LDL Uptake Mechanism, Regulation, LDLR Structure and Relation to Hypercholesterolaemia

Question 1

What is the function of an LDLR?

Answer 1

LDLs are taken up by hepatocytes and steroidogenic glands via LDL Receptor mediated endocytosis to provide cholesterol and cholesteryl esters for the liver/adrenals.

Question 2

How do LDLRs internalise LDLs?

Answer 2

LDLs bind via ApoB100 to the LDLRs in a clathrin coated pit which forms an acidic internal vesicle (made so by proton pumps) that merges with the endosome.

Question 3

What happens to the internalised LDLs/LDLR receptors?

Answer 3

The acidic endosome causes the LDL to dissociate from the LDLR.

The LDLRs are then segregated into recycling vesicles and sent back to the membrane.

The acidic vesicle merges with the lysosome, where the LDL is lysed and the cholesteryl esters are broken down/fatty acids released.

Question 4

What are the potential fates of cholesterol taken up from the lysed LDLs?

Answer 4

The cholesterol is then either transported to the plasma membrane or, in adipose cells, re-esterified by the enzyme ACAT (Acetyl-coA-Cholesterol AcylTransferase) and deposited in the lipid droplet.

Question 5

Aside from uptake from lipoproteins, how can hepatocytes get cholesterol?

Answer 5

De Novo synthesis from Acetyl CoA in the Mevalonate pathway, a pathway whose rate is regulated by HMG CoA Reductase (enzyme for the RLS).

Question 6

What did experiments in skin fibroblasts show about LDLR expression and activity?

Answer 6

Clathrin coated pits accounted for around 2% of the cell surface, meaning that there were around 50,000 receptors expressed.

Each LDLR was found to be capable of taking up one LDL at a time, with around 270 being taken up in the lifetime of the receptor.

It took around 5 minutes to internalise an LDL and 3 minutes to recycle the receptors.

Question 7

At what level is LDL uptake regulated and why?

Answer 7

LDL receptors (LDLRs) are saturated at normal plasma LDL concentrations, so the cell controls its intake by regulation of turnover of the receptor though promoting transcription or increasing degradation.

Question 8

What regulatory effects does excess cholesterol have on the hepatocytes in relation to obtaining cholesterol?

Answer 8

Inhibition of the mevalonate pathway by inhibition og HMG CoA Reductase (decreases transcription, increases degradation).

Decreases LDLR expression through the SCAP/SREBP pathway (also decreases the transcription of HMG-CoA-R)

Question 9

Give a brief overview of how CH levels change the activation state of the SCAP/SREBP Pathway

Answer 9

When CH is high binds directly to SCAP (SREBP Cleavage Activating Protein), allowing SCAP to bind to a transmembrane protein called insig, which causes SCAP to be retained in the ER.

When cholesterol levels are low this does not occur and the complex is trafficked to the ER and the pathway is activated.

Question 10

What are the structural components of SCAP and their function?

Answer 10

Cholesterol levels are sensed by a sensing domain formed from the 8 N-terminal transmembrane helices, which works by sensing the changes in membrane fluidity due to cholesterol content.

The C-terminal WD domains on SCAP interact with the C-terminal regulatory domain of SREBP.

Question 11

What is Insig 1/2 and what regulates it?

Answer 11

Insig1/2 is an intramembrane protein that binds to five of the transmembrane domains of SCAP to cause its retention in the ER in response to high cholesterol.

This inhibitory binding is promoted when insig binds of oxysterols - a product of high CH

Question 12

How are oxysterols produced?

Answer 12

They are formed by oxygenases as well as by a non-enzymatic autoxidation of cholesterol during particularly high concentrations.

Question 13

What is the first thing that occurs when SCAP is not inhibited by insig/high CH conditions?

Answer 13

Upon activation, SCAP binds to SREBP (sterol regulatory element binding protein) which allows the complex to recruit proteins that direct it to the Golgi apparatus.

Question 14

How is the SCAP/SREBP complex trafficked to the golgi?

