Cell-Interactions and ECM Flashcards
The intestinal epithelial lining?
- Epithelial cells(connected by tight junctions)
- Basement Membrane
- Connective tissue filled with fibroblasts
- Two layers of smooth muscle(circular/longitudinal)(responsible for contraction)
Occluding junctions in vertebreates vs invertebrates?
Vertebrates: Tight junctions
Invertebrates: Septate junctions
Where are tight junctions located?
Close to the apical surface
Glucose transport into epithelial cells of the gut?
After a meal glucose in the gut must be taken up. However, there is typically a higher concentration of glucose in the epithelial cells compared to the lumen of the gut. This means glucose preferes diffusing into the lumen. To get the glucose into the cells a sodium/glucose symport transport protein is used
Concentration of Na+ in the gut lumen and epithelial cells?
Concentration of Na+ in the gut lumen is high and low in the epitherlial cells
How does the Na+/Glucose symport protein work?
- PM protein binds glucose, Na+ has diffusion pressure that pushes the Na+ into the cell due to the concentration gradients
- As Na+ is pushed into the epithelial cell it brings the glucose along with itW
Where are the Na+/Glucose symport proteins located?
Only on the apical membrane toward the gut lumen of epithelial cells
Passive Glucose carrier proteins?
These are located on the basolateral side of the epithelial cells
-This protein does not require energy and it releases the glucose from inside the cells into the ECF
Importance of tight junctions in the epithelial cells of the gut?
The tight junctions prevent the glucose transport proteins from going to the wrong side of the epithelial membrane
What can be seen with a LM?
Cannot see anything smaller than 400 nm (ie. bacterial organelles, phages, atoms, lipids, proteins, H2O)
What does Atomic number represent?
The number of protons in a molecule
Why does EM require an ultralow pressure vacuum?
Cannot have air in the electron microscope because it would absorb the electrons and then the image would no longer be focused
Why does EM require 100000V?
This voltage needs to be across the anode/cathode so that the electrons are pulled out of the cathode and are accelerated
Cathode vs anode in EM?
Cathode: Electron source
Anode: Acceleration
Why is osmium tetroxide used in EM?
For contrast, this helps scatter the electron beam so we can see the specimen better
T/F; An ultra thin specimen is required for EM?
True, the specimen must be between 50 and 100nm in thickness
EM vs LM Similarities?
-Both use various lenses(but EM uses magnetic lenses rather than glass)
-Lenses in both cases serve the ame purpose, in EM they focus the electron beam and in LM they focus the light beam
What happens when the voltage is increased in EM?
Higher the voltafe the lower the wavelength and the better the resolution of the image
What molecules are used to examine tight junctions?
Tracer molecules
Example of a tracer molecule?
Lanthanum, the EM electron beam cannot go through it which causes it to be black on the EM. Tight junctions can be seen because the black will stop at the tight junction because the lanthanum can no longer diffuse
Freeze fracture EM?
- Rapid freezing of cells
- Frozen cells are cleaved along the hydrophbic core of the lipid bilayer(between phospholipids)
Tight junction proteins + which is larger?
- Claudins(smaller)
- Occludins(larger)
Claudins?
-Have N and C termini in the cytosol
-Can recognize othe rclaudins
-Provide tight binding
Occludins?
-C and N terminus are both in the cytosol
-Intracellular loops can connect to occludin on the other cells
-Localized to TJs through phosphorylation
-Two isoforms(alternative splicing)
How are tight junctions anchored to cytoskeleton ?
Zonula occludens attach the tight junctions to intracellular actin filaments
How many amino acids does a 65 kDA protein have?
The average mass of an amino acid is 100 daltons
65kDA = 65 000 Da
65000/100 = 650 amino acids
Three major types of cytoskeletal fibers?
- Actin
- Intermediate filaments
- Microtubules
What cytoskeletal fibers are involved in cell junctions?
Only actin and intermediate filaments
Claudin 16 mutations?
Cause a loss of Mg2+ in the blood(hypomagnesemia) and high Ca2+ in the urine(hypercalciuria)
Anchoring junctions two types?
- Actin filament attachment sites
-Cell-cell junctions(adherens)
-Cell-matrix junctions(focal adhesions) - Intermediate filament attachment sites
-Cell-cell junctions(desmosomes)
-Cell-matrix(hemidesmosomes)
Importance of cell anchoring junctions in epithelium?
