Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

The cytosolic tails of sortilin and APP interact via the FLVHRY motif of sortilin with the YNPTYKFFE motif in APP, which overlaps with the ARH binding consensus YENPTY.

“Their cytosolic tails interact via the FLVHRY motif of sortilin with the YNPTYKFFE motif in APP, which overlaps with the ARH binding consensus YENPTY (26,27).”

• cites [26] Garcia, 2001, Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein, Science sourceNot checked — no full text of the source
• cites [27] He, 2002, ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2, J. Biol. Chem sourcePartly supported

  The Results (page unavailable) identify an APP peptide containing YENPTY, and the Discussion reports that ARH binds the LDL receptor tail through an NPVY-containing sequence. The paper does not establish YENPTY as an ARH-binding consensus or report an interaction between the cytosolic tails of sortilin and APP via FLVHRY and YNPTYKFFE. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Sortilin and APLP2 co-precipitate in the presence of NH4Cl.

“Notice the co-precipitation of sortilin and APLP2 in the presence of NH 4 Cl.”

Overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin, APLP2, and sAPLP2 in HEK293 cells.

“The data show that overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin (A), APLP2 (B), and sAPLP2 (C) in HEK293 cells.”

In HepG2 and Huh7 cells, APLP2 knockdown does not block exogenous PCSK9-induced LDLR degradation.

“Importantly, under significantly reduced levels of APLP2, exogenous PCSK9 (input media; Fig. 1C) still induced degradation of up to 70% of the LDLR in HepG2 (Fig. 1A) and Ն90% in Huh7 cells (Fig. 1B).”

• Also stated in: § Results · plain

  “In conclusion, a substantial APLP2 deficiency did not block the ability of extracellular PCSK9 to induce the degradation of the LDLR in HepG2 and Huh7 cells.”

Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms.

“Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms.”

APLP2 and sortilin interact strongly with each other.

“Surprisingly, although APLP2 and sortilin did not bind Sec24, they interacted strongly with each other, as also confirmed by co-immunoprecipitation (Fig. 7).”

Sortilin stabilizes APLP2.

“Interestingly, sortilin was shown to target APP for lysosomal degradation (55), whereas our data revealed that sortilin stabilizes APLP2 (Fig. 4B), suggesting a functional difference between the interaction of sortilin with APP or APLP2.”

Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation.

“Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation.”

Sec23B binds weakly to sortilin and APLP2.

“From this assay, we found that Sec24A did not bind APLP2 or sortilin, whereas Sec23B bound weakly to sortilin and APLP2, with the latter interaction best seen when Sec23B has the Gluc-1 domain at its N terminus (Fig. 7B).”

Exogenous PCSK9 does not degrade sortilin or APLP2.

“Unexpectedly, the intracellular levels of sortilin and APLP2 were dramatically decreased in HEK293 cells co-expressing PCSK9 (Ϫ90 and Ϫ40%, respectively; Fig. 4A), whereas neither protein was degraded in the presence of exogenous PCSK9 (not shown).”

The colon, small intestine, retina, olfactory tract, and cerebellum express high levels of PCSK9, APLP2, and sortilin.

“In situ hybridization in adult mouse liver or 10-day-old whole body revealed that the colon, small intestine, retina, olfactory tract, and cerebellum express high levels of PCSK9, APLP2, and sortilin (Fig. 8, left panel), whereas in the mouse (Fig. 8, right panel) and human (56) liver, sortilin expression is rather poor.”

Sortilin binds APLP2.

“Sortilin binds APLP2.”

APLP2 has no effect on sortilin levels.

“Conversely, APLP2 had no effect on the levels of sortilin (Fig. 4B, lanes 1 and 4).”

Exogenous PCSK9 reduces LDLR levels similarly (30-40%) in primary hepatocytes from WT and Aplp2-/- mice.

“Furthermore, the LDLR levels were similarly reduced (30 -40%) by exogenous PCSK9 (2-or 16-h incubations) in primary hepatocytes isolated from both genotypes (Fig. 2E).”

Aplp2-/- mice have similar plasma total cholesterol levels to WT mice.

“However, similar plasma total cholesterol levels were observed in both genotypes (116 versus 123 mg/dl in WT mice).”

WT PCSK9 and ER-retained PCSK9-KDEL degrade sortilin, APLP2, and sAPLP2.

“Western blot analysis showed that WT PCSK9 and ER-retained PCSK9-KDEL were able to degrade sortilin, APLP2, and sAPLP2.”

Sort1-/- mice have similar plasma total cholesterol levels to WT.

“In addition, plasma total cholesterol (Fig. 3D) and PCSK9 (Fig. 3E) levels were similar in WT and Sort1 Ϫ/Ϫ littermates.”

