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Altered processing of amyloid precursor protein in cells undergoing apoptosis.

Fiorelli T, Kirouac L, Padmanabhan J - PLoS ONE (2013)

Bottom Line: Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases.Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results.Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25-35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.

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Bioinformatic prediction of potential proteolytic sites involved in generation of the novel APP fragments.The figure shows a schematic depicting known proteolytic sites on APP, and table identifying these various sites on APP770 isoform. Each site is assigned a number starting from the N-terminus, with a putative caspase cleavage site (DEVD* E563) identified by a support vector machine-based caspase substrate predictive model identified using an asterisk. The right side of the table is a grid illustrating the predicted molecular weights of fragments generated by cleavage at one or two of the sites identified. Each cell contains the predicted molecular weight for a fragment generated by cleavages at the site identified in the left side of the table and by number in the top row of the grid. The light gray cells on the diagonal, those where both row and column represent the same site, indicate cleavage at a single site with predicted molecular weight of the N-terminal and C-terminal fragments listed, respectively. The white cells show the molecular weight of the internal fragment generated by cleavage at two different sites. The molecular weights of fragments containing the 6E10 epitope (Aβ1–16) are bolded and underlined. Note that sites in the near extracellular domain, specifically the putative caspase site and the MT-MMP site, could potentially generate fragments similar in size and immunoreactivity to the fragments we observed.
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pone-0057979-g006: Bioinformatic prediction of potential proteolytic sites involved in generation of the novel APP fragments.The figure shows a schematic depicting known proteolytic sites on APP, and table identifying these various sites on APP770 isoform. Each site is assigned a number starting from the N-terminus, with a putative caspase cleavage site (DEVD* E563) identified by a support vector machine-based caspase substrate predictive model identified using an asterisk. The right side of the table is a grid illustrating the predicted molecular weights of fragments generated by cleavage at one or two of the sites identified. Each cell contains the predicted molecular weight for a fragment generated by cleavages at the site identified in the left side of the table and by number in the top row of the grid. The light gray cells on the diagonal, those where both row and column represent the same site, indicate cleavage at a single site with predicted molecular weight of the N-terminal and C-terminal fragments listed, respectively. The white cells show the molecular weight of the internal fragment generated by cleavage at two different sites. The molecular weights of fragments containing the 6E10 epitope (Aβ1–16) are bolded and underlined. Note that sites in the near extracellular domain, specifically the putative caspase site and the MT-MMP site, could potentially generate fragments similar in size and immunoreactivity to the fragments we observed.

Mentions: The novel APP fragments observed in lysates from cells undergoing apoptosis immunoreacted with 6E10 and the C-terminal caspase cleavage site-specific antibodies. The only fragment generated by secretase cleavage of APP that would immunoreact with both of these antibodies would be the C99 fragment that is truncated by cleavage at VEVD*A740. Such a fragment would have a predicted molecular weight of ∼8 kDa. As this is considerably smaller than the molecular weight of the fragments generated under apoptotic conditions, we scanned APP for other known proteolytic sites, such as those cleaved by calpains, caspases, and MT-MMPs, to determine whether APP fragments with molecular weights similar to those we observed could be generated by proteases other than the secretases. Figure 6 shows a ball-and-chain model of APP with various proteolytic sites numbered. The grid towards the right of Figure 6 shows the predicted molecular weight of fragments generated by cleavage at a single site (light gray boxes, showing MW of N-terminal and C-terminal fragments respectively), or the internal fragment generated by cleavage at two sites identified by the intersection of a row and column. For example, cleavage at the β-secretase site (row 5) and the γ-secretase site (column 7) generates a fragment with a predicted molecular weight of ∼4.5 kDa, Aβ. These estimated molecular weights are based on the amino acid structure at neutral pH with no post-translational modifications and are calculated using the APP770 isoform. For fragments containing the 6E10 epitope, the molecular weight is bolded and underlined.


