Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment.
The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.
Kim
Hangun
H
College of Pharmacy, School of Biological Sciences and Technology, Chonnam National University, Bldg. 1-211, 300 Yongbong-dong, Gwangju 500-757, Korea..
Ki
Hyunkyoung
H
Park
Hee-Sae
HS
Kim
Kwonseop
K
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D053764:Presenilin-1; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D053764:Presenilin-1; D051880:Receptors, Notch; C413604:nicastrin protein; D049452:Green Fluorescent Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse