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APH1 polar transmembrane residues regulate the assembly and activity of presenilin complexes.

Pardossi-Piquard R, Yang SP, Kanemoto S, Gu Y, Chen F, Böhm C, Sevalle J, Li T, Wong PC, Checler F, Schmitt-Ulms G, St George-Hyslop P, Fraser PE - J. Biol. Chem. (2009)

Bottom Line: Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity.Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1.These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, Toronto, Ontario M5S 3H2, Canada.

ABSTRACT
Complexes involved in the gamma/epsilon-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-beta precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive gamma-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

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γ-Secretase activity in APH1−/− cells complemented with APH1 mutants. A, processing of recombinant FLAG-tagged C100 to generate the AICD fragment was used to monitor activity of the complexes from normal mouse fibroblasts (MEF WT) at 4 and 37 °C and in the presence of the inhibitor, difluoroketone-167 (DFK). Western blotting with antibodies directed to the APP-CTF (α-APP-CTF) and FLAG-tag (α-FLAG) indicated AICD generation by wild-type APH1 (APH1-WT) and T200V control proteins. A synthetic peptide corresponding to the AICD fragment (without FLAG tag) was also used as a positive control. No processing of the C100 to produce the AICD fragment was observed for any of the APH1 His-mutants (H171A/K, H197A/K/D). B, examination of Aβ peptides generated from the C100 substrate indicated significant amounts in normal mouse fibroblast (MEF WT) and APH1 knock-out cells expressing wild-type APH1 or T200V, but levels in cell expressing His-mutants (H171A/K, H197A/K/D) were not significantly elevated over background.
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Figure 7: γ-Secretase activity in APH1−/− cells complemented with APH1 mutants. A, processing of recombinant FLAG-tagged C100 to generate the AICD fragment was used to monitor activity of the complexes from normal mouse fibroblasts (MEF WT) at 4 and 37 °C and in the presence of the inhibitor, difluoroketone-167 (DFK). Western blotting with antibodies directed to the APP-CTF (α-APP-CTF) and FLAG-tag (α-FLAG) indicated AICD generation by wild-type APH1 (APH1-WT) and T200V control proteins. A synthetic peptide corresponding to the AICD fragment (without FLAG tag) was also used as a positive control. No processing of the C100 to produce the AICD fragment was observed for any of the APH1 His-mutants (H171A/K, H197A/K/D). B, examination of Aβ peptides generated from the C100 substrate indicated significant amounts in normal mouse fibroblast (MEF WT) and APH1 knock-out cells expressing wild-type APH1 or T200V, but levels in cell expressing His-mutants (H171A/K, H197A/K/D) were not significantly elevated over background.

Mentions: Given their effects on γ-secretase proteins and complex assembly, the activities associated with His-171 and His-197 mutants were assessed using a cell-free assay. Membrane preparations from knock-out fibroblasts expressing the APH1 mutants were combined with recombinant FLAG-tagged C100, and processing to AICD and Aβ was determined. Knock-out fibroblasts expressing wild-type APH1, control T200V mutant, or normal mouse fibroblasts produced significant levels of the AICD fragment after incubation of membrane preparations at 37 °C (Fig. 7A). This processing was prevented by the addition of difluoroketone-167, which is a substrate-based difluro ketone inhibitor of the γ-secretase (34). The TM3 mutants (Q83A, E84A, and R87A) also display activity levels similar to wild-type APH1, which is consistent with their ability to restore PS1 endoproteolysis and assembly into a mature γ-secretase complex (supplemental Fig. 5).


APH1 polar transmembrane residues regulate the assembly and activity of presenilin complexes.

Pardossi-Piquard R, Yang SP, Kanemoto S, Gu Y, Chen F, Böhm C, Sevalle J, Li T, Wong PC, Checler F, Schmitt-Ulms G, St George-Hyslop P, Fraser PE - J. Biol. Chem. (2009)

γ-Secretase activity in APH1−/− cells complemented with APH1 mutants. A, processing of recombinant FLAG-tagged C100 to generate the AICD fragment was used to monitor activity of the complexes from normal mouse fibroblasts (MEF WT) at 4 and 37 °C and in the presence of the inhibitor, difluoroketone-167 (DFK). Western blotting with antibodies directed to the APP-CTF (α-APP-CTF) and FLAG-tag (α-FLAG) indicated AICD generation by wild-type APH1 (APH1-WT) and T200V control proteins. A synthetic peptide corresponding to the AICD fragment (without FLAG tag) was also used as a positive control. No processing of the C100 to produce the AICD fragment was observed for any of the APH1 His-mutants (H171A/K, H197A/K/D). B, examination of Aβ peptides generated from the C100 substrate indicated significant amounts in normal mouse fibroblast (MEF WT) and APH1 knock-out cells expressing wild-type APH1 or T200V, but levels in cell expressing His-mutants (H171A/K, H197A/K/D) were not significantly elevated over background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713549&req=5

Figure 7: γ-Secretase activity in APH1−/− cells complemented with APH1 mutants. A, processing of recombinant FLAG-tagged C100 to generate the AICD fragment was used to monitor activity of the complexes from normal mouse fibroblasts (MEF WT) at 4 and 37 °C and in the presence of the inhibitor, difluoroketone-167 (DFK). Western blotting with antibodies directed to the APP-CTF (α-APP-CTF) and FLAG-tag (α-FLAG) indicated AICD generation by wild-type APH1 (APH1-WT) and T200V control proteins. A synthetic peptide corresponding to the AICD fragment (without FLAG tag) was also used as a positive control. No processing of the C100 to produce the AICD fragment was observed for any of the APH1 His-mutants (H171A/K, H197A/K/D). B, examination of Aβ peptides generated from the C100 substrate indicated significant amounts in normal mouse fibroblast (MEF WT) and APH1 knock-out cells expressing wild-type APH1 or T200V, but levels in cell expressing His-mutants (H171A/K, H197A/K/D) were not significantly elevated over background.
Mentions: Given their effects on γ-secretase proteins and complex assembly, the activities associated with His-171 and His-197 mutants were assessed using a cell-free assay. Membrane preparations from knock-out fibroblasts expressing the APH1 mutants were combined with recombinant FLAG-tagged C100, and processing to AICD and Aβ was determined. Knock-out fibroblasts expressing wild-type APH1, control T200V mutant, or normal mouse fibroblasts produced significant levels of the AICD fragment after incubation of membrane preparations at 37 °C (Fig. 7A). This processing was prevented by the addition of difluoroketone-167, which is a substrate-based difluro ketone inhibitor of the γ-secretase (34). The TM3 mutants (Q83A, E84A, and R87A) also display activity levels similar to wild-type APH1, which is consistent with their ability to restore PS1 endoproteolysis and assembly into a mature γ-secretase complex (supplemental Fig. 5).

Bottom Line: Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity.Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1.These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, Toronto, Ontario M5S 3H2, Canada.

ABSTRACT
Complexes involved in the gamma/epsilon-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-beta precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive gamma-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

Show MeSH
Related in: MedlinePlus