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P2‐275: βCTF is an inefficient substrate for proteolysis by gamma‐secretase
Author(s) -
Funamoto Satoru,
Nobuhara Mika,
Ishihara Seiko,
Ihara Yasuo
Publication year - 2010
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2010.05.1325
Subject(s) - proteolysis , gamma secretase , chemistry , biochemistry , sf9 , subtilisin , recombinant dna , amyloid precursor protein secretase , peptide sequence , peptide , substrate (aquarium) , enzyme , biophysics , microbiology and biotechnology , biology , amyloid precursor protein , spodoptera , medicine , ecology , disease , pathology , alzheimer's disease , gene
Background: gamma-Secretase hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Over 50 molecules have been reported as gamma-secretase substrate, which indicates that inhibition of gamma-secretase activity to prevent Abeta production results in harmful side effects. However it remains unclear whether substrate specificity exists on gamma-secretase. Given that gamma-secretase possesses substrate specificity, it may lead to substrate specific gamma-secretase modulation. The aim of this study is to examine substrate specificity of gamma-secretase. We established a methodology to generate recombinant gamma-secretase substrates with bona fide N-terminal sequence. Methods: gamma-Secretase substrates, such as betaCTF, alphaCTF, S2-cleaved Notch and alpha-cleaved APLP2, were N-terminally fused with APP signal peptide followed by subtilisin-recognition sequence. After expressed in sf9 cells, those recombinant proteins were isolated by affinity purification with S189 subtilisin immobilized resin. Once captured by the resin, the recombinant proteins were precisely cleaved by addition of fluoride ion at the C-terminal of the subtilisin-recognition sequence, which generated gamma-secretase substrates carrying bona fide N-terminal sequence. The gamma-secretase substrates were incubated with CHAPSO-solubilized microsomal fraction of CHO cells that exhibited high specific activity (Kakuda et al., JBC 2006). We measured intracellular domain of the substrates cleaved by gamma-secretase by means of quantitative western blot. Results: Apparent Km values for betaCTF, alphaCTF, S2-cleaved Notch and alpha-cleaved APLP2 were in a range of 790-820 nM, whereas apparent Vmax values were 1.85 nM/min for betaCTF and 8.11-9.06 nM/min for the other substrates. The Vmax/Km value for betaCTF was approximately one-fifth of that for the other substrate. We also examined kinetics of AICD production from alphaCTF in the presence of S2-cleaved Notch. Various concentrations of alphaCTF were incubated with gamma-secretase in the presence of defined amounts of S2-cleaved Notch. Double reciprocal plot of the AICD production in the presence of S2-cleaved Notch generally displayed a pattern characteristic of competitive inhibition. Conclusions: Among substrates tested, betaCTF is an inefficient substrate for proteolysis by gammasecretase.