MP5: Cell interactions

Question 1

Why is neonatal MFS more severe than classical MFS?

Answer 1

Neonatal MFS is a rare and severe form of MFS that presents in infants, while classical MFS typically presents in childhood or adolescence. It’s thought to be more severe due to characteristic mutations that occur in the ‘neonatal region’. Through GFP-fluorescence experiments, it was shown that nMFS mutants did not incorporate into the microfibrils, whereas cMFS did. This is likely because the neonatal region is an assembly site and so mutations in it interfere with the assembly pathway that occurs outside of the cell, resulting in no lateral assembly.

Question 2

What are the 4 possible functions of microfibrils?

Answer 2

• Structural/mechanical
• Sequester TGF-beta for controlled growth signalling
• Anchors the lens to the ciliary body in the eye
• Helps with stability during embryonic tissue development, and may even induce tensile forces that shape the embryo

Question 3

How can MFS be both haploinsufficient or dominant negative?

Answer 3

If the mutation arises in a premature stop codon in the FBN1 gene, is will cause nonsense mediated decay and hence haploinsufficiency. Missense mutations can cause functional haploinsufficiency due to severe misfolding, but a lack of NMD.
Dominant negative mechanisms occur when NMD doesn’t occur and the mutated protein interferes with WT protein function. This can arse in either disruption of microfibril function, or impairment of WT assembly and a severe loss of microfibrils (neonatal MFS).
Both mechanisms result in a loss of microfibril function, and hence MFS.

Question 4

How is TGF-beta implicated in MFS?

Answer 4

Reduced Fibrillin-1 results in less sequestration of TGF-beta, and also an increase in release of matrix-degrading enzymes. There’s a possibility these enzymes are a result of increased TGF-beta signalling, and hence inhibitors are being used to try and block the signal and reduce the enzymes.

Question 5

What are the key domains within the Fibrillin-1 protein?

Answer 5

• TGF-beta binding domain (sequesters TGF-beta)
• Calcium-binding EGF domain (stabilises protein structure and mediates protein-protein interactions)

Question 6

How are skin specific disorders a consequence of FBN1 mutations? How do their modes of pathogenesis differ to MFS?

Answer 6

A subset of FBN1 mutations in TGF-beta binding domains 4 and 5 are associated with skin specific disorders, such as stiff skin syndrome (SSS). These mutations don’t affect microfibril assembly, but instead are thought to impact integrin binding which potentially causes defective interactions with cells.
MFS results in a reduction of microfibril quantity/quality (structural defect), whereas SSDs cause altered microfibril-cell interactions (signaling defect).

Question 7

Describe the process of elastogenesis and the associated genetic disorders.

Answer 7

1. Tropoelastin (core structural monomer of elastic fibers) is secreted from the cell.
2. It binds fibulin-4 and fibulin-5 which aid in interactions with microfibrils.
3. Microfibrils form an array.
4. The tropoelastin assembly forms the elastin core within the microfibril array.

• Microfibrils: MFS
• Fibulin 4: autosomal recessive cutis laxa (ARCL)
• Fibulin 5: ARCL and age-related macular degeneration (AMD)
• Elastin: supra valvular aortic stenosis (SVAS) and autosomal dominant cutis laxa (ADCL)

Question 8

What is a missense mutation?

Answer 8

When a single base pair substitution alters the amino acid codon.

Question 9

What is a nonsense mutation?

Answer 9

Conversion of a codon into a premature stop codon.

Question 10

What is the role of lysyl oxidase in the ECM?

Answer 10

Lysyl oxidase (LOX) is critical in the formation and repair of the ECM by oxidizing lysine residues in elastin and collagen, thereby initiating the formation of covalent cross linkages. It is bound by fibulin-4 and fibulin-5.

Question 11

What’s the difference between a pathogenic and polymorphic variant? Relate this to age-related macular degeneration.

Answer 11

Pathogenic means the variant is responsible for causing the disease with ample scientific evidence.

Biochemical evidence for AMD shows two missense mutations causing protein misfolding, thus making them pathogenic. However, only one of these examples has sufficient strong evidence (S227P).

Another missense mutation doesn’t cause structural changes and the resulting amino acid is often found at this position in other cbEGF domains, so is probably a polymorphism.

Question 12

What is an osteoblast?

Answer 12

Bone cells that are primarily responsible for synthesizing bone matrix proteins and minerals. They’re primarily composed of type I collagen, proteoglycans and glycoproteins.

Question 13

What is the process called for the formation of bone? How do intramembranous processes differ to endochondral processes?

Answer 13

Bone formation in vertebrates occurs through a process called ossification, which involves the deposition of mineralized tissue, calcium and phosphorous onto a collagen matrix.