Reinforces cells to make them more stable and prevents cells from flaking off the BM
Adherens?
-Adherens connect to microfilaments (actin) inside the cell via intracellular anchor proteins
Desmosomes?
-Desmosomes connect to intermediate filaments inside the cell via intracellular anchor proteins
Focal adhesions?
-Focal adhesions are found on the basolateral side of the cell and they connect to the ECM and intracellularly they connect to actin filaments
Hemidesmosomes?
-hemidesmosomes are found on the basolateral side of the cell and they connect to the ECM and intracellularly they connect to IF
Where are adherens located?
-Located just under the tight junctions
-They form an adhesion belt
Adaptor proteins of adherens?
-Catenins
-Vinculin
These connect to the transmembrane adherens and to the intracellular actin
Transmembrane adheren protein?
-Caherins
These form homodimers with cadherins on the other cell
-These are hydrophilic
-These are calcium dependent require Ca++ for binding/stability
Epithelial sheet during embryogenesis?
Adhesion belt is formed between epithelial cell sheet
-During embryogenes the sheet forms an invagination caused by an organized tightening along adhesion belts
-The epithelial tube(neural tube) then pinches off from the sheet which will later give rise to the spinal cord
How does the epithelial sheet fold into an invagination?
The adherence junctions are attached to actin intracellularly, the actin filaments with myosin contract the epithelial sheet
How is myosin activated?
-Myosin has 2 heavy chains with head domains that can only interact with actin when the light chains are phosphorylated
-Myosin light chain kinase which depends on ATP phosphorylates the light chains activating the myosin and allowing it to bind actin
How does Actin-Myosin lead to the invagination of the epithelial sheet during embryogenesis?
Once myosin has been activated MLK, the myosin head domains bind actin. Half the myosin will have their heads pointing one direction and the others will have their heads pointing in the other direction with their tails connection, this forms a bipolar filament. The bipolar filament will then hydrolyze ATP causing a power stroke which pulls the actin filament toward the tails of the myosin
What type of intermediate filaments do desmosomes connect to?
-Keratin in the epithelia
-Desmin in the heart
Where are desmosomes needed?
Everywhere where there are large forces, desmosomes help reinforce the cells
Desmosome transmembrane proteins?
Part of the cadherin family:
- Desmoglein
-Desmocollin
These form homodimers with the same protein on the other cell
Anchoring proteins of desmosomes?
-Plakoglobin
-Desmoplakin
-Plakophilin
These bind to the transmembrane proteins(demoglein/desmocollin) and intracellulary bind to intermediate filament
Pemphigus vulgaris?
Caused by autoantibodies to desmoglein(not genetic/mutations). The immune system attaacks the desmoglein proteins via antibodies which results in severe blistering
Plakoglobin?
One of the anchoring proteins of desmosomes
It bind both desmoglein/desmocollin and provides binding for desmoplakin which binds the IFs
Desmoplakin?
Anchoring protein of desmosomes, that binds the transmembrane protein through plakoglobin and binds IFs
Structure of desmoplakin?
Desmoplakin has a coiled-coil rod domain between its two binding head groups. This allows the protein to attach both IFs and plakoglobin
How are coiled-coil interactions stabilized(3)?
- Coiled coild often form and have hydrophobic residues in their centers away from aqueous environments
- Hydrophilic amino acids are on the outside of the coiled-coils these can form polar interaction with the aqueous environment
- Often charged amino acids will form ionic interaction to stabalize the coiled-coil
WHat coiled coils used for?
To multimerize proteins within the cell in the cytosol or in the ECM
Focal adhesions?
Form cell-matrix interactions with actin
Transmembrane protein of focal adhesions?
Integrins
These bind to the ECM and bind the actin via adaptor proteins
Adaptor proteins of focal adhesions?
Alpha-actinin, talin or filament bind the integrin and then bind to actin with the help of vinculin
Integrins?
-Transmembrane focal adhesion proteins
-Heterodimers(consist of alpha/beta subunits)
-Heterodimer binds to ECM, which can be fibornectin(collagen)
-Also binds anchoring proteins which connect to actin filaments
Hemidesmosomes?
-Cell-matrix junction
-Found on the baslateral side of the cell
-These are attached to the BM(which ECM)
-Inside the cells these are attached to IFs
What cells use hemidesmosomes?
