HMG CoA reductase

What reaction is catalysed by HMG CoA reductase?

Conversion of HMG CoA to mevalonate during the production of cholesterol

What is HMG CoA?

3-hydrox-3-methylglutaryl CoA

What is a healthy mean cholesterol level?

4-5 mmol/L

What risks increase with and increase in cholesterol?

Increased risk of coronary heart disease and cardiovascular death

When are statins often prescribed?

When cholesterol reaches around 6 mmol/L

How can HMG CoA reductase be inhibited by drug action?

Use of compactin or mevinolin, which can act as competitive inhibitors.

What is the action of statins?

Inhibits HMG CoA reductase

What is the rate limiting step of cholesterol synthesis?

The action of HMG CoA reductase

What size is HMG CoA reducatse?

60 kDa determined from SDS PAGE, full size is 120 kDa with a transmembrane region

What is the structure of HMG CoA reductase?

Cytosolic N and C-terminals.
8 TM domains form sterol sensing domain and a 60 kDa cytosolic catalytic domain.
N-linked glycosylation site between TM spans 7 and 8

What molecule can be used to test how cholesterol affects cells?

25-hydroxycholesterol

How does cholesterol affect HMG CoA reductase?

Leads to rapid decrease in HMG CoA reductase, causes degradation.

How can the presence of HMG CoA reductase be detected experimentally?

β-galactosidase domain added to replace the catalytic domain. X-gal added, cultures containing β-galactosidase domain will become blue.
Addition of 25-hydroxycholesterol can see how β-galactosidase expression changes

Which region of HMG CoA reductase senses cholesterol?

Sterol sensing domain

What animals were used to determine the regions of the sterol sensing domain required for changes due to cholesterol?

Sea Urchin and hamsters.
Hamsters HMG CoA reductase action is similar to humans.
Sea Urchin HMG CoA reductase levels don't change due to cholesterol

How was Sea Urchin and Hamster HMG CoA reductase used to determine which regions of the sterol sensing domain are required?

Regions of HMG CoA reductase sterol sensing region in Hamster where spliced with Sea Urchin DNA to determine regions that respond

Which regions of the sterol sensing domain are responsible for cholesterol sensing?

Transmembrane spans 2-6

How was the position of the HMG CoA reductase gene promoter identified?

Regions of the gene and before the gene were cloned until the distance of the promoter from the gene was determined due to the removal of bases leading to no transcription

How far from the start of the HMG CoA reductase gene was the promoter found?

with 180 base pairs from the start of the gene

How were the bases involved in transcription binding for the transcription of the HMG CoA reductase gene determined?

Each base within the 180 base region was changed and transcription measured.
Determined that the region for TF binding was the Shine-Dalgarno sequence

What region of DNA is involved in HMG CoA reductase production?

Sterol response element (SRE)

How was the protein that binds to the sterol response element determined?

Shine-Dalgarno sequence of the gene immobilised on a bead and nuclear protein added. Unbound proteins washed away and the gene for the protein that binds to the SRE was determined after purification

How does the SRE-binding protein size differ in the cloned version and the purified version?

Cloned version is much larger than the purified version

What is the transcription factor that binds to the sterol response element?

SRE-binding protein (SREBP)

What is the structure of SRE-binding protein?

N-terminal acidic domain.
Basic H-L-H domain.
Two transmembrane segments.
Regulatory C-terminal

What is the function of the acidic domain of SREBP?

Helps interactions with other proteins

What is the function of the basic helix-loop-helix (bHLH) in SREBP?

Helix inserts into DNA, zipper contains leucine that allows the insertion of the HLH into the DNA

What cleavage sites are present in SREBP?

LS region (inside ER) and DRSR regions (cytosolic)

How does the two step cleavage of SREBP act?

Regulation of cleavage at LS region.
Allows slight conformational change for DSRS region to be accessed by unregulated protease.

What similarities are there between SCAP and HMG CoA reductase?

Both contain TM domains 2-6 with similar structures, act as sterol sensing domains

What molecule activated cleavage of the LS region of SREBP?

SREBP-cleavage activating protein (SCAP).
Not a protease but activates cleavage

How do cholesterol levels affect SCAP?

Low cholesterol is sensed leading to the cleavage of SREBP at the LS region

How do proteins interact with SCAP?

Via the WD40 domain

How can the proteins that bind to SCAP be determined?

Use bead conjugated antibody that binds to SCAP.
Centrifugation causes SCAP and any bound protein to be present in the pellet

What proteins are known to interact with SCAP?

SREBP2, almost always bound in low cholesterol

How can the localisation of SCAP be determined?

Replace WD40 domain with GFP

How does the localisation of SCAP change in high cholesterol levels?

Moves to the far end of the ER, away from SREBP.

How does the localisation of SCAP change in low cholesterol levels?

Moved from RER to Golgi, important in SREBP movement to Golgi

Where is the protease required for site 1 cleavage of SREBP located?

In the Golgi, constituently active

How was the translocation of the protease for site one cleavage of SREBP to the Golgi prevented, what did it show?

KDEL motif added, prevents transport from RER to Golgi.
Protease still acted at site 1, showing that the localisation of the protease and SREBP are important for regulation

What is INSIG?

Insulin induced gene

How does cholesterol levels affect INSIG-SCAP interactions?

High cholesterol causes INSIG binding to SCAP, prevents movement to Golgi due to binding to the sterol sensing domain of SCAP, cholesterol can also bind to the sterol sensing domain of SCAP

What does SCAP interact with in low cholesterol concentrations?

Interacts with Sec24

What are the components of the COPII coat?

Sar1, Sec23, Sec 24

What is the function of the Sar1/Sec23/Sec24 complex?

Involved in the transport of SREBP and SCAP to the Golgi

How does INSIG prevent the transport of SREBP and SCAP to the Golgi?

Binds to the sterol sensing domain of SCAP, preventing interactions with Sec24 as MELADL region is inaccessible

What region of SCAP interacts with Sec24?

Interacts via MELADL sequence in loop 6 of SCAP

How does cholesterol prevent SREBP and SCAP transport to Golgi?

Binds to sterol sensing domain on SCAP, preventing interaction with Sec24 as SCAP then binds INSIG

How do oxysterols prevent SREBP and SCAP transport to Golgi?

Binds to sterol sensing domain of INSIG which then binds SCAP

What is required for the prevention of SCAP transport?

High cholesterol and INSIG presence

How does cholesterol affect INSIG levels?

Low cholesterol leads degradation of INSIG to ensure a large enough increase in cholesterol when required.
INSIG promoter binds SREBP, so as cholesterol levels increase so do INSIG levels

What step of cholesterol synthesis is the point where cholesterol synthesis is the only option?

Once Lanosterol has been produced

Which sterols can bind to the sterol sensing domain of HMG CoA reductase?

Cholesterol doesn't bind, Lanosterol can bind as can INSIG

How can INSIG binding lead to HMG CoA reductase ubiquitination?

Lanosterol triggers INSIG binding to HMG CoA reductase. INSIG also binds Gp78 (E3 ubiquitin ligase), causes ubiquitination of HMG CoA reductase

How is ubiquitinated HMG CoA reductase degraded?

ATP used to remove HMG CoA reductase from the membrane by VCP action and then degraded in the cell by the 26S protease

How can oxysterols cause a decrease in HMG CoA reductase levels?

Binds to INSIG causing binding to SCAP.
Can also inhibit SCAP and SREBP action