Sortilin and APLP2 interact strongly in the split-luciferase assay.

“However, the assay unraveled an unsuspected strong interaction between sortilin and APLP2 (Fig. 7B, upper right corner).”

In HepG2 and Huh7 cells, 50 μg/ml pure PCSK9 enhances LDLR degradation independently of APLP2.

“Our data showed that this high concentration of pure PCSK9 in the medium similarly enhanced the degradation of the LDLR independently of APLP2 (not shown).”

Aplp2-/- mice have slightly but significantly reduced circulating PCSK9 levels by 20% in males and 27% in females compared with WT.

“we first analyzed the circulating levels of PCSK9 in Aplp2 ϩ/ϩ (WT) and Aplp2 Ϫ/Ϫ mice, and found them slightly but significantly reduced by 20 and 27% in Aplp2 Ϫ/Ϫ males and females, respectively (Fig. 2A).”

ER-retained PCSK9-KDEL enhances the degradation of APLP2, sAPLP2, and sortilin, likely in the ERAD pathway.

“This is further evidenced by the ability of ER-retained PCSK9-KDEL to enhance the degradation of APLP2, sAPLP2, and sortilin likely in the ERAD pathway (Fig. 5).”

APLP2 and sortilin interact with each other.

“Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other”

• Also stated in: § Abstract · plain

  “However, using a proximity assay based on a split luciferase (37), we showed that APLP2 and sortilin can interact with each other in HEK293 cells.”

• Also stated in: § PCSK9 Convertase-mediated LDLR Degradation · boosted

  “These data demonstrate that in HEK293 cells sortilin and APLP2 can bind to each other.”

Co-expression of PCSK9 reduces intracellular sortilin levels by ~90% in HEK293 cells.

“Unexpectedly, the intracellular levels of sortilin and APLP2 were dramatically decreased in HEK293 cells co-expressing PCSK9 (Ϫ90 and Ϫ40%, respectively; Fig. 4A), whereas neither protein was degraded in the presence of exogenous PCSK9 (not shown).”

Surface LDLR levels in the liver of Sort1-/- and Sort+/+ mice were similar.

“Surface LDLR levels in the liver of Sort1 Ϫ/Ϫ and Sort ϩ/ϩ mice were similar.”

WT and Aplp2-/- primary hepatocytes secrete similar levels of endogenous PCSK9.

“Finally, WT or Aplp2 Ϫ/Ϫ primary hepatocytes secreted similar levels of endogenous PCSK9 (98.1 Ϯ 4.2 versus 74.3 Ϯ 20.3 ng/ml for WT mice; n ϭ 3 and p ϭ 0.12), suggesting that lower circulating levels of PCSK9 in Aplp2 Ϫ/Ϫ male and female mice (Fig. 2A) are not due to a secretion defect of PCSK9 by hepatocytes.”

Overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells.

“overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells.”

Sec24A does not bind APLP2 or sortilin.

“From this assay, we found that Sec24A did not bind APLP2 or sortilin, whereas Sec23B bound weakly to sortilin and APLP2, with the latter interaction best seen when Sec23B has the Gluc-1 domain at its N terminus (Fig. 7B).”

APLP2 and sortilin do not bind Sec24.

“Surprisingly, although APLP2 and sortilin did not bind Sec24, they interacted strongly with each other, as also confirmed by co-immunoprecipitation (Fig. 7).”

Sort1-/- mice have similar plasma PCSK9 levels to WT.

“In addition, plasma total cholesterol (Fig. 3D) and PCSK9 (Fig. 3E) levels were similar in WT and Sort1 Ϫ/Ϫ littermates.”

Pcsk9-/- mice showed a 2.5-fold higher level of LDLR.

“The latter showed a 2.5-fold higher level of LDLR.”

Co-expression of PCSK9 reduces intracellular APLP2 levels by ~40% in HEK293 cells.

“Unexpectedly, the intracellular levels of sortilin and APLP2 were dramatically decreased in HEK293 cells co-expressing PCSK9 (Ϫ90 and Ϫ40%, respectively; Fig. 4A), whereas neither protein was degraded in the presence of exogenous PCSK9 (not shown).”

Liver APLP2 levels were not affected in Pcsk9-/- mice.

“Note that liver APLP2 levels were not affected in Pcsk9 Ϫ/Ϫ mice (last lane).”

In mouse and human liver, sortilin expression is rather poor.