There are two types of ossification:
1. Intramembranous ossification is the process by which bone forms directly from stem cells that condense into a membrane-like structure. Occurs in the skull.
2. Endochondral ossification is the process by which bone forms from a pre-existing cartilage model. Chondrocytes enlarge and produce a calcified extracellular matrix that serves as a scaffold for bone formation. Osteoblasts replace the cartilage with bone tissue/collagen. Forms long bones.

Question 14

Describe the structure of type I collagen. What genes encode the peptides involved?

Answer 14

Type I collagen is synthesized from two gene products, COL1A1 and COL1A2. The procollagen fibre is formed from 2 chains from COL1A1 and 1 from COL1A2.
Each mature chain consists of a repeating sequence composed of glycine, proline and hydroxyproline, which provide the structural stability and strength of the triple helix.
After the triple helix formation is complete, the C-terminal propeptide is cleaved, leaving the mature collagen molecule that can undergo PTMs such as glycosylation and hydroxylation.

Question 15

Compare osteogenesis type I to types II-IV

Answer 15

Type I: caused by COL1A1 haploinsufficiency due to premature stop codons (mild disease).
Types II-IV: both COL1A1 or COL1A2 can have amino acid substitutions, causing dominant negative pathogenesis. Most of these mutations affect major ligand binding regions, disrupting collagen-matrix interactions (severe disease).

Question 16

What is the function of fibroblasts?

Answer 16

Cells that secrete collagen in the formation of connective tissue.

Question 17

What is the 3-hydroxylation complex?

Answer 17

A complex in the ER that is responsible for the 3-hydroxylation of some proline residues in collagen. Composed of:
- Prolyl 3-hydroxylase
- Cartilage-associated protein
- Cyclophilin B

Question 18

Why is the ECM difficult to study? How is the ECM studied?

Answer 18

ECM proteins are rapidly assembled outside of the cell, making studying PTMs and processing hard.
- Difficult to track
- Insoluble
- Not concentrated
- Lack of model organisms
- Large genes

Multidisciplinary methods are used:
- organism studies (disease phenotypes)
- cell isolation
- dissection of the large proteins for recombinant expression
- NMR and XRC
- Homology modeling

Question 19

What confers the ECM’s rope-like, gel-like, elastic and ‘sticky’ properties?

Answer 19

Rope-like: fibrous proteins
Gel-like: GAGs/proteoglycans
Elastic: elastin
‘Sticky’: fibronectin and laminin

Question 20

How is the ECM able to influence cellular behaviour?

Answer 20

1. mechanical cues e.g., ECM stiffness via integrin-mediated adhesion
2. biochemical cues e.g., GFs and cytokines
3. biophysical cues e.g., orientation and alignment of ECM fibers

Question 21

Give the functions and biological importance of the ECM.

Answer 21

Functions:
- structural support
- adhesion
- bioavailability of growth factors e.g., TGFb

Biological importance:
- genetic disorders
- maintenance of stem cell niche
- tumor microenvironment

Question 22

What is the ECM composed of? Give examples of each.

Answer 22

GAGs (hyaluronan)
Proteoglycans (heparin sulfate)
Structural proteins (collegens, fibrillin)
Adhesive proteins (fibronectin, laminin)

Question 23

What are the typical features of fibrous proteins found in the ECM?

Answer 23

Modular
Multiple forms (alternative splicing, etc)
Fibrous…
PTMs
Self-assemble to form fibrils
‘Sticky’

Question 24

Compare the composition of the basement membrane to the ECM of connective tissue.

Answer 24

Basement membrane:
- type IV collagen
- laminins
- perlecan
- integrins

Connective tissue:
- collagen
- elastin
- fibroblasts
- tissue macrophages

Question 25

Describe the structure and function of integrins.

Answer 25

Integrins are TM proteins that form ECM-cell and cell-cell interactions in a DYNAMIC fashion.

They’re composed of two subunits, alpha and beta, that are non-covalently linked to form a heterodimeric complex. Their tails interact with many intracellular proteins to initiate different signaling pathways.

Question 26

What is the importance of the integrin-ECM ligand pair, a5b1 and fibronectin? What happens when this doesn’t occur?

Answer 26

It mediates fibronectin fibril formation and ECM assembly, which is fundamental to vertebrate development. Lack of a5b1 or fibronectin is early embryonic lethal in mouse models.

Question 27

What are focal adhesion kinases?

Answer 27

Non-receptor tyrosine kinases, activated by integrin-mediated adhesion to the ECM which transduces signals from the ECM to the cell interior.

Question 28

What are talins?

Answer 28

Cytoskeletal proteins that link integrins to the actin cytoskeleton, forming focal adhesions.