Answer 14

Through COPII anterograde transport. This membrane trafficking mechanism begins with Sar1 recruiting Sec23/24. Sec 24 recognises a MELADL sequence in loop 6 of SCAP, which can only happen when it is exposed by the low cholesterol content.

Question 15

What is the first thing to happen to the SCCAP/SREBP complex in the golgi?

Answer 15

Here the complex can recruit a membrane bound subtilisin-like serine protease called S1P (with a DSH catalytic triad) that cleaves a loop in SREBP to separate the still membrane bound Sterol Regulatory Element (SRE) from the regulatory domain.

Question 16

What releases the SRE TF from the membrane?

Answer 16

This 68kDa fragment is cleaved from the membrane region by S2P, a hydrophobic in-membrane metalloprotease with a HEXXH catalytic motif that cleaves within the transmembrane region.

Question 17

What is the structure of SRE?

Answer 17

A basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor.

Question 18

What happens when the SRE is released from the golgi?

Answer 18

It is transported to the nucleus where is acts to upregulate the LDLR and HMG CoA Reductase genes.

SRE is quickly degraded in the nucleus, so cholesterol levels must remain low to keep the pathway and hence LDL-R expression turned on.

Question 19

What is responsible for the protein-level regulation of LDLRs?

Answer 19

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9)

Question 20

How was PCSK9 discovered?

Answer 20

Through discovery of two autosomal dominant mutations, S127R and F216L, that lead to hypercholesterolaemia – responsible for 2% of genetic cases.

Question 21

What is the role of the PCSK9 subtilisin domain?

Answer 21

Contrary to original belief, PCSK9 does not cleave LDLRs with its protease domain, but instead the activity for this appears to be limited to autocatalytic cleavage of the pro domain – a process necessary for secretion.

Question 22

How does PCSK9 regulate LDLRs?

Answer 22

PCSK9 facilitates the decrease in LDLR through secretion into the blood where it binds to the N-terminal region of the EGF-A domain in a Ca2+ dependent manner by its catalytic domain.

Binding to the LDLR is thought to direct it to the lysosome, stimulating degradation indirectly and hence reducing the LDL uptake.

Question 23

What is the nature of the S127R and F216L mutations?

Answer 23

Overexpression the gene was shown to decrease the level of expressed LDLR, so these mutations were assumed to be gain-of-function.

They are thought to increase the autocatalysis, upregulating the secretion and so activity of the enzyme.

Question 24

How is PCSK9 related to therapy goals?

Answer 24

The inhibition of PCSK9 is the target of many pharmaceutical projects, through inhibition of the autocatalysis or disruption of the PCSK9-LDLR interaction.

Methods being tried include small molecules, peptides or antibodies, antisense/siRNA treatments and even CRISPR-Cas9 gene therapy.

Question 25

What is the protein size and genetic structure of the LDLR?

Answer 25

These 860AA, 93kDa proteins that are formed of five domains, from the N-terminal extracellular domain to the C-terminal intracellular ones.

The gene is made up of 18 exons spanning 45kbp, producing a 5.3kbp transcript.

Question 26

What is domain I of LDLRs?

Answer 26

The N-terminal binding region has 7 40-AA Class A repeats (~50% identical), each with 6 cysteine residues that form disulphide bonds.

The original deletion studies showed that ApoB100 (hence LDL) binding requires only repeats 2-7, while only repeat 5 is needed for ApoE. However, this has now been contested, as it has been shown that deletion of repeats 1 and 2 confers familial hypercholesterolaemia.

Question 27

What is domain II of LDLRs?

Answer 27

This is a 400AA epidermal growth factor (EGF) precursor homology domain, named due to the ~30% homology with the EGF precursor gene.

This includes three “growth factor” repeats, A, B and C. A and B have binding activity, and are separated from C by a YWTD repeat region that forms a beta-propeller motif important for receptor recycling and releasing bound LDL in response to lower pH in the endosome.

Question 28

What is domain III of LDLRs?

