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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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Histidine charge reversal and translocations mutants. A, expression of the negatively charged H197D mutant or shifting the His-197 residue down the transmembrane helix failed to rescue NCT maturation and PS1 endoproteolysis to generate the amino-terminal fragment (PS1-NTF). B, immunoprecipitation of APH1 indicated a reduced binding of H197D and the translocation mutants (H197V/V193H, H197L/L190H) to NCT and the absence of PS1 interactions as compared with the wild-type or the control T200V mutant. C, PS1 immunoprecipitation revealed that the His-197 mutants did not bind PS1. m, mature; im, immature.
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Figure 5: Histidine charge reversal and translocations mutants. A, expression of the negatively charged H197D mutant or shifting the His-197 residue down the transmembrane helix failed to rescue NCT maturation and PS1 endoproteolysis to generate the amino-terminal fragment (PS1-NTF). B, immunoprecipitation of APH1 indicated a reduced binding of H197D and the translocation mutants (H197V/V193H, H197L/L190H) to NCT and the absence of PS1 interactions as compared with the wild-type or the control T200V mutant. C, PS1 immunoprecipitation revealed that the His-197 mutants did not bind PS1. m, mature; im, immature.

Mentions: Reversing the histidine charge by substitution with aspartic acid or relocating the side chain vertically along the TM domain was also investigated to further understand the contributions of these residues to APH1 function. Reversing the charge in an H197D mutant did not have an appreciable effect on expression (Fig. 5A). However, no rescue of NCT maturation or increase in PS1 endoproteolysis was observed. Quantification of the PS1-NTF indicated that cells expressing the H197D mutant were not statistically different from the background APH1 knock-out fibroblasts (supplemental Fig. 2B). Assembly of the γ-secretase complex was also compromised by the charge reversal as shown by co-immunoprecipitation studies. Precipitation of the H197D mutant recovered the APH1 monomers and dimers, but the amount of associated mature as well as immature NCT was reduced and no binding to PS1 was observed (Fig. 5B). Lack of binding to PS1 was confirmed by PS1 immunoprecipitation where virtually none of the H197D mutant was detected (Fig. 5C). This may be due in part to the low level of the PS1-NTF (70% reduction), which is significantly different from H197A. However, the complete failure of the H197D mutant to associate with PS1-NTF suggests that the charged His side chain plays a role not only in PS1 endoproteolysis but also in the assembly of a normal γ-secretase complex.


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)

Histidine charge reversal and translocations mutants. A, expression of the negatively charged H197D mutant or shifting the His-197 residue down the transmembrane helix failed to rescue NCT maturation and PS1 endoproteolysis to generate the amino-terminal fragment (PS1-NTF). B, immunoprecipitation of APH1 indicated a reduced binding of H197D and the translocation mutants (H197V/V193H, H197L/L190H) to NCT and the absence of PS1 interactions as compared with the wild-type or the control T200V mutant. C, PS1 immunoprecipitation revealed that the His-197 mutants did not bind PS1. m, mature; im, immature.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Histidine charge reversal and translocations mutants. A, expression of the negatively charged H197D mutant or shifting the His-197 residue down the transmembrane helix failed to rescue NCT maturation and PS1 endoproteolysis to generate the amino-terminal fragment (PS1-NTF). B, immunoprecipitation of APH1 indicated a reduced binding of H197D and the translocation mutants (H197V/V193H, H197L/L190H) to NCT and the absence of PS1 interactions as compared with the wild-type or the control T200V mutant. C, PS1 immunoprecipitation revealed that the His-197 mutants did not bind PS1. m, mature; im, immature.
Mentions: Reversing the histidine charge by substitution with aspartic acid or relocating the side chain vertically along the TM domain was also investigated to further understand the contributions of these residues to APH1 function. Reversing the charge in an H197D mutant did not have an appreciable effect on expression (Fig. 5A). However, no rescue of NCT maturation or increase in PS1 endoproteolysis was observed. Quantification of the PS1-NTF indicated that cells expressing the H197D mutant were not statistically different from the background APH1 knock-out fibroblasts (supplemental Fig. 2B). Assembly of the γ-secretase complex was also compromised by the charge reversal as shown by co-immunoprecipitation studies. Precipitation of the H197D mutant recovered the APH1 monomers and dimers, but the amount of associated mature as well as immature NCT was reduced and no binding to PS1 was observed (Fig. 5B). Lack of binding to PS1 was confirmed by PS1 immunoprecipitation where virtually none of the H197D mutant was detected (Fig. 5C). This may be due in part to the low level of the PS1-NTF (70% reduction), which is significantly different from H197A. However, the complete failure of the H197D mutant to associate with PS1-NTF suggests that the charged His side chain plays a role not only in PS1 endoproteolysis but also in the assembly of a normal γ-secretase complex.

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