Question 29

What is mechanobiology, and what two processes does it involve?

Answer 29

The ability of the cell to exert, sense, decipher and respond to physical forces.

1. mechanosensing
2. mechanotransduction

Question 30

How can the mechanobiology of the ECM on cells give rise to fibrosis and cancer?

Answer 30

Fibrosis:
Tissue injury/inflammation causes fibroblasts to differentiate into myofibroblasts which deposit ECM, resulting in stiffer ECM. Stiff ECM induces myofibroblast differentiation further, creating a feedforward loop.

Cancer:
ECM stiffness can promote cancer cell proliferation, migration and survival.

Question 31

How does talin respond to varying tensions i.e., act as a mechanosensitive signalling hub?

Answer 31

Talin contains domains that can adopt a number of conformations under different forces. The more force applied, the more unfolded the protein and hence the more domains are available to exert positive or negative effects.

Question 32

How might integrin mediate the activation of TFGb?

Answer 32

TGFb is non-covalently bound in a latent form in the ECM - it’s in a cage. Binding of TGFb to integrin an subsequent application of force causes a conformational change that opens the cage and releases TGFb.

Question 33

Which pathways are activated by focal adhesion kinases? What do these pathways lead to?

Answer 33

1. MAPK –> cell proliferation and migration
2. RhoA, a small GTPase that can regulate actin cytoskeleton organization.
3. Hippo pathway –> cell proliferation and reinforcement of FAK signaling

Question 34

Describe the Hippo pathway.

Answer 34

Increased ECM stiffness is sensed by focal adhesions that activate a serine/threonine kinase cascade. This activation causes activation of transcriptional co-activators YAP and TAZ which promote the expression of genes involved in cell proliferation, as well as reinforcement of the FAK signals.

Question 35

What sort of mutations cause MFS?

Answer 35

Premature stop codons = haploinsufficiency
Missense mutations on structural residues
cbEGF mutations.

Question 36

What is so special about TGFb binding domains 4 and 5 on FBN1? What types of proteins bind at each of these sites?

Answer 36

TB4 and TB5 are critical for sequestration and regulation of TGFb activity and mutations in these can lead to decreased sequestration, contributing to connective tissue disorders such as MFS.

TB4: Integrins
TB5: Heparan sulfate proteoglycans
This signaling may feedback from the damaged microfibrils of MFS, back into the cell.

Question 37

ADCL and SVAS are both caused by mutations in the ELN gene. How do they have distinct phenotypes?

Answer 37

ADCL is associated with C-terminal mutations and SVAS by others.

The majority of these mutations cause PTC insertion. If this occurs upstream of the C-terminus, NMD removes the polypeptide and results in haploinsufficiency. This results in the SVAS phenotype.

NMD only occurs if you have a downstream exon that’s intact, so when PTC insertion occurs at the C-terminus, the mutated transcript can’t be removed and results in a dominant-negative mutation. This results in the ADCL phenotype.

Question 38

How does lysyl oxidase interact with fibulin 4 and fibulin 5?

Answer 38

LOX can cross-link fibulin to elastin fibers, helping to stabilize the ECM and maintain tissue structure.

Question 39

What is the pathogenic mechanism behind the S227P substitution in the cbEGF domain of fibulin-5 that causes AR cutix laxa?

Answer 39

S227 is a conserved residue with cbEGF domains. The structural change to a proline is likely highly disruptive, potentially affecting interactions with enzymes such as LOX that cross-link fib5 with other proteins.

Question 40

Why are fibulin-4 and -5 mutations recessive, whilst those of elastin and fibrillin-1 are dominant?

Answer 40

Elastin and fib-1 are core structural ECM proteins with a stoichiometric requirement, hence their dominant inheritance.

Fib-4 and -5 are accessory proteins with only qualitative requirements, as shown by heterozygous patients not displaying severe phenotypes.

Question 41

Describe common diseases associated with defects in elastogenesis. What mutations cause them?

Answer 41

1. Cutis laxa - loose skin. Mutations in the elastin gene are autosomal dominant (SVAL), but mutations in fib-4 or fib-5 are autosomal recessive (ARCL).
2. MFS - mutations in fibrillin-1

Question 42

What is AMD characterized by? What might be the cause of it?

Answer 42

Age-related macular degeneration is a common eye disorder that’s characterized by the build-up of drusen deposits under the retina.

Missense mutations in fibulin-5 are thought to cause rare forms of AMD, caused by possible protein misfolding. Protein misfolding due to G267S causes haploinsufficiency in the eye (autosomal dominant) whereas S227P causes an absence of fibulin-5 with profound effects on all elastic tissues (autosomal recessive) e.g., ARCL.

