This paper presents data indicating that palmitoylation of APP in the early secretory pathway regulates its processing by BACE1. The findings are novel and offer a previously unknown aspect of APP biology that could be targeted for therapeutic purposes. The conclusions are supported by a combination of biochemical experiments in both cellular and animal models of the disease. The findings are intriguing and open a new area of research. Indeed, a few important points will require further evaluation:
1) The formation of disulfide bonds seems to reduce the palmitoylation of APP. The basic information for the folding of a protein is contained in the primary amino acid sequence. However, co- and post-translational modifications can assist and improve the efficiency of folding. The formation of disulfide bonds is a co-translational event. It occurs during translation and helps the folding of the nascent polypeptide. In contrast, palmitoylation is a post-translational event. It occurs after the folding of the nascent polypeptide. Perhaps palmitoylation is being used here to “mark” unfolded/misfolded APP. In fact, a possible scenario could be that correctly folded APP (with disulfide bonds) is not palmitoylated while unfolded/misfolded APP (without disulfide bonds) is palmitoylated. If this is the case, then the preferential β cleavage of the palmitoylated (and still immature) version of APP in the early secretory pathway could be envisioned as an attempt to remove unfolded/misfolded APP.
2) Ceramide and cholesterol esters (CEs) have been previously associated with the pathogenesis of AD. Both are synthesized at the ER and use palmitoyl-CoA as one of the substrates. In the case of ceramide, palmitoyl-CoA is fused to serine to generate sphingosine, which then is used to synthesize ceramide. In the case of CEs, palmitate is one of the fatty acids that can be attached to the cholesterol moiety. It would be interesting to determine whether there is cross-talk between the above lipid biosynthetic pathways and the palmitoylation of a polypeptide.
The paper by Kovacs and colleagues reports the discovery that a significant proportion of APP in the cell is palmitoylated and enriched in lipid rafts. APP palmitoylation increased the colocalization of APP with BACE1, the β-secretase. Consequently, BACE1 cleavage of APP was increased, as was the generation of Aβ. Using both genetic and pharmacologic approaches, they show a direct relationship between APP palmitoylation and Aβ production. Moreover, the group discovered that ACAT inhibition also reduced APP palmitoylation, lipid raft localization, and Aβ generation.
The authors present a very thorough, rigorous study that convincingly demonstrates the role of APP palmitoylation in lipid raft localization and BACE1-mediated cleavage of APP and Aβ generation. Further, their observations strongly suggest that inhibition of either palmitoylation or ACAT should prove effective for lowering cerebral Aβ levels in AD. Kovacs and colleagues present an intriguing therapeutic alternative to direct inhibition of BACE1, which could be associated with mechanism based toxicities. Thus, further investigation of inhibition of APP palmitoylation as a therapeutic approach for AD is clearly warranted.
For many years, evidence accumulated that AD has extensive links to lipid metabolism. Dora Kovacs' group was one of the first to identify such a link on the molecular level with their ACAT targeted experiments. There is also extensive evidence suggesting APP processing, especially Aβ generation, is very sensitive to neuronal cholesterol and other lipids. APP may even serve as a receptor for some lipidated lipoproteins and function directly or indirectly as a sensor for lipid levels. Now, the Kovacs group has identified an even more direct link—APP is covalently bound to palmitoyl at the cysteine residues 186 and 187. Palmitoylation is one way to target proteins into lipid rafts, a specific membrane domain that is strongly implicated with amyloidogenic APP processing. Only a small fraction of APP was found to be palmitoylated, which is in good agreement that only a small fraction of total APP is found in lipid rafts. Without palmitoylation (or disulfide bridging), APP cannot be effectively exported from the ER. Interestingly, this appears to provide some explanation for the original ACAT results, because ACAT inhibition reduces APP palmitoylation and therefore impaired APP processing eventually resulting in decreased Aβ generation. Luminal S-palmitoylation for transmembrane proteins, as it is the case for APP, is thus far unprecedented (maybe a reason why it took so long to be discovered) and clearly offers a whole new range of potential ways to interfere with this modification and possibly some therapeutic options. Moreover, it is a clear example that not all APP is equal. Rather, the amyloidogenic fate of some APP molecules may already be foretold in the endoplasmic reticulum.
