Lecture 11 trafficking case study 2 Flashcards
What did mutation data show about apoB and E?
Repeats 2-7 are required to bind apoB but only repeat 5 was necessary for binding of apoE.
What experiment showed ligand release was caused by a change in pH from 7 to 5?
WT has LB, EGFP, O-linked sugar region, cytoplasmic tail. Then deleted EGFP mutation which binds to ligand fine but doesnt release ligand. they dont recycle back to cell surface. Suggests EGFP is important for ligand release (YWTD).
What is the structural difference of LDLR at pH 7 and 5?
At pH5 EGF-C is close to YWTD. EGFA and EGFB are further away. 7 repeats of LA repeats. Compact structure. Explains why it can’t bind to LDL. Still don’t know pH7 structure.
What is the predicted structure for pH7?
Open structure explains gel filtration data. LBD are available.
What is the molecular suggestion for ligand release?
After binding and being internalised goes to endosome system. They are maturing from rab5 early to rab7 late for example.
This leads to a drop in pH as they are maturing to lysosomes. The presumption is there is a competition for binding to the LA repeats between ligand and beta propellor. At pH 5 the beta propellor out competes the LDL ligand vice versa and pH7. LDL ligand is then released.
Is there evidence for this model?
Top surface from space imaging shows at pH7 when you map the predicted surface charge largely acidic patches and this hasnt got sufficient positive charge at the beta propellor in the same way that the ligand has positive charge. At pH 5 appearance of increased positive charge underneath the predicted binding surface contributed by histidines. This positive charge is now sufficient to bind to negative charge on LA repeat surface. Close enough that can out compete the ligand on LDL to drive LDL off and closed structure is adopted.
What is the YWTD-LA interaction conserved to?
Similar to RAP-LA4/5
Give some examples of mutations affecting the YWTD domain.
R4: W144, R5: H190, W193 Beta propellor: H562, H586, K582. Histidines protonated at acidic endosomal pH leading to confirmational change leading to ligand release. The inability to release ligand leads to recycling failure and lysosomal LDLR degradation.
What is PCSK9?
a novel regulator of LDLR
What does PCSK9 stand for?
Pro-protein convertase subtilisin kexin type 9.
What do mutations in PCSK9 cause?
ADH.
What do the different mutations cause?
Gain of function mutants lead to raised LDL-C. Loss of function mutants lead to lowered LDL-C.
How does PCSK9 act?
Enzyme activity is not required. Acts as chaperone-directing LDLR degradation.
How does PCSK9 function?
Travels through ER and golgi. Mature protein is secreted from the cell and is in the ECM to bind to the LDLR. When it binds it appears to cause an interactional knot with the LDLR and LDL. When this is internalised the receptor itself can be trafficked to lysosome of the ligand.
How does PCSK9 interact with the receptor?
Has an EGF-A domain like the LDLR. This is the interaction point.
How do mutations affect cellular activity?
Mutations that increase circulating cholesterol:
1. Increased affinity for LDL receptor. Shows decreased number at cell surface as more goes to lysosome for degradation.
2. Impaired degradation. Make proteins more stable in ECM. Concentration at ECM increases.
3. Self association. Increased oligomerisation. Demonstrate a higher affinity for the receptor.
All lead to impaired clearance of LDL cholesterol.
What does PCSK9 not interfere with?
Acid dissociation of LDL cargo.
What does the D374Y mutation cause?
As D aspartic acid is -ve charge forms an interaction with the positive charge histidine on the EGFA domain. All in the PCSK9 protein.
What is PCSK9 regulated by?
Statins, bile acids, cholesterol and many other things so very useful in pharma industry.
What is IDOL?
Inducible degrador of LDLR. RING E3. Expression controlled by cellular cholesterol status. Has a FERM domain which binds the LDLR through the phospho tyrosine binding motif in this domain. Modifies and ubiquitinates it.
What does the G15S IDOL mutation lead to?
Causes decreased IDOL dimerisation, which causes decreased IDOL autoubiquitylation, increased sorting of the LDLR to the lysosome for degradation and therefore increased circulating LDL cholesterol.
Where does the dimerisation function come from?
The RING domain.
What can IDOL recruit?
E2 for ubiquitin attachment. This changes the recycling of the receptor into trafficking to the lysosome.