Question 43

What is the proposed molecular basis of severity scales in osteogenesis imperfecta?

Answer 43

OI is caused by mutations in COL1A1 and COL1A2. Amino acid substitutions which affect triple helix assembly and haploinsufficiency are likely to be more severe since there will be much less collagen secreted.

Some substitutions will allow assembly and normal secretion, but give rise to dominant negative effects.

Question 44

What is Alport syndrome? What is it caused by?

Answer 44

Alport syndrome is a disease that affects the kidneys, ears, and eyes.

It’s caused by mutations in genes encoding type IV collagen, a major component of the basement membrane. Normally there’s a switch from early embryonic type IV collagen to adult. In X-linked Alport syndrome, the developmental switch doesn’t occur and the embryonic form isn’t sufficient to function in the adult.

Question 45

What is the general process that occurs in a Notch signaling pathway?

Answer 45

1. Notch receptor binds its ligand
2. 3 proteolytic cleavages occur
3. the intracellular domain of the Notch receptor (NICD) is released
4. NICD translocates to the nucleus where it interacts with transcriptional regulators for gene activation.

NB: There’s no signal amplification!

Question 46

What are the 3 modes that Notch signaling can cause in terms of cell fate regulation?

Answer 46

1. Lateral inhibition (e.g., maintaining neural progenitor cells and preventing differentiation)
2. Asymmetrical inheritance of Notch regulators
3. Boundary formation in Drosophila

Question 47

How is Notch signaling involved in DV boundary formation in Drosophila? What is the role of Fringe?

Answer 47

Serrate and Delta are both Notch ligands - they’re single-pass TM proteins, so the cells expressing them must be adjacent to the notch expressing cell for signaling to occur.

Dorsal expresses Fringe, a glycosyltransferase that adds O-linked glycans to the extracellular domains of Notch receptors. This modification increases the response to Delta, but restricts the response to Serrate.

This means that Serrate can only drive Notch signalling in ventral cells.

This aids in the formation of the dorsal-ventral boundary.

Question 48

What is a catch bond? What role do these play in Notch receptor activation?

Answer 48

A type of noncovalent bond whose lifetime increases with tensile force applied to the bond.

Recent studies have suggested that catch bonds play an important role in the activation of the Notch receptor. In the absence of mechanical force, the interaction between the Notch receptor and its ligand is weak and transient. However, when mechanical force is applied, such as through shear stress or tension, the bond between the Notch receptor and its ligand can become stronger and more stable.

This is potentially because the pulling opens up the cleavage site for NICD release.

Question 49

What is the significance of the Jagged C2 domain in Notch signaling?

Answer 49

The C2 domain binds calcium and shows calcium-dependent phospholipid binding. This gives new possibilities for cell-surface organisation due to a potential lipid binding domain at the N-terminus i.e., the protein may fold back on itself and bind the membrane. Could this prevent Notch signaling?

Question 50

Describe Alagille syndrome. What is it caused by? What is the pathogenic mechanism?

Answer 50

Alagille syndrome is commonly associated with a lack of normal bile ducts in the liver. It’s caused by mutations in the JAG1 or NOTCH2 genes.

All mutations cause alterations in protein folding, resulting in retention of the mutant protein and hence haploinsufficiency. However, each mutation can have it’s own mechanism of action - some reduce binding of Notch to Jagged, whilst others reduce Notch activation.

Question 51

Describe T-ALL (T-cell acute lymphoblastic leukemia). How is it associated with Notch signaling?

Answer 51

In T-ALL, immature T-cells grow and divide uncontrollably, leading to the accumulation of cancerous cells in the blood and bone marrow.

Some T-ALL patients have chromosomal translocation mutations that fuses the 3’ of the NOTCH1 gene to a promoter-enhancer region. Other mutations cause Notch activation in the absence of ligand e.g., if the cleavage site is constantly available without the need for mechano-pulling.

These result in constitutive activation of Notch signaling, leading to uncontrolled cell growth and division. Hence Notch is acting as an oncogene.

Question 52

How can Notch be both a tumor suppressor and an oncogene?

Answer 52

In some tissues and contexts, the activation of Notch signaling has been shown to suppress tumor growth and act as a tumor suppressor. This is thought to be due to the ability of Notch signaling to promote cell differentiation and inhibit cell proliferation and survival, which can prevent the development of cancerous cells.

On the other hand, in other contexts and tissues, the activation of Notch signaling has been shown to promote tumor growth and act as an oncogene. This is thought to be due to the ability of Notch signaling to promote cell proliferation and inhibit apoptosis (programmed cell death), which can lead to the development and progression of cancer.