This paper reported intriguing findings that amyloid precursor protein (APP) is palmitoylated at Cys186 and Cys187, and this palmitoylated form is cleaved by BACE1 in lipid rafts. Palmitoylation of transmembrane proteins usually occurs inside or close to the transmembrane domain or in the cytoplasmic domain (Charollais and Van der Goot, 2009), but in the case of APP, the palmitoylation sites are located in the N-terminal luminal domain. The authors showed that APP mutants with Cys-Ser or Cys-Ala substitutions at these sites do not undergo normal maturation and are retained in the ER. Because these cysteine residues are important for disulfide bond formation and only a small part of APP is palmitoylated, the ER retention of the APP mutants can be explained by improper folding of APP, but not by the lack of palmitoylation. It can be assumed that when APP is palmitoylated at these Cys residues, disulfide bonds will not be formed, which could affect APP maturation. In this regard, it seems that the property of palmitoylated APP differs from APP without palmitoylation and palmitoylated APP might be more unstable, as suggested in Dr. Puglielli’s comments.
In addition, APP is mostly distributed in non-raft membrane domains, as is BACE1. We have previously published experimental data suggesting that BACE1 cleaves APP mainly outside lipid rafts in neurons (Motoki et al., 2012).
Although this study revealed an interesting aspect of APP, further characterization of palmitoylated APP appears necessary to clarify its role in amyloidogenic processing.
References:
Charollais J, Van Der Goot FG.
Palmitoylation of membrane proteins (Review).
Mol Membr Biol. 2009 Jan;26(1):55-66.
PubMed.
Motoki K, Kume H, Oda A, Tamaoka A, Hosaka A, Kametani F, Araki W.
Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant.
Brain Behav. 2012 May;2(3):270-82.
PubMed.
Comments
University of Wisconsin-Madison
This paper presents data indicating that palmitoylation of APP in the early secretory pathway regulates its processing by BACE1. The findings are novel and offer a previously unknown aspect of APP biology that could be targeted for therapeutic purposes. The conclusions are supported by a combination of biochemical experiments in both cellular and animal models of the disease. The findings are intriguing and open a new area of research. Indeed, a few important points will require further evaluation:
1) The formation of disulfide bonds seems to reduce the palmitoylation of APP. The basic information for the folding of a protein is contained in the primary amino acid sequence. However, co- and post-translational modifications can assist and improve the efficiency of folding. The formation of disulfide bonds is a co-translational event. It occurs during translation and helps the folding of the nascent polypeptide. In contrast, palmitoylation is a post-translational event. It occurs after the folding of the nascent polypeptide. Perhaps palmitoylation is being used here to “mark” unfolded/misfolded APP. In fact, a possible scenario could be that correctly folded APP (with disulfide bonds) is not palmitoylated while unfolded/misfolded APP (without disulfide bonds) is palmitoylated. If this is the case, then the preferential β cleavage of the palmitoylated (and still immature) version of APP in the early secretory pathway could be envisioned as an attempt to remove unfolded/misfolded APP.
2) Ceramide and cholesterol esters (CEs) have been previously associated with the pathogenesis of AD. Both are synthesized at the ER and use palmitoyl-CoA as one of the substrates. In the case of ceramide, palmitoyl-CoA is fused to serine to generate sphingosine, which then is used to synthesize ceramide. In the case of CEs, palmitate is one of the fatty acids that can be attached to the cholesterol moiety. It would be interesting to determine whether there is cross-talk between the above lipid biosynthetic pathways and the palmitoylation of a polypeptide.
View all comments by Luigi PuglielliNorthwestern University Feinberg School of Medicine
The paper by Kovacs and colleagues reports the discovery that a significant proportion of APP in the cell is palmitoylated and enriched in lipid rafts. APP palmitoylation increased the colocalization of APP with BACE1, the β-secretase. Consequently, BACE1 cleavage of APP was increased, as was the generation of Aβ. Using both genetic and pharmacologic approaches, they show a direct relationship between APP palmitoylation and Aβ production. Moreover, the group discovered that ACAT inhibition also reduced APP palmitoylation, lipid raft localization, and Aβ generation.
