Activity-dependent isolation of the presenilin- gamma -secretase complex reveals nicastrin and a gamma substrate.
Presenilin heterodimers apparently contain the active site of gamma-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-beta precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional gamma-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (beta-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated gamma-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor protein substrate of gamma-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active gamma-secretase complex, indicate that beta-catenin, calsenilin, and presenilin-associated protein are not required for gamma activity, and suggest an unprecedented mechanism of substrate-protease interaction.
Esler
William P
WP
Center for Neurologic Diseases, Brigham and Women's Hospital and Program in Neuroscience, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Kimberly
W Taylor
WT
Ostaszewski
Beth L
BL
Ye
Wenjuan
W
Diehl
Thekla S
TS
Selkoe
Dennis J
DJ
Wolfe
Michael S
MS
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D053764:Presenilin-1; D053766:Presenilin-2; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D053764:Presenilin-1; D053766:Presenilin-2; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse