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Terms in this set (121)
ECM is an interlocking meshwork of ________________ macromolecules.
-Structural proteins (collagen, elastic fibers)
-Glycosaminoglycans (GAGs) (polysaccharide)
The 2 main types of Collagen in our bodies:
Name 2 Most abundant structural proteins of the ECM:
_______________ gives connective tissues their tensile strength
_______________ gives connective tissues their elastic properties
pulling on a cable vs rubber band
Basic unit of Collagen is called _________________
Explain what's so unusual and unique about Collagen's amino acid composition:
-Every 3rd residue is Glycine (small steric constrains & flexible a.a.)
-Very abundant Glycine + Proline (each takes up 1/3 a.a. composition)
-Hydroxyproline (many prolines post-translationally modified & hydroxylated)
-One of the MOST heavily post-translationally modified proteins (HYP most common)
Tropocollagen (chemical structure) has a shape and looks like
-Triple-helix: 3 intertwined polypeptide chains (1000 residues each)
-May be homotrimer or heterotrimer
-Narrow & slim & LONG structure (like me)
-Each polypeptide is a tightly wound helices (NOT alpha helix).
-Very Unique amino acid composition
30% of collagen a.a. composition is made of ______________ ;
Another 30% is made of ______________.
30% = Glycine
Another 30% = HYP + Proline
_____________ amino acid in collagen is the point of flexibility where it bends easily due to _______________.
-lack of side chain, few steric hindrance
______________ amino acid in COLLAGEN has a unique Cyclic structure and can adopt least conformations b/c of its steric constraints.
Name 3 most abundant amino acids that make up Collagen protein :
Every time the 3 polypeptide chains come together to form Collagen triple-helix, The CENTER of this supercoiled helix can only accommodate _______________ residues.
(Other residues cause steric crash)
Osteogenesis imperfecta affects ____________ amino acids of collagen, destroying the integrity of the structure of collagen.
Collagen hides the ___________ from the aqueous environment and exposes ___________ on the surface.
2 major driving forces of Protein conformation:
1. Hydrophobic core in the center and Hydrophilic residues exposed on the outside
2. Formation of Hydrogen bonds
Collagen predominantly adopts (alpha helix/ Beta sheet) conformation
It has a UNIQUE structure that doesn't resemble the majority of other proteins
Every 3rd residue in collagen is ________________.
_____________ are the dihedral angles of the polypeptide chain in Collagen - indicates how atoms are arranged.
Phi and psi angles
______________ residue allows a wide range of Phi and Si bonds in Collagen.
In collagen, Only ___________ residues can fit in the crowded center of the helix.
______________ residues promote unusual conformation of Collagen protein- tightly twisted extended helix with 3 residues per turn.
Proline + Hydroxyproline (HYP)
fibrils of collagen triple helix are further strengthened by
-Fibrils further strengthened by intrachain & Interchain crosslinks
+ stereoelectronic effects stabilize the collagen triple-helix
-Interchain H-bonds + Pro/Hyp stereoelectronic effects stabilize this helix
Collagen triple helix has how many residues per turn?
Collagen triple-helix structure is strengthened & stabilized by ________________.
1. Interchain Hydrogen bonds + Pro/Hyp stereoelectronic effects
2. Intrachain + interchain CROSSLINKS.
describe the Hierarchical organization of Collagen fibers:
1. Precursor α chain
2. Procollagen (Triple helix w/ loose ends)
3. Collagen molecule (Triple helix)
4. Collagen fibril
5. Collagen fiber
3 Major Post-translational modifications of Collagen:
1. Hydroxylation of proline and lysine residues
2. Proteolytic processing in EC space: removal of N-terminal & C-terminal domains of procollagen
3. Collagen cross-linking by lysyl oxidase
3 enzymes required for Collagen Hydroxylation:
(adding an -OH)
Which post-translational modification of Collagen occurs inside the cell?
Which post-translational modification of collagen occurs OUTSIDE the cell, after it's been secreted out?
