7-11: Connective Tissues Flashcards
What actually is the ECM?
The Extracellular Matrix is a dynamic, multiprotein polymer, which makes up the majority of our organs and tissues
Name 2 key (general) functions of the ECM
- Organisation: Cells within organised tissue compartments have different functions, and are ORGANISED BY THE ECM
- Tissue Strength: Cells are linked to the ECM to resist mechanical forces of tension + compression
What is the significance of collagen (and its different types) in the body?
It is the most abundant protein in the body and plays a key role in holding tissues together
There are 28 types (though more than 80% is Col1), which form different types of ECM structure and determine its properties
What causes Scurvy?
A lack of vitamin C in the diet:
Vitamin C is a cofactor for the enzymes Proline Hydroxlase and Lysyl Hydroxylase, both of which form intermolecular H bonds between collagen chains
Lack of Vitamin C hinders the formation of stable collagen fibres
State the steps of Fibrillar Collagen Formation
- Pro-alpha collagen chain synthesised as a single polypeptide and co-translationally imported into ER
- Modifications of amino acids (especially Hydroxylation of Pro and Lys, and Glycosylation of Hydroxylysines)
- Three Pro-alpha chains assemble into a triple helix - driven by C-terminal, non-collagenous domains that lack the Gly-X-Y repeat
- Collagen enters a secretory vesicle; N and C domains are cleaved to leave just the triple-helical collagen molecule
- Collagen triple-helices self-assemble into fibrils outside the cell
What is the Repeating Sequence found in collagen, and what is the significance of this repeat for the function of collagen?
A repeating, Proline-rich sequence, defined by the Gly-X-Y repeat (either Gly-Pro-Y or Gly-X-Hydroxyproline)
- Proline has a cyclical side chain which restricts rotation in the polypeptide backbone
- Glycine is very flexible (as its R group is just H)
- Glycine wraps around, Pro forces it into a STABLE HELIX STRUCTURE
What is the Basement Membrane?
The BM is a specialised form of ECM, containing Col4, that organises cell layers in metazoans
Give 5 examples of tissues where an important basement membrane separates different cell layers
- Kidney (GBM)
- Muscles
- Blood Vessels (Muscle/Epithelium)
- Epithelium (either at Lumen or External Surface)
- Neuromuscular Synapse
Describe how the structure of Col4 differs from Col1, and the consequences of this for the polymers it forms
- In Col4, the N and C domains (i.e. non-collagenous regions) are NOT cleaved
- C domain drives triple-helix formation via end-end interactions, and drives hexamer (“dimer of trimer”) formation
- N domain cross-links 4 triple-helices to form tetramers
- Interruptions within the Gly-X-Y repeats of the coiled-coil allows increased flexibility and sites for cross-linking
Overall, these interactions drive self-assembly of Col4 into a 2D mat, rather than a 3D network like Col1
How many heterotrimers of Col4 are there (and what drives this specificity)?
Only 3 heterotrimers form in practice:
a1a1a2
a3a4a5
a5a5a6
Even though 6 genes for Type 4 Collagen gives 56 theoretical combinations
The specificity of NC1 interactions drives specificity of trimer and hexamer formation
Also specificity BETWEEN trimers too (i.e. a1a1a2 will ONLY tetramerise with a1a1a2)
Where are the different isoforms of Col4 found
Most BMs contain a1a1a2, including Bowman’s Capsule
The GBM also contains a1a1a2 in development, but in adults forms a THICK BM containing a3a4a5 to act as a filtration barrier between endothelial cells and podocytes
Not sure for a5a5a6
What is Alport’s Syndrome and what causes it?
An X-linked Nephritis disease due to mutations in COL4A5 (or also COL4A3/4)
Can’t properly form a3a4a5 collagen, so a1a1a2 still expressed in GBM in adults, which forms less inter/intra-chain crosslinks and is thus less protected from physical pressure and proteolysis
-> Gradual degradation of GBM leads to disease
What factors can affect the severity and onset of Alport’s Syndrome?
- Much more severe in Hemizygous males than heterozygous females (because X-linked)
- Most severe disease caused by large gene rearrangements, nonsense mutations and splice-site-induced truncations
- Later onset disease caused by Gly-X-Y missense mutations, or conserved Cys residues that affect crosslinking
How are knock-out or knock-in mice created for transgenic model organism experiments?
Introduce DNA into embryonic stem cells, inject these ESCs into an embryo (-> hybrid embryo), implant this embryo back into a mouse, SOME tissues in the offspring will have the transgene
Identify which offspring have the mutation in their germ cells -> breed these
(Also these days CRISPR allows more specific editing, e.g. mimic 1-AA mutations)
What happens if we knock out COL4A3 in a mouse, and what is the use of this?
Progressive kidney failure, or “glomerulopathy” (as no longer correct Col4 isoform in GBM) - phenocopies Alport’s in humans
Can use these mice to test treatments, e.g., ACE inhibitors
Are ACE inhibitors and Gene Therapy effective in treating mice that phenocopy Alport’s syndrome?
They seem to extend the lifespan, as they reduce blood pressure on the GBM, so it takes longer for collagen to wear down -> this could extend the time to find a suitable organ donor
There is also some evidence that Gene Therapy to rescue COL4A3 in mice causes repair and return to function of the defective GBM
What is Laminin?