“In situ hybridization in adult mouse liver or 10-day-old whole body revealed that the colon, small intestine, retina, olfactory tract, and cerebellum express high levels of PCSK9, APLP2, and sortilin (Fig. 8, left panel), whereas in the mouse (Fig. 8, right panel) and human (56) liver, sortilin expression is rather poor.”

• cites [56] Petersen, 1997, Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography, J. Biol. Chem sourceNot checked yet

LDLR, PCSK9, and SREBP-2 mRNA expressions in WT and Sort1-/- livers are comparable.

“Additionally, LDLR, PCSK9, and SREBP-2 mRNA expressions in WT and Sort1 Ϫ/Ϫ livers were comparable (not shown).”

Sort1-/- mice show no difference in liver total LDLR levels compared with WT.

“Thus, in the liver, neither total LDLR levels examined by Western blotting (Fig. 3B) nor surface levels examined by immunohistochemistry (Fig. 3C) were affected.”

APLP2 and sortilin were targeted for degradation in both the ER and acidic compartments by PCSK9 in HEK293 and Huh7 cells.

“Even though we concluded that APLP2 or sortilin did not significantly affect PCSK9 activity on the LDLR, we were surprised by the fact that APLP2 and sortilin were targeted for degradation in both the ER and acidic compartments by PCSK9 in HEK293 and Huh7 cells (Figs. 456).”

Pcsk9+/- hepatocytes have ~30% increased LDLR levels.

“increased LDLR levels on Pcsk9 ϩ/Ϫ hepatocytes (ϳ30% increase; Fig. 3F)”

Primary hepatocytes from WT and Aplp2-/- littermates exhibit the same total LDLR levels.

“Primary hepatocytes isolated from three WT and three Aplp2 Ϫ/Ϫ littermates also exhibited the same levels of total LDLR (Fig. 2D).”

Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2.

“Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2.”

Sortilin depletion alone or in combination with APLP2 has no significant impact on LDLR levels in Huh7 cells.

“Knockdown of sortilin, APLP2, or both revealed that lack of sortilin, alone or in combination with APLP2, had no significant impact on LDLR levels, suggesting that PCSK9-triggered LDLR degradation was not sensitive to lower levels of these transmembrane proteins in Huh7 cells.”

Co-expression of sortilin with APLP2 results in higher APLP2 levels.

“Interestingly, co-expression of sortilin with APLP2 resulted in higher levels of the latter, suggesting that sortilin binding stabilizes APLP2 and partially rescues it from degradation.”

Heterozygous Pcsk9+/- mice exhibit a ~70% decrease in circulating PCSK9 levels compared with WT mice.

“Heterozygous Pcsk9 ϩ/Ϫ mice exhibit a ϳ70% decrease in circulating PCSK9 levels as compared with WT mice (29.7 Ϯ 11.0 versus 94.2 Ϯ 11.0 ng/ml, respectively; n ϭ 6 -7 mice; p ϭ 5 ϫ 10 Ϫ6 ).”

PCSK9 expression pattern is tissue-specific with moderate to high level mRNA concentration in the eye retina inner nuclear layer, brain olfactory tract and cerebellum, spleen, liver, small intestine, and colon.

“B, tissue-specific PCSK9 expression pattern with moderate to high level mRNA concentration in the eye retina inner nuclear layer, brain olfactory tract and cerebellum, spleen, liver, small intestine, and colon.”

In the liver of transgenic mice overexpressing PCSK9, PCSK9 did not target APLP2 and sortilin for degradation.

“However, this did not happen in the liver of transgenic mice overexpressing PCSK9 (not shown).”

PCSK9 can target APLP2 to endosomes/lysosomes for degradation.

“PCSK9 can target APLP2 to endosomes/lysosomes for degradation.”

Sort1-/- mice show no difference in liver surface LDLR levels compared with WT.

“Thus, in the liver, neither total LDLR levels examined by Western blotting (Fig. 3B) nor surface levels examined by immunohistochemistry (Fig. 3C) were affected.”

APLP2 and sortilin do not regulate PCSK9 convertase-mediated LDLR degradation.

“Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other”

• Also stated in: § Abstract · plain

  “Our data revealed that, in hepatocyte cell lines and in the liver, the ability of extracellular and intracellular PCSK9 to enhance the degradation of the LDLR, ex vivo and in vivo, does not require the presence of APLP2 or sortilin.”

Neither APLP2 nor sortilin modify the activity of PCSK9 on the LDLR.

“Thus neither APLP2 nor sortilin modify the activity of PCSK9 on the LDLR.”

APLP2 can partially regulate the levels of circulating PCSK9.

“However, Aplp2 Ϫ/Ϫ mice exhibited a 20 -27% decrease in plasma PCSK9 levels, suggesting that APLP2 can partially regulate the levels of circulating PCSK9 (Fig. 2A).”