-Epithelial and intestinal cells
Transmembrane proteins of hemidesmosomes?
-alpha6 beta4Integrins
-Heterodimer
-Essential to tether BM to ECM
-Integrins binds adaptor proteins in the cell that bind IFs
-Integrins attach to ECM in the BM
Anchoring proteins of hemidesmosomes?
-Plectin
-These are found inside the cell and bind IFs such as keratin in the epithelial cells
What do hemidesmosome integrins bind to inside the BM?
-Lamins and Collagen type IV these make up the ECM in the BM
Epidermolysis bullosa simplex?
-The least severe form of the disease
-BM is still intact
-Just some aspects going wrong with keratins binding to hemidesmosomes
-BM still intact
Epidermolysis bulls hemidesmosomal?
-Hemidesmosomes lose the ability to connect to the BM
-More severe then simplex form
alpha6beta4 integrin and plectin are most likely affected preventing the connection between the kkeratins and binding to BM
Epidermolysis bullosa junctional?
-BM proteins are affected
-Similar severity to hemidesmosomal
-Lamins are affected affects binding of hemidesmosomes to the BM
-BM still intact
Epidermolysis bullosa dystrophic?
-Most severe case
-Mutations in type 7 collagen which normally anchor the BM to plaques in the dermis
-The BM peels off completely
Communicating junctions?
- Gap junctions
- Chemical synapses
- Plasmodesmata(plants)
Gap Junctions?
-Between two cells
-Channels where molecules can diffuse through depending on their size/mass
What can diffuse through gap junctions?
Up to 1000 dalton molecules can freely diffuse through the channels
-Proteins cannot pass through
Why are gap junctions important?
-Provides coupling between cells
-Can allow for electric coupling since electrons can go through the pores
Proteins that make up the gap junctions?
-Connexins
-Four pass transmembrane protein
-6 connexins form one connexon(half a channel)
-Channels form between connexons
How many different connexins are there?
21 different connexin genes and connexins
Gap that forms between two cells due to the gap junctions?
2-4 nm
Are connexons homomeric or heteromeric?
They can be formed by different or identical connexins
Concentration of Ca2+ in cytosol, Er and ECM?
Cytosol: 100 nM(lowest)
ER: 1 mM(highest due to storing)
ECM: 1 mM(roughly the same as ER but can be higher)
How does Ca2+ regulate gap junctions?
- When there is high cytosolic Ca2+ or low pH the gap junction is shut
- When there is low cytosolic Ca2+ or high pH the gap junction is open
How does the gap junction open due to high Ca2+ concnetration in the cytosol?
Ca2+ binds directly to connexins of gap junctions through a protein called calmodulin which open the channel
What causes a high Ca2+ concnetration in the cytosol during cell death?
During cell death the cell can no longer maintain the PM which means it can also no longer maintain the gap junctions. This means Ca2+ will enter into the cell cytosol
Why do neigbouring cells gap junctions close when a cell dies?
To prevent the Ca2+ from the dying cell from entering the healthy and functional cell
T/F: Gap junctions are heterogenous in terms of their size?
True, gap junctions be made of many connexins or just few
Name the cell junctions from most apical to most basal?
- Tight junctions
- Adherins(cell-cell)
- Desmosomes(cell-cell)
- Gap junctions(close to BM)
- Hemidesmosomes(cell-BM)
- Focal adhesions
Cell junctions?
-permanent
-Stability
-Communication
Cell adhesions?
-Transient anchorage
-Cell-cell recognition
-often before cell junctions are established
-Partially overlapping molecules(cadherins, integrins)
Cell adhesion molecules and their role in embryogenesis?
- Cells at the top of the neural tube become neural crest cells
- Neural crest cells migrate to a new location and differentiate into peripheral ganglia
- This is all controlled by CAMs
T/F: Each part of the developing embryo expresses a different cadherin?
True, and since cadherins tend to bind to the same type of cadherins cells expressing the same cadherins tend to be held together
Describe embryonic tissue?
Flimsy tissue with no structure
What happens when you mix cells with EDTA and trypsin(protease)? c
Can easily dissociate cells of the embryonic tissue using a protease which degrades proteins such as cadherins which are holding the cells together. Using EDTA also help dissociate cells because it binds to metals such as Ca2+ ripping them out of proteins
Disassociation constant of EDTA for Ca2+?