Answer 28

This domain contains 2 N-linked and 18 O-linked oligosaccharides, which appear to have little function, as attested by its deletion, but may act as a spacer to ensure the binding region is beyond the extracellular matrix.

Question 29

What is domain IV of LDLRs?

Answer 29

This is the single helical transmembrane domain, comprising a chain of 22 hydrophobic amino acid residues.

Question 30

What is domain V of LDLRs?

Answer 30

The internal C-terminal domain (50 residues) contains a signal sequence (NPXY) essential for receptor-ligand (LDL) internalization. This interacts with clathrin, concentrating the LDLRs in the coated pits.

Question 31

What mediates the pH dependent release of LDLs from LDLRs in the acidic endosome?

Answer 31

The β-propellor region of the EGF precursor domain contains a large number of histidine residues that allow it to act as a sensor for the acidity.

These have no net charge at physiological pH (7.4) but in the endosome (pH 5.3) they are protonated, leading to a conformational change in the domain.

A crystal structure of the extracellular domain at pH 5.3 showed the β-propellor domain interacting with the N-terminal Binding Region (repeat 4 and 5), hence it is thought that the change in conformation allows it to displace the LDL.

Question 32

What is Familial Hypercholesterolaemia (FH)?

Answer 32

This is a family of genetic disorders characterised by incredibly high levels of plasma LDL, which leads to early onset CVD/CHD/atherosclerosis and xanthomas (deposition of LDL-derived cholesterol under the skin and in tendons).

In 90% of cases this is caused by mutations in the LDLR gene preventing proper uptake of LDLs.

Question 33

What is the increase in LDL levels seen in FH?

Answer 33

Those heterozygous for the autosomal dominant disorder tend to have 2-4 fold higher blood LDL levels, with 5-10 fold higher in homozygous individuals.

Question 34

What is the incidence of FH in europe?

Answer 34

1 in 500 (htz) and 1 in 106 (hmz); though recent next-gen sequencing studies suggest that it may in fact be as high as 1 in 200 and 160,000 respectively.

Question 35

What is the high frequency of FH thought to be a result of?

Answer 35

Alu repeats nearby and within the LDLR gene leading to instability through Alu-mediated recombination.

Question 36

What is the nature of LDLR gene mutation?

Answer 36

Around 90% of these are SNPs or small (

Question 37

What defines class I mutations of LDLRs?

Answer 37

No or negligible LDL-R synthesis in the endoplasmic reticulum (ER) – hence no surface expression.

Question 38

What defines class II mutations of LDLRs?

Answer 38

Defective transport to the Golgi body for glycosylation and sorting. Usually, the receptor movement is delayed, rather than halted.

About 50% of all LDL-R mutations are class 2.

Question 39

What are the subclasses of class II LDLR mutations?

Answer 39

Lack of trafficking to the golgi for sorting and sequestration within the cell is subclass A.

For a few mutants lacking the transmembrane domain the receptor is secreted. When this happens the mutation is labelled subclass 2B.

Question 40

What is an example of a Class 2A LDLR mutation?

Answer 40

The Lebanese Mutation, in which the receptor is truncated after amino acid 659 - a nonsense mutation cuts off everything after the β-propeller region exposing two cysteine residues from glycan repeat C.

Question 41

What are the consequences of the Lebanese mutation?

Answer 41

This prevents trafficking from the ER to the Golgi due to recruitment of ER ‘gatekeeper proteins’, resulting in degradation of the receptor protein without stimulation of an immune response.

Question 42

What is the incidence of the Lebanese Mutation?

Answer 42

The mutation is so known because it occurs in the Lebanon in higher frequencies, 1 in 100,000 rather than 1,000,000.

Question 43

What defines class III mutations of LDLRs?

Answer 43

Mutations in the ligand binding repeats prevent binding of LDL. This can be tested for through the use of antibodies raised to wildtype LDLRs.

Question 44

What defines class IV mutations of LDLRs?

Answer 44

Here binding of LDL is normal, but the mutant receptors are unable to internalise receptor-ligand complexes due to disruption of the 50-residues cytoplasmic domain (790-839).

