Notch and the amyloid precursor protein are cleaved by similar gamma-secretase(s).
Gamma-secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the gamma-secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP gamma-like proteolysis. In an effort to resolve this issue directly, we established an in vitro gamma-secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and N100Flag. Analysis with specific gamma-secretase inhibitors, dominant-negative gamma-secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same gamma-secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).
Kimberly
W Taylor
WT
Center for Neurologic Diseases, Brigham & Women's Hospital, and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Esler
William P
WP
Ye
Wenjuan
W
Ostaszewski
Beth L
BL
Gao
Jun
J
Diehl
Thekla
T
Selkoe
Dennis J
DJ
Wolfe
Michael S
MS
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics; D053764:Presenilin-1; D053766:Presenilin-2; D017434:Protein Structure, Tertiary / Q000235:genetics; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D009842:Oligopeptides; C508853:PSEN1 protein, human; C508861:PSEN2 protein, human; D010446:Peptide Fragments; D010455:Peptides; D053764:Presenilin-1; D053766:Presenilin-2; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human