In the ER - Hydroxylation of Prolines+ Lysines
Extracellular - 1) Proteolytic cleavage of N-terminal and C-terminal domains of Procollagen
2) Cross-linking of collagen by lysyl oxidase
All enzymes of Collagen Hydroxylation require _____________ in order to properly carry out its function.
Fe, O₂, Ascorbic acid (vitamin C)
-use O₂ as substrate
→requires Ascorbic acid (Vitamin C) to maintain Fe in Fe⁺⁺ (II) state.
What role does Ascorbic acid have in Collagen?
-helps maintain Fe (Iron) in Fe⁺⁺ state by preventing it from being oxidized to Fe (III) which renders Hydroxylation enzymes INACTIVE.
think of a rusting iron ship with sailors on it, the oranges (ascorbic acid) help keep the sailors healthy (no scurvy) but the ship keeps rusting (keeps hydroxylation enzymes active)
Explain why Scurvy cause connective tissue dysfunction:
you need vitC to hydroxylate prolines and lysine on the procollagen inside the ER. a lack of vitC (scurvy) disables the hydroxylating enzymes for post-translational modification
Scurvy is due to long-term deficiency of Vitamin C in diet. Integrity of collagen requires Hydroxylating enzymes for PTM, and these enzymes require Ascorbic acid to remain functionally active.
The enzyme responsible for Collagen cross-linking is ________________
Enzymes in charge of Collagen hydroxylation are ________-dependent ________________ that use _________ as substrate.
Enzymes in charge of Collagen crosslinking are ________-dependent _______________ that use _________ as substrate.
Fe⁺⁺-dependent Prolylhydroxylase & Lysyl hydroxylase ; O₂
Cu-dependent Lysyl oxidase ; O₂
The "rope-winding machine" of collagen is located at _________________
N-terminal and C-terminal domains of Tropocollagen
(Which later gets cleaved when Tropocollagen gets secreted extracellularly b/c no longer needed)
Tropocollagen is also referred to as ___________________
Explain the mechanism of how Collagens are Crosslinked:
Lysyl oxidase enzyme kicks out amine group (deamination) and allows formation of reactive aldehyde groups in lysine residues →These react with each other and with other residues to form extensive covalent cross-linking within collagen fibers
(It is NOT the enzyme creating the cross-linking; rather it is the reactive aldehyde groups ,created by the enzyme, that react to form crosslinks)
Explain the overall process of collagen Biosynthesis and Assembly:
1 Translation on ribosome
2. Hydroxylation of Proline + Lysines
3. Release from ribosome + addition of ER sugars
4. Folding of globular domains and formation of TRIPLE HELIX
5. Secretion from cell
6. Removal of N- and C-terminal domains
7. Deamination of Lysine residues to form aldehydes and form CROSSLINKs.
→assembled into higher order structure
3 Example diseases associated with Collagen:
-Osteogenesis imperfecta (mild, severe form)
Pattern of inheritance for Osteogenesis imperfecta is predominantly _________________.
Hydroxylation of prolines and lysines take place in _______________ intracellularly.
(Secreted protein from ribosome is inserted into ER where PTM takes place)
Formation of _______________ chemical group is important in creating cross-links within collagen fibers.
Explain how Osteogenesis imperfecta cause abnormality of collagen at molecular level:
Gly to Ala missense mutation leading to replacement of even one Glycine in the repeating Gly-X-Y sequence can cause structural pertubation and clinical consequence.
2 forms of Osteogenesis imperfecta:
Mild - Type I
Severe - Type II, III, IV
What are the 2 Different mechanisms that lead to Mild type vs. Severe type of Osteogenesis imperfecta:
Mild type - Total LOSS of gene product
Severe type - Missense mutation of glycine to alanine
Total loss of gene product of collagen causes (mild / severe) form of Osteogenesis imperfecta
Describe why the total loss of gene product leads to Mild form of OI, where as a small missense mutation of glycine to alanine causes the Severe form of OI:
Even though Total loss of gene product leads to an allele being absent, there's ANOTHER copy of allele that is still functional and capable of making collagen --> doesn't affect the integrity of collagen at all, it just delivers HALF the amount of proteins than normal --> Mild phenotype
With Missense mutation, HALF the proteins are perfectly functional, but the other HALF are dysfunctional due to mutation --> "every good protein is connected to bad protein", bad connection --> collapse of entire structure --> severe phenotype.
