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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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APH1 mutants and rescue of γ-secretase phenotype in knock-out fibroblasts. A, APH1 knock-out fibroblasts (MEF APH1−/−) expressing wild-type (WT) or mutated APH1 H171A, H197A, Q83A, E84A, and R87A were subjected to immunoblotting analysis of NCT, PS1, PEN2, and APH1. Mutant APH1 proteins showed modest rescue of NCT maturation and PEN2 expression relative to wild-type protein but varied in the amount of PS1 amino-terminal fragment (PS1-NTF) generated. B, quantification of the level of PS1 endoproteolysis as measured by levels of the PS1-NTF, indicating the most significant changes were observed for the H171A and H197A substitutions. Mutation of the polar residues in TM3 did not affect PS1 processing. m, mature; im, immature; Tub, tubulin used as loading control.
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Figure 2: APH1 mutants and rescue of γ-secretase phenotype in knock-out fibroblasts. A, APH1 knock-out fibroblasts (MEF APH1−/−) expressing wild-type (WT) or mutated APH1 H171A, H197A, Q83A, E84A, and R87A were subjected to immunoblotting analysis of NCT, PS1, PEN2, and APH1. Mutant APH1 proteins showed modest rescue of NCT maturation and PEN2 expression relative to wild-type protein but varied in the amount of PS1 amino-terminal fragment (PS1-NTF) generated. B, quantification of the level of PS1 endoproteolysis as measured by levels of the PS1-NTF, indicating the most significant changes were observed for the H171A and H197A substitutions. Mutation of the polar residues in TM3 did not affect PS1 processing. m, mature; im, immature; Tub, tubulin used as loading control.

Mentions: Mutant APH1 proteins containing alanine substitutions of the two potentially charged (E84A and R87A) or polar (Q83A) residues located in TM3 were expressed at levels similar to wild-type APH1 (Fig. 2A). Similar replacements of His-171 (H171A) or His-197 (H197A) also did not affect expression with monomeric and dimeric forms being observed for all APH1 variants. Dimerization has been previously reported for cells expressing high levels of APH1 (28). Although expressed at comparable levels, the APH1 mutants displayed different abilities to rescue the phenotypic characteristics of the knock-out fibroblast. Expression of the TM3 mutants (E84A, R87A, Q83A) led to a partial rescue of NCT maturation as well as PEN2 expression and restored PS1 endoproteolysis to the same level as that seen for wild-type APH1 (Fig. 2A). In contrast, the two histidine substitutions, H171A and H197A, were less efficient in rescuing the γ-secretase deficiencies associated with the APH1 knock-out fibroblasts. Maturation of NCT was slightly restored, but a significant decrease in PS1 endoproteolysis was observed (Fig. 2A). Quantification of PS1-NTF levels indicated that loss of His-171 resulted in an ∼30% reduction in PS1 cleavage, and the H197A substitution had a more pronounced effect, leading to an ∼50% decrease in PS1-NTF compared with wild-type APH1 (Fig. 2B).


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)

APH1 mutants and rescue of γ-secretase phenotype in knock-out fibroblasts. A, APH1 knock-out fibroblasts (MEF APH1−/−) expressing wild-type (WT) or mutated APH1 H171A, H197A, Q83A, E84A, and R87A were subjected to immunoblotting analysis of NCT, PS1, PEN2, and APH1. Mutant APH1 proteins showed modest rescue of NCT maturation and PEN2 expression relative to wild-type protein but varied in the amount of PS1 amino-terminal fragment (PS1-NTF) generated. B, quantification of the level of PS1 endoproteolysis as measured by levels of the PS1-NTF, indicating the most significant changes were observed for the H171A and H197A substitutions. Mutation of the polar residues in TM3 did not affect PS1 processing. m, mature; im, immature; Tub, tubulin used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: APH1 mutants and rescue of γ-secretase phenotype in knock-out fibroblasts. A, APH1 knock-out fibroblasts (MEF APH1−/−) expressing wild-type (WT) or mutated APH1 H171A, H197A, Q83A, E84A, and R87A were subjected to immunoblotting analysis of NCT, PS1, PEN2, and APH1. Mutant APH1 proteins showed modest rescue of NCT maturation and PEN2 expression relative to wild-type protein but varied in the amount of PS1 amino-terminal fragment (PS1-NTF) generated. B, quantification of the level of PS1 endoproteolysis as measured by levels of the PS1-NTF, indicating the most significant changes were observed for the H171A and H197A substitutions. Mutation of the polar residues in TM3 did not affect PS1 processing. m, mature; im, immature; Tub, tubulin used as loading control.
Mentions: Mutant APH1 proteins containing alanine substitutions of the two potentially charged (E84A and R87A) or polar (Q83A) residues located in TM3 were expressed at levels similar to wild-type APH1 (Fig. 2A). Similar replacements of His-171 (H171A) or His-197 (H197A) also did not affect expression with monomeric and dimeric forms being observed for all APH1 variants. Dimerization has been previously reported for cells expressing high levels of APH1 (28). Although expressed at comparable levels, the APH1 mutants displayed different abilities to rescue the phenotypic characteristics of the knock-out fibroblast. Expression of the TM3 mutants (E84A, R87A, Q83A) led to a partial rescue of NCT maturation as well as PEN2 expression and restored PS1 endoproteolysis to the same level as that seen for wild-type APH1 (Fig. 2A). In contrast, the two histidine substitutions, H171A and H197A, were less efficient in rescuing the γ-secretase deficiencies associated with the APH1 knock-out fibroblasts. Maturation of NCT was slightly restored, but a significant decrease in PS1 endoproteolysis was observed (Fig. 2A). Quantification of PS1-NTF levels indicated that loss of His-171 resulted in an ∼30% reduction in PS1 cleavage, and the H197A substitution had a more pronounced effect, leading to an ∼50% decrease in PS1-NTF compared with wild-type APH1 (Fig. 2B).

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