The authors present a very thorough, rigorous study that convincingly demonstrates the role of APP palmitoylation in lipid raft localization and BACE1-mediated cleavage of APP and Aβ generation. Further, their observations strongly suggest that inhibition of either palmitoylation or ACAT should prove effective for lowering cerebral Aβ levels in AD. Kovacs and colleagues present an intriguing therapeutic alternative to direct inhibition of BACE1, which could be associated with mechanism based toxicities. Thus, further investigation of inhibition of APP palmitoylation as a therapeutic approach for AD is clearly warranted.
View all comments by Robert VassarUniversity of the Saarland
For many years, evidence accumulated that AD has extensive links to lipid metabolism. Dora Kovacs' group was one of the first to identify such a link on the molecular level with their ACAT targeted experiments. There is also extensive evidence suggesting APP processing, especially Aβ generation, is very sensitive to neuronal cholesterol and other lipids. APP may even serve as a receptor for some lipidated lipoproteins and function directly or indirectly as a sensor for lipid levels. Now, the Kovacs group has identified an even more direct link—APP is covalently bound to palmitoyl at the cysteine residues 186 and 187. Palmitoylation is one way to target proteins into lipid rafts, a specific membrane domain that is strongly implicated with amyloidogenic APP processing. Only a small fraction of APP was found to be palmitoylated, which is in good agreement that only a small fraction of total APP is found in lipid rafts. Without palmitoylation (or disulfide bridging), APP cannot be effectively exported from the ER. Interestingly, this appears to provide some explanation for the original ACAT results, because ACAT inhibition reduces APP palmitoylation and therefore impaired APP processing eventually resulting in decreased Aβ generation. Luminal S-palmitoylation for transmembrane proteins, as it is the case for APP, is thus far unprecedented (maybe a reason why it took so long to be discovered) and clearly offers a whole new range of potential ways to interfere with this modification and possibly some therapeutic options. Moreover, it is a clear example that not all APP is equal. Rather, the amyloidogenic fate of some APP molecules may already be foretold in the endoplasmic reticulum.
View all comments by Tobias HartmannNational Institute of Neuroscience, NCNP
This paper reported intriguing findings that amyloid precursor protein (APP) is palmitoylated at Cys186 and Cys187, and this palmitoylated form is cleaved by BACE1 in lipid rafts. Palmitoylation of transmembrane proteins usually occurs inside or close to the transmembrane domain or in the cytoplasmic domain (Charollais and Van der Goot, 2009), but in the case of APP, the palmitoylation sites are located in the N-terminal luminal domain. The authors showed that APP mutants with Cys-Ser or Cys-Ala substitutions at these sites do not undergo normal maturation and are retained in the ER. Because these cysteine residues are important for disulfide bond formation and only a small part of APP is palmitoylated, the ER retention of the APP mutants can be explained by improper folding of APP, but not by the lack of palmitoylation. It can be assumed that when APP is palmitoylated at these Cys residues, disulfide bonds will not be formed, which could affect APP maturation. In this regard, it seems that the property of palmitoylated APP differs from APP without palmitoylation and palmitoylated APP might be more unstable, as suggested in Dr. Puglielli’s comments.
In addition, APP is mostly distributed in non-raft membrane domains, as is BACE1. We have previously published experimental data suggesting that BACE1 cleaves APP mainly outside lipid rafts in neurons (Motoki et al., 2012).
Although this study revealed an interesting aspect of APP, further characterization of palmitoylated APP appears necessary to clarify its role in amyloidogenic processing.
References:
Charollais J, Van Der Goot FG. Palmitoylation of membrane proteins (Review). Mol Membr Biol. 2009 Jan;26(1):55-66. PubMed.
Motoki K, Kume H, Oda A, Tamaoka A, Hosaka A, Kametani F, Araki W. Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant. Brain Behav. 2012 May;2(3):270-82. PubMed.
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