Why does a TOTAL loss of gene product STILL lead to a phenotype even though Osteogenesis imperfecta is a Autosomal dominant disease?
OI manifests Haploinsufficiency. (half genes present in important gene, not enough= phenotype)
-Normally heterozygous for LOF allele often have no phenotype, because the consequences are masked by presence of one WT allele. However, in proteins that are normally abundant & critical to cellular functions are EXCEPTIONS to the rule, whereby a deletion/ inactivation of a single gene copy leads to an abnormal phenotype - b/c the remaining functional copy of gene is NOT ADEQUATE to produce normal function (50% level of function is not sufficient)
How are the Collagen products affected as a result of total loss of gene product vs. Missense mutation of glycine to alanine?
<Total loss of gene product>
-functional peptides produced, but only 50% of normal amount generated --> don't have enough to build all the CT in our body. Still normal function.
-50% have defective proteins, which make connections with the other half functional proteins --> perturbation of integrity --> disturbance of packing & Disassembly.
______________ is a phenomena in which a single functional copy of a gene is insufficient to maintain normal function.
Structure of Elastic fiber:
-Basic functional unit : "Elastin"
-Consist of core region of Elastin surrounded by a thin outer envelope of Microfibrils
-Elastin has alternating stretch of hydrophobic + hydrophilic regions
-Microfibrils composed of glycoprotein + fibrillin
-Covalent bond between hydrophilic regions of LYSINE residues
In Elastic fibers, there are abundant covalent bonds made inbetween _________________
Hydrophilic Lysine residues
Main components of microfibrils of Elastin :
Marfan syndrome is a CT disorder caused by mutation in _________________
(component of Microfibrils, a thin outer envelope of elastic fiber)
_______________ in Elastic fibers plays a role in organizing the structure that make up elastic fibers.
What shape do Glycosaminoglycan polymers have?
(some uronic acids have COO⁻& some NAGlu, NAGal have SO₄⁻ that give dense negative charge → linear, extended conformation to minimize repulsive forces)
_________________ enables the Elastic fibers to extend/ stretch easily by breaking up the bond.
Noncovalent interactions of tropoelastin subunit.
Etiology of Marfan's syndrome:
-mostly DE NOVO (from new) mutation
-No family history
(Fibrillin has a very long protein, prone to de-novo mutations)
What makes Marfan's syndrome and OI have a prevalent de novo mutations that make these diseases so common in population?
-Very very long amino acid sequence (longer it is more targets for mutation)
-Containing amino acids that are UNIQUE in structure also makes them prone to de novo mutations. For example:
-Fibrillin has a LOT of cysteines, a unique amino acid that no other residues can substitute.
-Collagen's amino acid sequence has a strict adherence to having Glycine every third position, and no other residues can replace this position.
-Collagen also has Unusual Prolines that no other amino acid can replicate
--> A small mutation can be lethal in functionality due to how unique collagen is.
Fibrillin in Elastin has abundant ________________ amino acids.
While _____________ resist stretching, ________________ provide complementary function, resisting compression.
________________ is a family of Linear polymers composed of repeating disaccharide units.
Structure of Glycosaminoglycans:
-Linear polymer composed of repeating Disaccharide units
-Uronic acid (glucuronic acid-GlcA) paired with either N-acetylglucosamine or N-acetylgalactosamine (NAGs)
-1 or more Hydroxyls on amino sugar are usually esterified with sulfate
-Very high density of NEGATIVE charge from SO₄⁻ of amino sugar + COO⁻ of Uronic acid
-Pattern of sulfated & nonsulfated sugar residues → specific RECOGNITION by protein ligands that bind electrostatically
-Extended conformation to minimize repulsive forces between charged groups
Which monosaccharide contains the sulfate group?