EDTA has a very low disossiation constant for Ca2+. The lower the constant the stronger it binds to Ca2+ and is able to pull it out of proteins
What happens if you mix dissociated cells from two different embryonic organs(liver and retina)?
Liver cells will find each other and the kidney cells will find each other
Cadherin-dependent cell sorting?
If you mix E-cadherins with N-cadherins the cadherins will always sort out and separate from each other. Even if you mix cells expressing low levels of cadherins with cells expressing high levels of cadherins thos expressing high levels will associate and the lower ones will also associate
Most frequent type of cell-cell adhesion?
Homophilic binding (both molecules that are binding are identical)
Preferred mechansisms of cell-cell adhesion?
- Homophilic binding
- Heterophilic binding(one molecule on the cell surface recognizes another molecule on the other cell)
Cell-Cell adhesion through extracellular linker molecule?
-Ocasionally possible
-An adaptor protein connects two cell by binding molecules on both cells
Cell adhesion molecules and are they homophilic or heterophilic?
- Cadherin(homophilic)
- Ig-superfamily CAMs
- Selectins(heterophilic)
- Integrins(heterophilic)
Classical cadherins?
-Intracellular domain
-Transmembrane domain
-Cadherin extracellular domain(binds to other cadherins)
Fat-like cadherins?
-Have lots of extracellular cadherin domains and can reach out super far into the extracellular space to bind other cadherins
Seven-pass TM cadherins?
-Pass the PM 7 times
T-Cadherin?
Don’t have transmembrane domains instead they are tethered by GPI anchors to the membrane
Structure of the classical cadherins?
-INtracellular domain
-Transmembrane domain
-Extracellular domain made up of multiple cadherin repeats
-Between each of the cadherin domains are Ca2+ binding domains
-Three Ca2+ bind before the terminal repeat (three binding sites instead of two on the terminal repeat)
-Lots of beta structures make up the cadherin repeat domains
Function of Ca2+ in cadherins?
-Makes the protein more stable
-When you use EDTA to remove the Ca2+, the protein becomes floppy and sensitive to proteolytic cleavage
Homophilic interactions of cadherins?
-Homodimer interactions between cadherins occur at the terminal cadherin domains
-Ca2+ binding is necessary to provide the correct conformation
Affinity vs Avidity?
Affinity: Describes the interaction of 2 components (high affinity = spontaneously bind)
Avidity: Accumulated strengths of multiple affinities
Affinity and avidity of cadherins?
Affinity: Cadherins have a low affinity for each other but cadherins tend to cluster which results in a high avidity, all of the affinities accumulated to create a high avidity
Compaction of an early mouse embryo?
1.5 days: Cells not tightly bound
4 cell stage: cells still not tightly bound
8-16 cell stage: E-cadherin interactions start resulting in cells much closer together
3.5 days(32 cells): cells extremely tight
Exons encoding the constant region of cadherins?
-C1, C2 and C3 are all exons that encode the intracellular portion of cadherins
-This region is always the same
Exons encoding the variable region of cadherins?
-V1-V15 exons
-These are large exons more than 200bp in length that encode for the entire extracellular + TM portion of the cadherin
-Each exon has its own promoter
-The constant region is only encoded by one of these exons
How is protocadherin gene spliced with variable region V8?
- Protocadherin gene contains all the variable exons and all the constant exons
- Transcription of the gene starts at V8 and encodes from V8-V15 and then C1-C3
- Then the mRNA is spliced to remove genes V9-V15
- Now there is a mature mRNA with V8-C1-C2-C3 this is then translated into the functional protein
Selectins structure?
-Bind to oligosaccharides on other cells via a lectin domain
-Extracellular domain made up of selectin domains with lectin domain on the end
-Transmembrane region
-Intracellular egion connected through anchoring proteins to actin
Types of selections?
-L-selectin (lymphocytes)
-P-selectin (platelets, endothelial cells)
-E-selectin (activated endothelial cells)
All have to do with blood vessels
How do white blood cells deal with inflammation ?
They must come out of the blood and enter the tissue
How do white blood cells come out of the blood which is flowing rapidly?