Altered processing of amyloid precursor protein in cells undergoing apoptosis.

Fiorelli T, Kirouac L, Padmanabhan J - PLoS ONE (2013)

Bioinformatic prediction of potential proteolytic sites involved in generation of the novel APP fragments.The figure shows a schematic depicting known proteolytic sites on APP, and table identifying these various sites on APP770 isoform. Each site is assigned a number starting from the N-terminus, with a putative caspase cleavage site (DEVD* E563) identified by a support vector machine-based caspase substrate predictive model identified using an asterisk. The right side of the table is a grid illustrating the predicted molecular weights of fragments generated by cleavage at one or two of the sites identified. Each cell contains the predicted molecular weight for a fragment generated by cleavages at the site identified in the left side of the table and by number in the top row of the grid. The light gray cells on the diagonal, those where both row and column represent the same site, indicate cleavage at a single site with predicted molecular weight of the N-terminal and C-terminal fragments listed, respectively. The white cells show the molecular weight of the internal fragment generated by cleavage at two different sites. The molecular weights of fragments containing the 6E10 epitope (Aβ1–16) are bolded and underlined. Note that sites in the near extracellular domain, specifically the putative caspase site and the MT-MMP site, could potentially generate fragments similar in size and immunoreactivity to the fragments we observed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585261&req=5

pone-0057979-g006: Bioinformatic prediction of potential proteolytic sites involved in generation of the novel APP fragments.The figure shows a schematic depicting known proteolytic sites on APP, and table identifying these various sites on APP770 isoform. Each site is assigned a number starting from the N-terminus, with a putative caspase cleavage site (DEVD* E563) identified by a support vector machine-based caspase substrate predictive model identified using an asterisk. The right side of the table is a grid illustrating the predicted molecular weights of fragments generated by cleavage at one or two of the sites identified. Each cell contains the predicted molecular weight for a fragment generated by cleavages at the site identified in the left side of the table and by number in the top row of the grid. The light gray cells on the diagonal, those where both row and column represent the same site, indicate cleavage at a single site with predicted molecular weight of the N-terminal and C-terminal fragments listed, respectively. The white cells show the molecular weight of the internal fragment generated by cleavage at two different sites. The molecular weights of fragments containing the 6E10 epitope (Aβ1–16) are bolded and underlined. Note that sites in the near extracellular domain, specifically the putative caspase site and the MT-MMP site, could potentially generate fragments similar in size and immunoreactivity to the fragments we observed.
Mentions: The novel APP fragments observed in lysates from cells undergoing apoptosis immunoreacted with 6E10 and the C-terminal caspase cleavage site-specific antibodies. The only fragment generated by secretase cleavage of APP that would immunoreact with both of these antibodies would be the C99 fragment that is truncated by cleavage at VEVD*A740. Such a fragment would have a predicted molecular weight of ∼8 kDa. As this is considerably smaller than the molecular weight of the fragments generated under apoptotic conditions, we scanned APP for other known proteolytic sites, such as those cleaved by calpains, caspases, and MT-MMPs, to determine whether APP fragments with molecular weights similar to those we observed could be generated by proteases other than the secretases. Figure 6 shows a ball-and-chain model of APP with various proteolytic sites numbered. The grid towards the right of Figure 6 shows the predicted molecular weight of fragments generated by cleavage at a single site (light gray boxes, showing MW of N-terminal and C-terminal fragments respectively), or the internal fragment generated by cleavage at two sites identified by the intersection of a row and column. For example, cleavage at the β-secretase site (row 5) and the γ-secretase site (column 7) generates a fragment with a predicted molecular weight of ∼4.5 kDa, Aβ. These estimated molecular weights are based on the amino acid structure at neutral pH with no post-translational modifications and are calculated using the APP770 isoform. For fragments containing the 6E10 epitope, the molecular weight is bolded and underlined.

Bottom Line: Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases.Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results.Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25-35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.

Show MeSH
Related in: MedlinePlus