Which monosaccharide contains the carboxylate group?
N-acetylgalactosamine) NAGs= sulfate
Uronic acid = carboxylate
(aka glucuronic acid - GlcA)
What chemical feature of Glycosaminoglycans makes it adopt a LINEAR polymer conformation unlike proteins where they like to ball up and hide the hydrophobic residues?
Abundant negative charges repel each other --> extended shape.
Also makes it ideal for attracting positively charged molecules.
___________________ serve as a recognition tag by a variety of protein ligands that bind to glycosaminoglycans electrostatically.
Patterns of sulfated and non-sulfated sugar residues of glycosaminoglycans
Glycosaminoglycans assume extended conformation in order to ____________________
minimize repulsive forces between charged groups
______________ is the most abundant Glycosaminglycans in our body.
List all types of Glycosaminoglycans:
_______________ form highly viscous solutions that act as lubricants in synovial joints for example.
-very LONG glycosaminoglycan
-forms highly viscous solutions that function as lubricants
-Appear glassy & translucent
-resist compression in tissues
-contribute to tensile strength & elasticity of cartilage and tendons
-e.g. Synovial joint fluid, vitreous humor
_________________ can limit how quickly the water can diffuse through the ECM.
______________ has the longest polymer of glycosaminoglycans
Which glycosaminoglycan (GAG) is not linked to a core protein?
(All other ones can be covalently linked to proteoglycans)
_______________ contributes to tensile strength of cartilage, tendons, ligaments and walls of aorta.
(ingredients of joint supplement for dogs)
Function of Dermatan sulfate :
-contributes to pliability of SKIN
-found in blood vessels and heart valves
("Pliable" - easily bent, flexible)
______________ type of glycosaminoglycans have no uronic acids.
_______________ are found in horny structures like horns, hair, hoofs, nails, etc.
______________ is a natural Anti-coagulant made in mast cells
-made in mast cells
-released into blood where it binds to & stimulates the anti-coagulant protein antithrombin III.
_____________ is formed by glycosaminoglycans covalently bonded to core protein.
(Glycosaminoglycan/ Core protein) predominantly makes up the Proteoglycan and is often a main site of biological activity
(small contribution from core protein)
_______________ in a Proteoglycan is the main site of biological activity
In a Proteoglycan, Glycosaminoglycan is linked to a __________ in core protein by __________________.
serine residue ;
ECM proteoglycans come in 2 forms:
-Integral membrane proteins
-integral membrane proteoglycan
-3 heparan sulfates chains bind to a variety of extracellular ligands --> modify interactions between ligands and receptors on the cell surface
-often serves as a co-receptor (e.g. GCR)
______________ is a transmembrane proteoglycan bound by 3 heparan sulfate chains which then bind to many extracellular ligands.
-some proteoglycans form very large supramolecular assemblies of many core proteins all bound to a single molecule of Hyaluronate
In a classic example of Proteoglycan aggregates, one very long molecule of ________________ is bound by hundreds of ______________, each of which contains many covalently bound ________________.
-Aggrecan core protein
-Chondroitin sulfate + keratan sulfate chains
______________ mediates the Aggrecan core protein-Hyaluronate interaction in proteoglycan aggregates.
List all Adhesive proteins of ECM:
Interaction between _______________ help attach a cell to the extracellular matrix.
Fibronectin and Integrin proteins
What is the role of Fibronectin and Integrin proteins in ECM?
Fibronectin is OUTSIDE the cell that binds to collagen fibrils.
Integrins are embedded IN PLASMA MEMBRANE.
--> Integrin binds to Fibronectin and tether it to the Cytoskeleton INSIDE the cell, thus transmitting tension across plasma membrane
______________ is a molecule outside the cell that interact with ECM components, especially collagen fibrils.
There are about 20 different types of Fibronectin in our bodies, all of which arise by _________________.
Alternative RNA splicing of the SAME gene
List all diverse Functions of Fibronectin:
-help hold together the ECM (e.g. by linking collagen to cells)
-maintain cell's shape & grow in right direction
How does Collagen vs. Fibronectin differ in terms of how they express & produce 20 different subtypes of proteins?