- WBC start rolling along the endothelial layer to slow down(they do this by attaching and detaching from endothelial cells)
- They attach to endothelial cells because the endothelial cells express selectins and WBC have oligosaccharides on them that bind to the selectins
- In order to get into the tissue the WBC must stop rolling completely. They do this by forming a second cell adhesion
- Integrins form the second cell adhesion which allows WBCs to stop completely on the endothelial layer and migrate into the inflammed tissue
WBC: Weak adhesion and rolling vs atrong adhesion and emigration?
Weak adhesion and rolling:
- selectin dependent
Strong adhesion and emigration:
-Integrin-dependent
Structure of Ig-like cell adhesion proteins?
-Comes in different alternatively spliced varieties
Some have long intracellular domains some are GPI anchored.
-Fine-tuning interactions weaker than cadherins, they often coexpressed with cadherins to increase interaction
-Homophilic interactions
Linkage and signalling of classical cadherins in cell adhesion?
- Cadherin dimers form on separate cells
- Cells bind to eachtoher through cadherins. This triggers Rac1-GDP(inactive) to localize to the membrane on the cells
- As signalling progresses within the cells PI3K is activated which activates a GEF which activates Rac1-GTP
- Rac1-GTP activates Arp2/3 which facilitates actin filament formation
- Actin will then grow toward the cell membranes and push the two cell membranes together so they can bind through other cadherin dimers
Integrins are made up of what subunits to make heterodimers?
One alpha and one beta subunit
Are integrins found in prokaryotes?
No, they are strictly found in multicellular organisms
Caenorhabditis elegans integrin genes?
-Two alpha subunit genes
-One beta subunt gene
-Form two different integrins
How many alpha and beta subunit genes do integrins in humans have?
-18 alpha-subunit genes
-8 beta subunit genes
How many different hetrodimer integrins can form in vertebrates?
18 X 8
144 heterodimers, but the real number is actually only 24 heterodimers(not each subunit can pair up)
Cell-matrix adhesion integrin receptors?
-Collagen receptors(often pair in B1 and bind to various collagen types)
-Laminin receptors(typically B4)
-RGD receptors
Cell-cell adhesion integrin receptors?
Leukocyte-specific receptors bind to WBCs
Structure of integrin heterodimers?
-N-terminus in the ECM
-Most of the protein in the ECM
-C-terminus in the cytosol short region
-TM region (single span)
-Most integrins are connected intracellulaelt to anchor proteins which bind actin, excep alpha6 and beta4 which bind IFs
-Bind Ca2+ and Mg2+
Alpha6 and Beta4?
-Have a large intracellular terminus that binds IFs
-More flexible than those that bind actin
What type of TM protein are integrin heterodimers?
Type I TM proteins
Integrin-binding sequences?
-frequently involve either D(aspartic acid) or E(glutamic acid) residues
-These are acidic amino acids meaning up phsyiological pH they are negatively charged which helps the ligand bind to the integrin receptor
RGD receptor ligand sequence?
Ligands that bind RGD receptor of integrins often have the sequence arginine-glycine-aspartic acid in an exposed loop
What ligands bind RGD?
-Fibronectin, fibrinogen and some collagens
Aspartic acid binds to what in the beta head domain?
Aspartic acid is negative and bind Ca2+
Synergy sites?
Ligands often contain accessory sites for receptor binding
Fibronectin and its synergy site?
Fibronectin has an Fn10 domain that binds RGD receptors and an Fn9 domain that is a synergy site.
Fn9 binds the alpha subunit of the intergrin and mediates a stronger reaction between the fibronectin and the integrin
Two forms of fibronectin found in the body and where are they found/formed?
- Soluble fibronectin found circulation in the bloodstream and its produced in the liver and secreted constantly
- Cellular fibronectin formed by mesenchymal cells and forms a network of fibronectin
Are integrins always active?
No
When do we need blood platelet aggregation?
Normally, blood platelets circulate in the blood however to prevent blood loss due to a cut we need blood platelets to be activated and aggregate to form a clot
alphaIIbbeta3 integrin?
This is the integrin heterodimer present on platelets.
Normally it is present in the circulation in an inactive form.(not forming clots)
Activation of alphaIIbbeta3 integrin
- alphaIIbbeta3 integrin is activate when it comes in contact with ligands that it doesn’t normally
- These ligands can be things like collagen type IV or thrombin that come in contact with the integrin because of blood flowing in/out of the site of injury
- These ligands activate the integrin causing it to bind to fibrinogen and form a fibrinogen network(converts soluble fibrinogen into an insoluble fibrinogen polymer)
Two ways that integrin activation can occur by?
- Via extracellular ligand binding (outside in activation)
- From within the cell via the integrin cytoplasmic tail(inside out)
What is integrin activation important for?
- Cell aggregation(adhesion of circulating platelets to soluble matrix)
- Cell-cell adhesion(leukocytes and inflamation)
Integrin outside in activation?
- Extracellular ligand binds to the benf inactive conformation of the integrin(weak binding)
- Alpha and beta chains straighten
- Extracellular ligand will now bind much stronger to the integrin
- Conformational change in the beta subunit head domain
- Separation of the entire alpha and beta subunits(legs move apart)
- Active integrin
T/F: In the bent conformation the integrin has a very low affinity for ligands?
True
Salt bridge
An aspartic acid and arginine that bind ionically(non-covalent) to each other and hold the alpha and beta subunits together in a tight conformation in the talin binding region (intracellular)
What happens if you mutate one of the amino acids in the salt bridge?
The salt bridge will no longer form which will cause activation of the integrin
Talin binding region ?
Found on the beta subunit of the integrin
-intracellular region
-Binds to Talin protein
Two mechanisms that activate Talin?
- PI1,5P-2
- Calpain(protease)
Talin protein?
-Normally inactive in the cytosol
-inactive: the head domain binds the C-terminal domain which prevents it from binding the talin binding region on the beta subunit
How does PI4,5P-2 activate Talin protein?
- PI4,5P-2 is a lipid in the pM that can bind other lipids in the PM and results in the opening up of the talin protein to activate it
- This now makes the head domain accesible and allows it to bind to the integrin beta subunit
How does Calpain activate talin protein?
- Protease that cleaves the head domain from talin.
- The head domain then binds the talin binding region of the beta subunit
How does talin compete with the salt bridge?
When talin binds it pushes the alpha and beta subunit tails of the integrin apart. Meanwhile the salt bridge is trying to pull them together
Activated talin head?
-Binds to the beta subunit
-Disrupts the ionic salt bridge
-separation of alpha and beta subunits of the integrin
-Activation of the integrin
Disintegrins ?
-Large group of snake venom proteins containing a number of cysteine residues and RGD sequence
-Small proteins
-More than 40 different protein
What do disintegrins do?
-Block the function of some integrins
-Act as competitive inhibitor for integrin-fibrin interaction and block blood cagulation(block alphaIIbbeta3)
-Inhibit angiogenesis
How do the disintergrins block other integrins?
SInce they have an RGD sequence they are able to compete with the natural integrin and can bind their ligands
Tight knots of disintegrins?
Disintegrins form tight knots
-Due to their many cysteine residues the disintegrins are able to form disulfide bonds that cause the tight knots in the structure
T/F: disintegrins mimic the structure of integrins by having an exposed loop eith the RGD binding sequence
True
Integrins affinity vs avidity?
Integrins similar to cadherins have a low affinity for each other but have a high avidity because they tend to cluster together
Integrins connect fibronectin to actin?
Adaptor complexes bind integrins to the actin filament and the integrins bind to fibrnectin
T/F: The integrins help to cooling fibronectin and actin?
True, the two fiber networks tend to run in the same direction in cells
Cytochalasin?
A molecule that disrupts actin filaments, when actin is disrupted in the cell the fibronectin in the cell also becomes disrupted which indicated that there is a tight connection between the two fibers
20% of proteins in the body?
Collagens
Collagens?
-Composed of three polypeptide chains(alpha-chains)
-Form a collagen triple helix
-Exist as homotrimers or heterotrimers
-Form aggregates by association of triple helices
How many collagen genes in humans and how many collagen types?
46 collagen genes
28 collagen proteins
2 principal forms of collagen?
-Fibril-forming
-Sheet-forming
Characteristic amino acid sequence in collagen?
-Glycin-X(often proline)-Y(often hydroxyproline)
What happens if the characteristic AA sequence in collagen is mutated?
Disease
Right-handed vs left-handed collagen?
-Three left handed alpha chains come together to form one right-handed(clockwise) triple helix
Why is glycine always in the center of the triple helix?
Because it has no side chains therefore it is the only residue that can fit
What stabalizes the collagen triple helix?
H-bonds form between H2O and proline and between H2O and hydroxyproline
How are alpha-chains synthesized?
