linker protein-mediated communication)
- activation via outside in or inside out signalling
integrin structure
- transmembrane glycoproteins
- 2 polypeptides (alpha and beta)
- RGD/RGDS sequence on linker proteins binds specificially to integrin receptors
- activation of integrins can lead to clustering and formation of focal adhesions
Collagen basics
- structural role (stress bearing), cell attachment, differentiation, chemotaxis
- 1/4 of proteins in vertebrates in collagen by weight
- 28-30 types
Steps in collagen fiber formation (7)
- transcription
- translation
- post translational modification
- secretion as procollagen
- processing to tropocollagen (not all, type IV doesn’t)
- associate with ECM
- insolubilization into ECM
Collagen nomenclature
- 3 polypeptides: either homotrimers (Types II and III) or heterotrimers (Types I, IV, V)
- each polypeptide is alpha chain
- 1 for homotrimers; 1 and 2 or 1,2,3 for heterotrimers
- roman numerals for collagen type designation
ie. Type I collagen: alpha1(I), alpha1(I), alpha2(I)
Type II collagen: alpha1(II), alpha1(II), alpha1(II)
Collagen structure
- tertiary: left handed type II trans helix
- quaternary: right handed superhelix
- fibrous proteins
- form insoluble fibers with high tensile strength
- aa composition: 1/3 glycine; charged/positive aa (proline, Hyp/hydroxyproline, hydroxylysine); polar, basic aa also found
- largely repeats of gly-X-Y (often x=pro, y=hyp)—>regular repeating structure
Collagen gene transcription control
TGF-beta signal from outside cell to stimulate gene transcription via SMAD
IL-1beta signal from outside cell to stimulate gene transcription
TNF-alpha signals from outside cell to inhibit gene transcription
Several transcription factors interact with enhancer/repressor elements in promoter region
collagen gene transcription nomenclature
Eg
COL1A1, COL1A2, COL3A1
Collagen translation
-synthesized on membrane bound ribosomes as larger precursor
collagen post translational modifications
- occur cotranslationally
- include hydroxylation of prolyl and lysyl residues, and glycosylation of hydroxylysyl residues
prolyl hydroxylase
- hydroxylates prolyl residues in collagen
- has Fe++ ion at active site
- dioxygenase
- requires ascorbate
- proline hydroxylated at C4 only if pro is on amino side of gly (different enzyme if on carboxyl side)
Scurvy
- dietary deficiency of vitamin C (ascorbate)
- poor calcification of developing teeth (inhibition of collagen formation of dentin & cementum)
lysyl hydroxylase
- hydroxylates lysyl residues
- free lysine not a substrate
- hydroxlation of lysine only if on amino side of glysine
- requires ascorbate
Ehler’s-Danlos Type VI
- lysyl hydroxylase dificiency
- lowered crosslinking of collagens
Collagen glycosylation
- n linked or O linked
- unique to collagens: glycosylated hydroxylysyl residues
- glucose/galactose disaccharide
- degree of glycosylation varies with tissue type
procollagen
-after post translational modification two alpha1(I) and one alpha2(I) procollagen associate to form triple helical procollagen
-formed in lumen of ER, packaged and secreted by passing through Golgi
-h bonding between N-H of Gly and O of suceeding reside of other chain in 3 polypeptide chains
-vertically staggered so gly-x-y on same level along helix axis
STRONG
Collagen stability
depends on:
- h bonds between N-H of Gly and C=O of second residue in triplet on other chain
- gly as 3rd aa (makes close packing of aa)
- Hyp content (more is more stable, -OH groups also participate in H bonding)
- total imino acid content (more is more stable)
Animals with higher body temperature need higher Tm so more stability.
Tm for measuring collagen stability
measure temperature vs viscosity
Tm is temperature when 1/2 helical structure is lost
OI (osteogenesis imperfecta)
- mutation of single glycine to cysteine at residue 988
- triple helix disrupted
- skeletal deformities, brittle bones
Ehler’s-Danlos Type IV
- defects in type III collagen (gly, skip exon)
- thin, translucent skin
- bruise easily
- rupture arteries
- perforate intestines
- rupture uterus
Collagen modification in ECM
- procollagen peptidases cleave N and C terminal of Types I, II, II (not IV) –> product called tropocollagen
- N terminal propeptides have intrachain disulfides, C terminal propeptides have interchain disulfides
- tropocollogen contains entire Gly-x-y repeat region and telopeptides
Ehler’s-Danlos Type VII
- from deficiency in procollagen peptidases
- hyperextensible skin that bruises easily
- dislocation of major joints
Collagen fibril assembly
Types I, II, III
tropocollagen molecules align with 1/4 stagger with 400A gaps between head and tail (site of cross link formation and nucleation site for calcium deposition in bone; also dark part in stains)
adjacent rows have 680A gap between them
Cross linking collagen into fiber
- oxidation of lysyl and hydroxylysyl (only in collagen) catalyzed by lysyl oxydases
- spontaneous condensations of products from above form covalent bonds between adjacent tropocollagen molecules
lysyl oxidase
- ε amino termini of lysyl residues converted to aldehydes in the presence of O2
- requires Cu++
Hydroxypyridinium
AKA pyridinoline
- cross link unique to collagen
- formed by cross link of two hydroxylysyl residues and a lysyl residue in collagen
Desmosine
cross link unique to elastin
- 3 lysines converted to allysines via lysine amino oxidase
- aldol condensations form desmosine (or isodesmosine) cross link
Collagenases
- cleave collagen
- in family MMP (matrix metalloproteinases)
- cleave in the triple helical region of collagens
- critical to tissue remodeling in vertebrates
- in vertebrates cleave ~1/4 from C terminus