Collagen - all 20 types encoded by DIFFERENT genes
Fibronectin - all encoded by SAME gene --> undergoes alternative RNA splicing
Bacteria Staphlococcus and Streptococcus have receptors for ______________, which increases their infectivity by ________________.
-by allowing fibronectin to penetrate ECM of host cells.
______________ of ECM play an important role in blood coagulation.
_______________ component of ECM is found in almost all tissues and BLOOD.
______________ is a dimer of 2 chains, held by disulfide bridges
How are Fibronectin content different in Tumor cells?
-LESS production of Fibronectin
--> cells assume more rounded shapes & Not well-anchored in ECM, therefore metastasize.
-exist as DIMER of 2 chains
-two dimers joined by TWO Disulfide bridge
-Each chain is folded into 5-6 domains
-"RGD sequence" & "Synergy sequence" involved in binding to cells
______________ are the receptors for extracellular matrix proteins
Several Integrins bind specifically to ________________ sequence of the Adhesive proteins (e.g. Fibronectin)
-consist of 2 polypeptide chains (α and β)
-embedded in PLASMA MEMBRANE
-MANY different kinds (at least 20)
-Integrins bridge across cell membrane between ECM proteins and cytoskeleton.
--> Function as Transmembrane linkers or linkers
_______________ are the "linker" or "bridge" across cell membrane between ECM proteins and Intracellular cytoskeleton.
ECM is made / secreted by _______________
Fibroblasts and other specialized cells of fibroblast family
(e.g. chondroblasts in cartilage, osteoblasts in bone)
What happens when pieces of embryonic tissue (containing fibroblasts) are placed on collagen gel? What does this imply?
Fibroblasts organize collagen into COMPACT BAND of aligned fibers
--> ECM is made and oriented by cells within it. (e.g. Fibroblasts )
Interaction between _______________ plays a key role in communicating what is happening in the cell interior and exterior.
Extracelluluar Fibronectin forms FIBERS on the cell surface --> aligns/ interacts with ACTIN fibers within the cell.
(red=actin; green=fibronectin; yellow=actin+fibronectin)
Rapid Matrix degradation allows the cells to _________________ during growth.
-CUT through extracellular matrix, enabling them to DIVIDE while embedded in matrix and to travel through it.
Extracellular matrix components are degraded by ______________.
Extracellular proteolytic enzymes (proteases)
2 major proteases that degrade ECM:
-MMP9 (Matrix metalloprotease)
-Plasmin (most well-known/studied serine protease)
How are Matrix proteases regulated to activate only when needed?
1. Confining the proteases to the cell surface by physically attaching them with anchoring proteins
2. Proteases secreted in inactive form, then catalytically activated by Membrane-associated activators close to cell surface
3. Protease inhibitors that prevent diffusion of proteins too far away.
Proteases that degrade ECM reside (inside the cell/ Outside of cell)
OUTSIDE of cell
Tumor cells generate pathway through basement membrane efficiently by __________________
Dysregulation of ECM degradation
2 Mechanisms by which Tumor cells dysregulate ECM degradation:
1. Secrete Urokinase plasminogen activator, which activates Plasmin & collagenase --> induce proteolysis of ECM
2. Secrete Procollagenases, which activate Collagenase --> induce proteolysis of ECM
________________ plays an important role in regulating activities of Secreted molecules (e.g. TGFB)
Explain the role of ECM in regulating secreted molecules:
-provides dense array of growth factor BINDING SITES --> generates large local reservoirs of growth factors, limiting their diffusion and focusing their actions on NEARBY cells.
Another MAJOR contributor to pathology of Marfan syndrome (other than mutation to fibrillin) :
Aberrant TGFB signaling, thus resulting in altered interaction between TGFB and fibrillin-1.
VEGF (Vascular endothelial GF) interacts with ____________________ component of ECM.
Heparan sulfate chains of Proteoglycans
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