This prevents the receptors from congregating in clathrin coated pits or from internalising.

Mutations that cause this generally occur early, as only the first 22 amino acids are sufficient for rapid internalisation

Question 45

What is the internalisation signal in LDLRs?

Answer 45

The internalisation signal is conserved across species as NPXY – with the final residue crucially aromatic (W, Y or P).

In humans this motif is specifically FDNPVY.

Question 46

What is an example of a Class IV LDLR mutation, and what are its consequences?

Answer 46

One mutation that is associated with FH is point mutation of 807 to a cysteine; Y807C.

This prevents phosphorylation and subsequent recognition by LDLRAP1, a protein involved in stimulating internalisation.

Question 47

What is LDLRAP1?

Answer 47

An adaptor protein that recruits the LDLRs to clathrin triskelions.

LDLRAP1 binds to LDLR NPXY motifs in the cytosolic domain by a 134-residue PT-binding domain, and to clathrin with its little pentapeptide clathrin box; LLDLE.

Question 48

What disorder arises from LDLRAP1 Mutation?

Answer 48

Autosomal Recessive Hypercholesterolaemia (ARH).

This creates identical symptoms to FH – elevated LDL levels and resulting xanthoma.

Question 49

How do LDLRAP1 mutations cause ARH?

Answer 49

Inability to recruit clathrin to the LDLRs prevents internalisation of the LDL-LDLR.

This effect is largely specific to the liver and lymphocytes, as many cell types (including fibroblasts) also use another adaptor protein, Dab 2, which can compensate.

Question 50

What defines class V mutations of LDLRs?

Answer 50

These receptors cannot recycle properly, which leads to a relatively mild phenotype as LDL-R is still present in the cell surface (though all must be newly synthesized).

Question 51

What does dysfunction of ApoB100 cause?

Answer 51

Familial Defective ApoB-100 (FDB), one of four monogenetic causes of hypercholesterolaemia

Question 52

What mutations of ApoB100 have been identified?

Answer 52

Over 20 rare mutations have been identified, most of which lead to hypobetalipoproteinaemia due to prevention of lipoprotein synthesis.

The most common mutation associated however is R3500Q, which has a frequency of 1 in 1,000.

Question 53

What is the structure of ApoB100?

Answer 53

The ApoB-100 protein encircles the protein, with the C-terminal end (the last 11%) forming a triangular loop that bends the end back over the equator at R 3500, crossing shortly after the binding site for LDLRs; Site B – 3359-3369.

This is stabilised by the interaction between Arg-3500 and Trp-4369.

Question 54

What is the effect of the ApoB100 R3500Q mutation?

Answer 54

It causes FDB by changing the conformation of the loop as it can no longer cross at the point at which it normally would (interaction between Arg-3500 and Trp-4369).

Instead, the C-terminal tail crosses back over site B and preventing binding to LDLRs.

This mechanism was verified through targeted mutation of W4369 to Y, creating the same effect.

Question 55

What are the four monogenetic causes of hypercholesterolaemia?

Answer 55

FH - Familial Hypercholesterolaemia (LDLR mutation)

FDB - Familial Defective ApoB-100 (ApoB100 mutation)

ARH - Autosomal Recessive Hypercholesterolaemia (LDLRAP1 Mutation)

PCSK9 Mutation

Question 56

After the LDLs have been broken down in the lysosome, what proteins extract the cholesterol?

Answer 56

Two proteins within the acidic sac: the membrane bound NPC1 (Niemann-Pick C1) and the soluble NPC2.

Question 57

How do NPC1 and NPC2 extract cholesterol from the lysosome?

Answer 57

Cholesterol first binds into NPC2 through hydrophobic interaction with a cavity in the protein and the cholesterol iso-octyl side chain, exposing the 3’-OH to the lysosome interior.

The cholesterol is then transferred to the NPC1, which instead binds it by its hydroxyl group, exposing the hydrophobic side chain and hence leading to insertion of the cholesterol into the lysosome membrane.

From here the cholesterol is trafficked with NPC1 to the ER for processing.