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Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.

ABSTRACT: Gamma-secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid beta-peptide (Abeta) from its precursor, APP. Gamma-secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of Abeta40 and Abeta42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of approximately 20-40 A in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, approximately 120-A-long complex within the membrane and revealed approximately 20-A pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.

SUBMITTER: Lazarov VK

PROVIDER: S-EPMC1458989 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.

Lazarov Vlado K VK Fraering Patrick C PC Ye Wenjuan W Wolfe Michael S MS Selkoe Dennis J DJ Li Huilin H

Proceedings of the National Academy of Sciences of the United States of America 20060424 18

Gamma-secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid beta-peptide (Abeta) ...[more]

PMID: 16636269

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Project description:BackgroundAlcadein proteins (Alcs; Alc?, Alc?and Alc?) are predominantly expressed in neurons, as is Alzheimer's ?-amyloid (A?) precursor protein (APP). Both Alcs and APP are cleaved by primary ?- or ?-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by ?-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTF? is initially cleaved at the ?-site to release the intracellular domain fragment (AICD) and consequently the ?-site is determined, by which A? generates. The initial ?-site is thought to define the final ?-site position, which determines whether A?40/43 or A?42 is generated. However, initial intracellular ?-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final ?-site position is determined remains unclear in Alcs.MethodologyUsing HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of ?-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal ?-cleavage site and Alc ICD possessing N-terminal ?-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ?-site position of all Alc?, Alc? and Alc?, and analyzed the relationship between the initially determined ?-site position and the final ?-cleavage position.ConclusionsThe initial ?-site position does not always determine the final ?-cleavage position in Alcs, which differed from APP. No additional ?-cleavage sites are generated from artificial/non-physiological positions of ?-cleavage for Alcs, while the artificial ?-cleavage positions can influence in selection of physiological ?-site positions. Because alteration of ?-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by ?-secretase, which may be induced by malfunction of ?-secretase itself or changes of membrane environment for enzymatic reaction.

Dataset Information

Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.

Publications

Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.

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