- Ribosomes dock at the ER and translate mRNA into a protein which is funneled into the ER lumen
- The alpha-chain is synthesized at the N-terminal and the C-terminal has a propeptide
- Hydroxylation of some proline and lysines by propel-hydroxylases and lysyl-hydroxylases
- Glycosylation of selected hydroxylysines
- Assembly of the three alpha-chains into a triple helix
Functions of the propeptides at the C-terminus?
- The propeptide has recognition sequence for other alpha-chains, which allows them to form the triple helix as soon as they recognize each other
- Propeptides prevent aggregation/assembly of the collagen molecules of the triple helix inside the cell
What happens if collagen molecules aggregate inside the cell?
The cell would burst
What happens once the triple helix is formed?
- Triple helix is secreted out of the cell
- The triple helix is secreted into deep envaginations of the cell to keep the collagens concentrated
2.Propeptides are cleaved - Self-assembly of triple helix into collagen fibrils
- Collagen fibrils can then further assemble into collagen fibers
Procollagen N-proteinase?
Cleaves the N-terminus propeptide
Ex. ADAMTS-2, -3, -14
Procollagen C-proteinease?
Cleaves the C-terminus propeptide
-Ex. Tolloid family
What are both of the proteineases dependent on?
Zinc
How long is the collagen triple helix?
300 nm in length, 1000 amino acids
Collagen microfibril?
-5 triple helices associated together
What is the driving force causing collagen alpha-helices to self-assemble?
Positive and negative charges align with each other on neighbouring alpha-helices
T/F: COllagen alpha-helices align in a stagerred pattern?
True, this causes a gap that leads to a banding pattern on EM
EM banding pattern of collagen fibrils?
Light band: Filled with protein
Dark band: Fills with osmium(this is the gap)
How do collagen triple helices assemble?
They assemble with a characteristic stagger, once a microfibril is formed the triple helices will then be added longitudianlly for gorwth
Purpose of hydrolyating lysines?
- Lysines with hydroxy are glycosylated
- Crosslinks
Purpose of hydroxylated prolines?
- Serves to form intermolecular H-bonds that stabalize the triple helix
How many genes encode the enzymes that hydroxylate lysine and proline residues?
3
What is needed to activate prolyl hydroxylase enzymes ?
Fe2+ (acts as one of its co-factors) and activates the enzyme
How is the proline residue hydrolxylated by prolyl hydroxylase?
The prolyl hydroxylase enzyme transfers an oxygen atom from alpha-ketoglutarate onto the proline, the alpha-ketoglutarate then becomes succinate
What happens when the proline substrate is absent?
The prolyl hydroxylase enzyme will oxidize Fe2+ to Fe3+ which makes the enzyme inactive
How does the enzyme reduce the Fe3+ co-factor back to Fe2+ to reactivate the enzyme?
Vitamin C reduces Fe3+ back to Fe2+
What happens when you lack vitamin C?
You get scurvy because the Fe3+ cannot be converted to Fe2+. The prolyl and lysyl hydrolxylases are no longer able to hydroxylate lysines and prolines
How does lysyl oxidase work?
- It takes either lysines or hydroxy lysines as a substrate
- It deaminates(removes the amine group) and converts the NH2 into a reactive group known as an aldehyde
What does lysyl oxidase require ?
O2, H2O and Cu2+
What does lysyl oxidase release from the reaction?
-NH3 and H2O2
What happens to the deaminated lysine?
The deaminated lysine spontaneously undergoes a condensation reaction releasing water and forming ALDOL crosslink
What is the ALDOL crosslink?
Protein that forms covalent bonds between two alpha-chains
These help stabalize the collagen fibers
Prolyl Hydroxylase?
-Adds hydroxyl group to some prolines
-IN the ER/Golgi
-Requires Vitamin C and Fe2+
Lysyl Hydroxylase?
-Adds hydroxyl group to some lysines
-In the ER/Golgi
-Requires vitamin C and Fe2+
N and C-propeptidases?
-Cleave the collagen propeptides outside the cell
-ECM
-Requires Zn2+
Lysyl Oxidases?
-Converts some lysines and hydroxylysines to allysines and hydroxy-allysines
-These reactive aldehydes react with free (hydroxy) lysines to form covalent cross-links
-Extracellular
-Cu2+ dependent
Ehlers-Danlos Syndrome?
Caused by mutations of collagens or collagen modifying enzymes
