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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.


Pro31–52 of PCSK9 has an autoinhibitory effect also when the C-terminal domain is deleted. WT-PCSK9 (WT) or WT-PCSK9 with residues 31–52 deleted (∆ 31–52WT) were purified from stably transfected HEK293 cells. The concentrations of the two purified PCSK9 preparations were adjusted by concentration/dilution (panel a). Similar procedures were performed for purified L455X-PCSK9 which lacks the C-terminal domain (L455X) and L455X-PCSK9 with residues 31–52 deleted (∆ 31–52L455X). Duplicates are shown. CHO T-REx cells stably transfected with an LDLR plasmid were incubated with equal concentrations of each of the four purified PCSK9 preparations shown in panel a. The amounts of the different PCSK9 preparations internalized in the CHO T-REx cells were determined by Western blot analyses of lysates using an antibody against the C-terminal V5 epitope tag (panel b).
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f0005: Pro31–52 of PCSK9 has an autoinhibitory effect also when the C-terminal domain is deleted. WT-PCSK9 (WT) or WT-PCSK9 with residues 31–52 deleted (∆ 31–52WT) were purified from stably transfected HEK293 cells. The concentrations of the two purified PCSK9 preparations were adjusted by concentration/dilution (panel a). Similar procedures were performed for purified L455X-PCSK9 which lacks the C-terminal domain (L455X) and L455X-PCSK9 with residues 31–52 deleted (∆ 31–52L455X). Duplicates are shown. CHO T-REx cells stably transfected with an LDLR plasmid were incubated with equal concentrations of each of the four purified PCSK9 preparations shown in panel a. The amounts of the different PCSK9 preparations internalized in the CHO T-REx cells were determined by Western blot analyses of lysates using an antibody against the C-terminal V5 epitope tag (panel b).

Mentions: To study whether deletion of Pro31–52 affected the binding and uptake of L455X-PCSK9 by the LDLR, CHO T-REx cells stably transfected with a WT-LDLR plasmid were cultured in the presence of equal amounts of purified Δ31–52-L455X-PCSK9 or L455X-PCSK9. As is shown in Fig. 1b, the amount of Δ31–52-L455X-PCSK9 in lysates was 5-fold higher than that of L455X-PCSK9. For comparison, the amount of Δ31–52-WT-PCSK9 in lysates was 3-fold higher than that of WT-PCSK9. Thus, deleting Pro31–52 from L455X-PCSK9 increased the uptake of L455X-PCSK9 by the LDLR in a fashion similar to that of deleting Pro31–52 from WT-PCSK9. These findings indicate that Pro31–52 does not exhibit its negative effect by interacting with the C-terminal domain of PCSK9.


Studies of the autoinhibitory segment comprising residues 31 – 60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations
Pro31–52 of PCSK9 has an autoinhibitory effect also when the C-terminal domain is deleted. WT-PCSK9 (WT) or WT-PCSK9 with residues 31–52 deleted (∆ 31–52WT) were purified from stably transfected HEK293 cells. The concentrations of the two purified PCSK9 preparations were adjusted by concentration/dilution (panel a). Similar procedures were performed for purified L455X-PCSK9 which lacks the C-terminal domain (L455X) and L455X-PCSK9 with residues 31–52 deleted (∆ 31–52L455X). Duplicates are shown. CHO T-REx cells stably transfected with an LDLR plasmid were incubated with equal concentrations of each of the four purified PCSK9 preparations shown in panel a. The amounts of the different PCSK9 preparations internalized in the CHO T-REx cells were determined by Western blot analyses of lysates using an antibody against the C-terminal V5 epitope tag (panel b).
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f0005: Pro31–52 of PCSK9 has an autoinhibitory effect also when the C-terminal domain is deleted. WT-PCSK9 (WT) or WT-PCSK9 with residues 31–52 deleted (∆ 31–52WT) were purified from stably transfected HEK293 cells. The concentrations of the two purified PCSK9 preparations were adjusted by concentration/dilution (panel a). Similar procedures were performed for purified L455X-PCSK9 which lacks the C-terminal domain (L455X) and L455X-PCSK9 with residues 31–52 deleted (∆ 31–52L455X). Duplicates are shown. CHO T-REx cells stably transfected with an LDLR plasmid were incubated with equal concentrations of each of the four purified PCSK9 preparations shown in panel a. The amounts of the different PCSK9 preparations internalized in the CHO T-REx cells were determined by Western blot analyses of lysates using an antibody against the C-terminal V5 epitope tag (panel b).
Mentions: To study whether deletion of Pro31–52 affected the binding and uptake of L455X-PCSK9 by the LDLR, CHO T-REx cells stably transfected with a WT-LDLR plasmid were cultured in the presence of equal amounts of purified Δ31–52-L455X-PCSK9 or L455X-PCSK9. As is shown in Fig. 1b, the amount of Δ31–52-L455X-PCSK9 in lysates was 5-fold higher than that of L455X-PCSK9. For comparison, the amount of Δ31–52-WT-PCSK9 in lysates was 3-fold higher than that of WT-PCSK9. Thus, deleting Pro31–52 from L455X-PCSK9 increased the uptake of L455X-PCSK9 by the LDLR in a fashion similar to that of deleting Pro31–52 from WT-PCSK9. These findings indicate that Pro31–52 does not exhibit its negative effect by interacting with the C-terminal domain of PCSK9.

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.