Lecture 10 trafficking case study

Question 1

How is cholesterol transported?

Answer 1

Either de novo (in our body) via HMG CoA reductase which is the rate limiting step. Or taken up from our diet.

Question 2

Where is exogenous cholesterol?

Answer 2

Exogenous cholesterol incorporated in chylomicrons.

Question 3

Where is de novo cholesterol incorporated?

Answer 3

Into the liver into very low density lipoproteins, VLDL.

Question 4

What do circulating particles do?

Answer 4

Give up cholesterol incorporated into VLDL.

Question 5

What does the LDL receptor do with cholesterol?

Answer 5

Mediates the uptake of low density lipoproteins from the bloodstream into cells.

Question 6

What do mutations in the LDL receptor do?

Answer 6

Lead to familial hypercholesterolaemia syndrome (FH). They are causal.

Question 7

What is the proportion of people carrying the mutation?

Answer 7

1 in 500 people carry mutations which cause FH. The mutations in the alleles are co dominant so additive effects depending on which alleles you have.

Question 8

What does FH lead to?

Answer 8

Atheresclerosis, coronary heart disease and death.

Question 9

What can LDLRs bind to?

Answer 9

Disparate ligands- apolipoprotein B and apolipoprotein E.

Question 10

What are disparate ligands?

Answer 10

Ligands that are structurally or functionally different but still bind to the same receptor.

Question 11

What are the cholesterol deposits called?

Answer 11

Characteristic xanthomas

Question 12

What are the 5 classes of LDLR mutations?

Answer 12

Class 1. Disruption of ER synthesis.
Class 2. Block ER to golgi transport (most common)
Class 2a (more severe defects phenotypically) and class 2b. Receptor doesnt fold properly so receptor cant be recruited by vesicle.
Class 3 LDLR reaches cell surface but fails to bind to ligand.
Class 4 LDLR fails to internalise correctly.
Class 5 blocks in LDLR-ligand dissociation essential for recycling. Clearance of particles decreases.

Question 13

What are the features of the LDL receptor?

Answer 13

Cell surface glycoprotein, 839 amino acids, undergoes extensive PTMs (N-linked glycosylation, O-linked glycosylation, extensive disulphide bind formation). Glycosylation increases molecular weight by 33%. Gene comprimises 18 exons.

Question 14

Where is the gene of LDLR located on the chromosomes?

Answer 14

Chromosome 19.

Question 15

Which of the exons show similarities to other proteins?

Answer 15

13 out of the 18 exons show similarity to other proteins. E.g., C9 component of the complement, EGF receptor, factor IX, factor X, protein C. LDLR has many domains which map to individual exons.

Question 16

Why are the conserved sequences in the cysteine residue important?

Answer 16

For maturation and folding of the receptor also for ligand binding.

Question 17

What is the YWDT domin?

Answer 17

Beta propellor domain. Essential for ligand release.

Question 18

What is exon 1 (a domain)?

Answer 18

Encompasses the signal sequence (21 amino acids).

Question 19

What are the exon 2-6 domains?

Answer 19

The LDL-A repeats. There are 7 repeats (around 40 amino acids each). Contains conserved cysteine residues. C-terminal SDE sequence element which is critical for ligand binding.

Question 20

What are exons 7-14 for?

Answer 20

EGF precursor homology. 400 amino acid sequence that is 33% identical to a portion of the human EGF precursor gene. Includes EGF repeats. Around 40 amino acid modules. A, B and C repeats in the domain.

Question 21

What is the difference between A,B and C?

Answer 21

Contiguous and separated from repeat C by the YWTD domain around 280 amino acids and required for the dissociation of LDLR ligands.

Question 22

Why are EGF repeats cysteine rich?

Answer 22

For disulphide cross linking but DONT contribute to ligand binding.

Question 23

What is exon 16 for?

Answer 23

Single transmembrane helix mainly hydrophobic.

Question 24

What are exon 17 and 18?

Answer 24

Cytoplasmic domain. 50 amino acid cytosolic region. Important for receptor-mediated endocytosis. Region also contains long 3’ UTR.

Question 25

What is the LDRL domain structure for?

Answer 25

LDL-A/LA repeats adopt conserved folds stabilised by S-S bonding and calcium co ordination. for disulphide bridge formation.

Question 26

Where do the co ordinations sites come from to form the octahedral confirmation?

Answer 26

Four coordination sites from 3 aspartates and 1 glutamate. 2 additional ones from the back bone carbonyls provided by conserved tryptophan and glycine. Commonly mutations in these which affect structure and ligand binding.

Question 27

What is the importance of the calcium binding?

Answer 27

It creates a distinct surface charge distribution in each of the 7 repeats. A large characteristic of a section of acidic charge (-ve) on the protein. Important for ligand binding and why they can bind to dispartite ligands.

Question 28

What is the beta propellor domain structure?

Answer 28

YWTD is a conserved tetrapeptide. Each of the 6 blades has one copy that sits in the second beta strand of each of the blades.

Question 29

How long does transport to the golgi take?

Answer 29

30 minutes as a complex protein with many disulphide bonds as they have to be rearranged as part of the folding process for the receptor.

Question 30

What is C7?

Answer 30

C7 is an antibody that only binds to correctly folded LDLR so is a confirmation antibody.

Question 31

What happens if LDLR is misfolded?

Answer 31

It gets de glycosylated and degraded via the endoplasmic reticulum associated degradation pathway (ERAD).

Question 32

What chaperones does LDR require for folding into ER and trafficking to golgi?

Answer 32

BiP, calnexin, PDIs (protein disulphide isomerases which rearrange disulphide bonds), RAP, Mesd/BOCA. These are nonspecific chaperones.

Question 33

What are the two specific chaperones which recognise LDLR?

Answer 33

RAP. Receptor adaptor protein. Has a C terminal KDEL motif. This is part of the retrieval pathway. The cells that make the receptor also make the ligand for that receptor so cell doesn't want ligand to be recognised via receptor in the cell. RAP interacts with LBD of LDLR blocks receptor ligand interaction.

Question 34

How does RAP interact with LA?

Answer 34

Conserved residue interacts with negative charge on LA domain. Lysine 256 interacts with LA repeat 4. Lysine side chain with basic charge. Stable interactions due to conserved tryptophans which has an acidic patch. This is all beyond me maybe watch again.

Question 35

What does Mesd chaperone?

Answer 35

LRP5/6 trafficking. Probably required for correct folding of YWTD domains. Mesd appears to contain central, structured domain and N/C terminal natively unstructured domains required for YWTD folding. Contains KDEL (or equivalent sequence).

Question 36

How does Mesd exist?

Answer 36

In open and closed forms. The acidic patch on the helix interacts with basic residues on beta propelllor blades causing them to close up. EGFC region interacts via hydrophobic interactions. This comes apart through pH drop for maturation of the receptor. Protonates basic residues of beta propellor. They come apart.

Question 37

What is essential for LDL receptor internalisation?

Answer 37

NPXY motif. autosomal recessive hypercholesterolaemia and DAB2 proteins are cargo adaptors required for LDLR internalisation.

Question 38

Why are NPXY mutations rare?

Answer 38

They are a small group.

Question 39

What do mutations in ARH, Dab1/2 cause?

Answer 39

Prevent effective internalisation. Same phenotype though of FH with increased plasma cholesterol levels.

Question 40

How are LDRs recycled to plasma membrane?

Answer 40

Requires an early endosomal protein SNX17, interacts with LDLR via its FERM domain. Overexpression of SNX17 causes increased LDLR recycling and decreased LDLc. Has potential treatment impacts.