Laminin is a high-MW glycoprotein with a cruciform structure, and is the second most abundant BM protein
Describe the structure of Laminin
Laminin is made up of three chains assembled into a cruciform structure
The long arm is a coiled-coil a-helix of the three chains, consisting of a 7-AA repeated sequence - these 7 include hydrophobic, hydrophilic, positive and negative AAs, allowing non-covalent interactions
The three N-terminal globular domains interact with other laminins, and with accessory proteins such as nidogen and perlecan - this promotes polymerisation into a 2D network (self assembly like collagen)
The C-terminal globular domain consists of 5 LG (laminin G-like) domains that interact with cell surface receptors: LG1-3 interact with integrins, while LG4-5 interact with Dystroglycan and Heparin
Small note: while electrostatic and hydrophobic interactions stabilise the coiled-coil, each end is stabilised by DISULPHIDE CROSS-LINKS
How many laminin trimers are there (and how does this compare to Col4)?
There are 15 heterotrimeric combinations of Laminin
A bit like Col4, there are 11 laminin genes, but interaction specificity means only 15 combinations
However, there is MORE tissue-specific variation than in col4 (as Col4 is almost all in the BM whereas laminins are found more widely)
SOME laminins are ubiquitous and essential (e.g., a1ß1y1), while others are tissue specific (e.g. a2 KO = MD, a3 KO = blistering of stratified epithelium)
What happens when the Laminin ß2 isoform is lost?
PIERSON SYNDROME - a rare lethal condition similar to the loss of GBM Col4 isoforms
Laminin11 (a5ß2y1) is expressed in GBM, eye, and synaptic BM, resulting in phenotype of nephrotic syndrome, eye abnormalities, AND muscular hypotonia
Which Laminin isoform is linked to a form of Epidermolysis Bullosa, and how/why?
Laminin 5 (a3ß3y2) - it links the epidermis to the dermis:
Lam5 links integrins (a6ß4) in the epidermis to the underlying Col4 in the BM, which is anchored by Col7 to the Col1 network in the dermis
Mutations in any of LAMA3, LAMB3 or LAMC2 cause Junctional Epidermolysis Bullosa (JEB), either Herlitz-type (lethal due to complete loss) or Non-Herlitz type (milder due to limited Lam5 function)
Can Gene Therapy be used to treat Non-Herlitz JEB?
YES - and here is how/why:
- A modified retrovirus is used to deliver a functional copy of (for example) LAMB3 to the patient’s UNaffected keratinocytes
- Grow them in culture, then graft this transgenic epidermis onto the patient
- Eventually the holoclones (containing proliferative stem cells) will make up the majority of the patient’s skin
What are the limitations of Gene Therapy for treating BM-related conditions
Limitations on gene size when using retroviruses to deliver (e.g., laminin gene is small enough to use, but dystrophin is NOT)
Also potential issues when randomly altering the genome of a cell that is being grafted back onto the patient
What do the cytoplasmic domains of integrins interact with?
MOST (e.g., in adherens junctions and FAs) interact with actin
BUT a6ß4 in hemidesmosomes link to Keratin Intermediate Filaments
Describe the tissue-specific distribution and effects of tissue-specific deletion of key ß integrin isoforms
ß4 = HEMIDESMOSOMES IN STRATIFIED EPITHELIUM (between epidermis and BM) - deletion leads to detachment of epidermis from BM, like Lam5 deletions
ß1 = Widely expressed - deletion is lethal
ß2+3 = Blood cells - deletion affects clotting + inflammation
If integrin ß4 is deleted, why does the epidermis lift off as 1 sheet instead of simply falling apart?
Because ß4 deletion results in loss of hemidesmosomes, but not cell-cell adhesions (AJs) that link to IFs
What is PA-JEB and what causes it?
[Junctional Epidermolysis Bullosa with Pyloric Atresia] is a rare, fatal, AutRecessive condition caused by the loss of a6ß4 integrin
It causes neonatal blistering and GI obstruction through loss of GI, Genitourinary, and Respiratory Epithelium
Why is the loss of integrin (e.g., in PA-JEB) more severe than the loss of Lam5 (in Herlitz or non-Herlitz JEB)?
Integrin ß4 is more widely expressed in tissues, and interacts with multiple Laminins
Laminin 5 is more specific in its integrin interactions, so its deletion has less widespread effects
Does loss of integrin ß4 gene also result in loss of a6 integrin expression?
YES - similar to Laminins and Col4, integrin interactions are specific
Why can BM formation still be seen when integrin ß is lost?
Lam 5 can be seen forming a continuous membrane, as the BM still forms, but just can’t attach to the layer underneath due to the lack of ß4
What is the result of a6 integrin deletion?
Same effect as loss of ß4 (in mice and humans)
What is EB Simplex and what is it caused by?
It is a milder form of EB, usually restricted to blisters on regions subject to mechanical stress
Often due to mutations in components of Keratin5/14 IFs…
OR in the cytoplasmic tails of integrin ß4
-> Cells are weakened internally, but the BM is still intact; therefore, cells can rip off but leave others still attached to BM
(Can also be caused by mutations in any of the adaptor proteins linking cytoplasmic domain of ß4 with IFs)
