Research Article: Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

Date Published: April 26, 2013

Publisher: Public Library of Science

Author(s): Yi Piao, Ayano Kimura, Satomi Urano, Yuhki Saito, Hidenori Taru, Tohru Yamamoto, Saori Hata, Toshiharu Suzuki, Stephen D. Ginsberg.


Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer’s β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-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 CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.

The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-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 γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.

Partial Text

The γ-secretase is comprised of four membrane proteins, presenilin 1 (PS1) or 2 (PS2), nicastrin (NCT), anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2) [1]. PS functions as the catalytic unit of this aspartyl protease complex [2]. Prior to intramembrane cleavage of type I membrane proteins by γ-secretase, the substrate membrane proteins are subject to primary extracellular/intraluminal cleavage at the juxtamembrane region by a sheddase such as a disintegrin and metalloproteinase (ADAM) [3]. This primary cleavage is essential for the subsequent intramembrane γ-cleavage, although the exact regulation of intramembrane cleavage by γ-secretase remains unclear.

In a previous study, the magnitude of Alcs γ-cleavage alteration in cells expressing FAD-linked PS1 mutants varied and differed from APP [17], suggesting that the determination of initial intramembrane ε-cleavage may not be necessarily prerequisite to cleave at a specific γ-cleavage site in the case of Alcs. We determined two to three ε-cleavage sites each in Alcα, Alcβ and Alcγ, as observed in APP and Notch [20]–[24]. One represented the major ε-site at which Alc CTF was predominantly cleaved. We found that some FAD-linked PS1 mutations affected the ratio of minor to major ε-cleavage, but this alteration to the ratio of minor to major γ-cleavage was not always apparent. Similarly, some FAD-linked PS1 mutations did not remarkably influence in the selection of ε-site but significantly affected the γ-cleavage site. These properties of Alcs intramembrane cleavage by γ-secretase differ from those of APP, in which changes to the ratio of minor to major γ-cleavage were consistent with changes to the ratio of minor to major ε-cleavage [10], [12], [25]. Therefore, we propose that the mechanism of intramembrane cleavage by γ-secretase is not regulated identically between Alcs and APP. Such relationship between the γ-site position and the ε-cleavage site in Alcs was demonstrated with several different types of experiment, and our current findings suggest that the endophenotype of γ-secretase malfunction appears to affect either γ-cleavage or ε-cleavage position in Alcs.