PCSK9 induces degradation of sortilin in the acidic compartments of endosomes/lysosomes.

“However, upon incubation of cells with both 5 mM NH 4 Cl and 10 g/ml of the cell permeable Cys-protease inhibitor E64d, we did observe a partial rescue of sortilin (not shown), suggesting that here also PCSK9 induces the degradation of sortilin in the acidic compartments of endosomes/lysosomes.”

The results on Sort1-/- mice in this study differ from those reported by Gustafsen et al. (35), who reported that Sort1-/- mice exhibit a ~30% reduction in circulating PCSK9 and ~2.2-fold increase in liver LDLR protein.

“Our results on Sort1 Ϫ/Ϫ mice differ from those of Gustafsen et al. (35) who reported that Sort1 Ϫ/Ϫ mice exhibit a ϳ30% reduction in the levels of circulating PCSK9 and ϳ2.2-fold increase in liver LDLR protein.”

• cites Gustafsen et al. [35] Gustafsen, 2014, The hypercholesterolemia-risk gene SORT1 facilitates PCSK9 secretion, Cell Metab sourceNot checked — no full text of the source

The higher than 50% decrease in circulating PCSK9 in Pcsk9+/- mice is likely due to increased LDLR levels on Pcsk9+/- hepatocytes, contributing to further clearance of circulating PCSK9.

“This higher than 50% decrease is likely due to increased LDLR levels on Pcsk9 ϩ/Ϫ hepatocytes (ϳ30% increase; Fig. 3F), thereby contributing to a further clearance of circulating PCSK9.”

There is a functional difference between the interaction of sortilin with APP or APLP2.

“Interestingly, sortilin was shown to target APP for lysosomal degradation (55), whereas our data revealed that sortilin stabilizes APLP2 (Fig. 4B), suggesting a functional difference between the interaction of sortilin with APP or APLP2.”

ER retention of the complex of PCSK9 with APLP2, sAPLP2, and sortilin likely leads to their degradation within the ERAD system.

“However, because PCSK9-KDEL is also able to degrade APLP2, sAPLP2, and sortilin (Fig. 5), this suggests that ER retention of the complex of PCSK9 with these proteins likely leads to their degradation within the ERAD system (50).”

• cites [50] Hampton, 2002, ER-associated degradation in protein quality control and cellular regulation, Curr. Opin. Cell Biol sourceNot checked — no full text of the source

PCSK9 could enhance degradation of APLP2 and sortilin by both the ERAD and endosomes/lysosome hydrolases.

“Thus, it is probable that PCSK9 could enhance the degradation of APLP2 and sortilin by both the ERAD and the endosomes/lysosome hydrolases.”

The 2.2-fold increased LDLR levels reported in Sort1-/- mice could not be primarily attributed to the loss of ~30% of circulating PCSK9.

“Thus, our data suggest that the 2.2-fold increased levels of LDLR reported in Sort1 Ϫ/Ϫ mice could not be primarily attributed to the loss of ϳ30% of circulating PCSK9 (35).”

• cites [35] Gustafsen, 2014, The hypercholesterolemia-risk gene SORT1 facilitates PCSK9 secretion, Cell Metab sourceNot checked — no full text of the source

APLP2 does not affect the ability of PCSK9 to enhance the degradation of the LDLR in cells and in vivo.

“Therefore, we conclude that APLP2 does not affect the ability of PCSK9 to enhance the degradation of the LDLR in cells and in vivo.”

Sortilin and APLP2 are new targets of intracellular PCSK9.

“This suggested that both sortilin and APLP2 are new targets of intracellular PCSK9.”

The transcription of Aplp2 or Sort1 is not regulated by SREBP2, unlike PCSK9, LDLR, and HMG-CoA reductase.

“This suggests that different for PCSK9, LDLR, and HMG-CoA reductase (57), the transcription of Aplp2 or Sort1 is not regulated by SREBP2.”

• cites [57] Dubuc, 2004, Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia, Arterioscler. Thromb. Vasc. Biol sourceNot checked — no full text of the source

The PCSK9-mediated degradation of APLP2 and sortilin in HEK293 and Huh7 cells likely involves the intracellular PCSK9 pathway.

“This likely involves the intracellular PCSK9 pathway, because incubation of these cells with exogenous PCSK9 had no effect on either protein (not shown).”

Both APLP2 and sortilin together with PCSK9 meet early in the secretory pathway and not at the cell surface.

“The above data and the fact that sortilin can stabilize APLP2 (Fig. 4B) suggest that both proteins together with PCSK9 meet early in the secretory pathway and not at the cell surface.”