Lecture 10 trafficking case study Flashcards
How is cholesterol transported?
Either de novo (in our body) via HMG CoA reductase which is the rare limiting step. Or taken up from our diet.
Where is exogenous cholesterol?
Exogenous cholesterol incorporated in chylomicrons.
Where is de novo cholesterol incorporated?
Into the liver into very low density lipoproteins, VLDL.
What do circulating particles do?
Give up cholesterol incorporated into VLDL.
What does the LDL receptor do with cholesterol?
Mediates the uptake of low density lipoproteins from the bloodstream into cells.
What do mutations in the LDL receptor do?
Lead to familial hypercholesterolaemia syndrome (FH). They are causal.
What is the proportion of people carrying the mutation?
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.
What does FH lead to?
Atheresclerosis, coronary heart disease and death.
What can LDLRs bind to?
Disparate ligands- apolipoprotein B and apolipoprotein E.
What are disparate ligands?
Ligands that are structurally or functionally different but still bind to the same receptor.
What are the cholesterol deposits called?
Characteristic xanthomas
What are the 5 classes of LDLR mutations?
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.
What are the features of the LDL receptor?
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.
Where is the gene of LDLR located on the chromosomes?
Chromosome 19.
Which of the exons show similarities to other proteins?
13 of 18 to C9 component of the complement, EGF receptor, factor IX, factor X, protein C. LDLR has many domains which map to individual exons.
Why are the conserved sequences in the cysteine residue important?
For maturation and folding of the receptor also for ligand binding.
What is the YWDT domin?
Beta propellor domain. Essential for ligand release.
What is exon 1 (a domain)?
Encompasses the signal sequence (21 amino acids).
What are the exon 2-6 domains?
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.
What are exons 7-15 for?
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 in the domain.
What is the difference between A,B and C?
Contiguous and separated from repeat C by the YWTD domain around 280 amino acids and required for the dissociation of LDLR ligands.
Why are EGF repeats cysteine rich?
For disulphide cross linking but DONT contribute to ligand binding.
What is exon 16 for?
Single transmembrane helix mainly hydrophobic.
What are exon 17 and 18?
Cytoplasmic domain. 50 amino acid cytosolic region. Important for receptor-mediated endocytosis. Region also contains long 3’ UTR.
What is the LDRL domain structure for?
LDL-A/LA repeats adopt conserved folds stabilised by S-S bonding and calcium co ordination. for disulphide bridge formation.
Where do the co ordinations sites come from to form the octahedral confirmation?
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.
What is the importance of the calcium binding?
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.
What is the beta propellor domain structure?
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.
How long does transport to the golgi take?
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.
What is C7?
C7 is an antibody that only binds to correctly folded LDLR so is a confirmation antibody.
What happens if LDLR is misfolded?
It gets de glycosylated and degraded via the endoplasmic reticulum associated degradation pathway (ERAD).
What chaperones does LDR require for folding into ER and trafficking to golgi?
BiP, calnexin, PDIs (protein disulphide isomerases which rearrange disulphide bonds), RAP, Mesd/BOCA. These are nonspecific chaperones.
What are the two specific chaperones which recognise LDLR?
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.
How does RAP interact with LA?
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.
What does Mesd chaperone?
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).
How does Mesd exist?
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.
What is essential for LDL receptor internalisation?
NPXY motif. autosomal recessive hypercholesterolaemia and DAB2 proteins are cargo adaptors required for LDLR internalisation.
Why are NPXY mutations rare?
They are a small group.
What do mutations in ARH, Dab1/2 cause?
Prevent effective internalisation. Same phenotype though of FH with increased plasma cholesterol levels.
How are LDRs recycled to plasma membrane?
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.
