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Studies of the autoinhibitory segment comprising residues 31 – 60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations

View Article: PubMed Central - PubMed

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and is internalized as a complex with the LDLR. In the acidic milieu of the sorting endosome, PCSK9 remains bound to the LDLR and prevents the LDLR from folding over itself to adopt a closed conformation. As a consequence, the LDLR fails to recycle back to the cell membrane. Even though it is the catalytic domain of PCSK9 that interacts with the LDLR at the cell surface, the structurally disordered segment consisting of residues 31–60 and which is rich in acidic residues, has a negative effect both on autocatalytic cleavage and on the activity of PCSK9 towards the LDLR. Thus, this unstructured segment represents an autoinhibitory domain of PCSK9. One may speculate that post-translational modifications within residues 31–60 may affect the inhibitory activity of this segment, and represent a mechanism for fine-tuning the activity of PCSK9 towards the LDLR. Our data indicate that the inhibitory effect of this unstructured segment results from an interaction with basic residues of the catalytic domain of PCSK9. Mutations in the catalytic domain which involve charged residues, could therefore be gain-of-function mutations by affecting the positioning of this segment.

No MeSH data available.


Related in: MedlinePlus

Amount of LDL internalized in HepG2 cells transiently transfected with PCSK9-containing plasmids with or without residues 31–52 deleted. HepG2 cells were transiently transfected with empty plasmid, the WT-PCSK9 plasmid or various mutant PCSK9 plasmids with or without residues 31–52 deleted (∆ 31–52). The amounts of fluorescently labelled LDL taken up by the transfected HepG2 cells were determined by flow cytometry. Mean (± SD) of three experiments is shown.
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f0015: Amount of LDL internalized in HepG2 cells transiently transfected with PCSK9-containing plasmids with or without residues 31–52 deleted. HepG2 cells were transiently transfected with empty plasmid, the WT-PCSK9 plasmid or various mutant PCSK9 plasmids with or without residues 31–52 deleted (∆ 31–52). The amounts of fluorescently labelled LDL taken up by the transfected HepG2 cells were determined by flow cytometry. Mean (± SD) of three experiments is shown.

Mentions: For all mutants studied, removal of Pro31–52 made the mutants more potent towards the LDLR as demonstrated by the reduced amounts of LDLRs (Fig. 2). To confirm these findings, the effect of removing Pro31–52 from the various mutant PCSK9s was also studied by flow cytometry. HepG2 cells transiently transfected with the different plasmids were incubated with fluorescently labelled LDL and the amount of LDL internalized was used as a measure of the amount of cell-surface LDLRs. As can be seen from Fig. 3, the full-length gain-of-function mutants involving arginines, had activities towards the LDLR that were similar to that of WT-PCSK9. In contrast, the D374Y-PCSK9 gain-of-function mutant was markedly more active than WT-PCSK9 leading to a 53% reduction in the amount of LDL internalized (Fig. 3). Deleting Pro31–52 from WT-PCSK9 reduced the amount of LDL internalized by 31%. For the gain-of-function mutants S127R-PCSK9, R215H-PCSK9, R218H-PCSK9 and R218S-PCSK9, deleting Pro31–52 made these mutants approximately 40% more active than the respective full-length mutants (Fig. 3) and approached the activity of that of the full-length D374Y-PCSK9 mutant. Thus, if these gain-of-function mutants involving arginines somehow led to deletion of residues 31–52 in vivo, they would have an activity similar to that of D374Y-PCSK9. Deleting Pro31–52 from the D374Y-PCSK9 mutant did not further increase the activity towards the LDLR (data not shown).


Studies of the autoinhibitory segment comprising residues 31 – 60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations
Amount of LDL internalized in HepG2 cells transiently transfected with PCSK9-containing plasmids with or without residues 31–52 deleted. HepG2 cells were transiently transfected with empty plasmid, the WT-PCSK9 plasmid or various mutant PCSK9 plasmids with or without residues 31–52 deleted (∆ 31–52). The amounts of fluorescently labelled LDL taken up by the transfected HepG2 cells were determined by flow cytometry. Mean (± SD) of three experiments is shown.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121147&req=5

f0015: Amount of LDL internalized in HepG2 cells transiently transfected with PCSK9-containing plasmids with or without residues 31–52 deleted. HepG2 cells were transiently transfected with empty plasmid, the WT-PCSK9 plasmid or various mutant PCSK9 plasmids with or without residues 31–52 deleted (∆ 31–52). The amounts of fluorescently labelled LDL taken up by the transfected HepG2 cells were determined by flow cytometry. Mean (± SD) of three experiments is shown.
Mentions: For all mutants studied, removal of Pro31–52 made the mutants more potent towards the LDLR as demonstrated by the reduced amounts of LDLRs (Fig. 2). To confirm these findings, the effect of removing Pro31–52 from the various mutant PCSK9s was also studied by flow cytometry. HepG2 cells transiently transfected with the different plasmids were incubated with fluorescently labelled LDL and the amount of LDL internalized was used as a measure of the amount of cell-surface LDLRs. As can be seen from Fig. 3, the full-length gain-of-function mutants involving arginines, had activities towards the LDLR that were similar to that of WT-PCSK9. In contrast, the D374Y-PCSK9 gain-of-function mutant was markedly more active than WT-PCSK9 leading to a 53% reduction in the amount of LDL internalized (Fig. 3). Deleting Pro31–52 from WT-PCSK9 reduced the amount of LDL internalized by 31%. For the gain-of-function mutants S127R-PCSK9, R215H-PCSK9, R218H-PCSK9 and R218S-PCSK9, deleting Pro31–52 made these mutants approximately 40% more active than the respective full-length mutants (Fig. 3) and approached the activity of that of the full-length D374Y-PCSK9 mutant. Thus, if these gain-of-function mutants involving arginines somehow led to deletion of residues 31–52 in vivo, they would have an activity similar to that of D374Y-PCSK9. Deleting Pro31–52 from the D374Y-PCSK9 mutant did not further increase the activity towards the LDLR (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and is internalized as a complex with the LDLR. In the acidic milieu of the sorting endosome, PCSK9 remains bound to the LDLR and prevents the LDLR from folding over itself to adopt a closed conformation. As a consequence, the LDLR fails to recycle back to the cell membrane. Even though it is the catalytic domain of PCSK9 that interacts with the LDLR at the cell surface, the structurally disordered segment consisting of residues 31–60 and which is rich in acidic residues, has a negative effect both on autocatalytic cleavage and on the activity of PCSK9 towards the LDLR. Thus, this unstructured segment represents an autoinhibitory domain of PCSK9. One may speculate that post-translational modifications within residues 31–60 may affect the inhibitory activity of this segment, and represent a mechanism for fine-tuning the activity of PCSK9 towards the LDLR. Our data indicate that the inhibitory effect of this unstructured segment results from an interaction with basic residues of the catalytic domain of PCSK9. Mutations in the catalytic domain which involve charged residues, could therefore be gain-of-function mutations by affecting the positioning of this segment.

No MeSH data available.


Related in: MedlinePlus