Analysis of the γ-secretase interactome and validation of its association with tetraspanin-enriched microdomains

Abstract

Gamma-secretase, an aspartyl protease that belongs to the iCLiPs (intramembrane cleaving proteases) family, is a multiprotein complex that consists of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (ref. 1). It is responsible for generation of the beta-amyloid peptide (Abeta), the primary component of senile plaques in the brains of patients with Alzheimer's disease. Although the four components are necessary and sufficient for gamma-secretase activity, additional proteins are possibly involved in its regulation. Consequently, we purified proteins associated with the active gamma-secretase complex from reconstituted PS-deficient fibroblasts, using tandem affinity purification (TAP) and identified a series of proteins that transiently interact with the gamma-secretase complex and are probably involved in complex maturation, membrane trafficking and, importantly, the tetraspanin web. Tetraspanins form detergent-resistant microdomains in the cell membrane and regulate cell adhesion, cell signalling and proteolysis. Association of the gamma-secretase complex with tetraspanin-enriched microdomains provides an explanation for the previously documented localization of gamma-secretase to raft-like domains. Thus, these studies suggest that maintenance of the integrity of tetraspanin microdomains contributes to the refinement of proteolytic activity of the gamma-secretase complex.

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

“…The regulation and maturation of the activity of the ␥-secretase complex could possibly be maintained by the G-protein scaffold protein, tetraspanin (15). Interestingly, this same study pulled out interactions of PS1 with components of the OST, in particular OST48 and ribophorin II.…”

Normal muscle regeneration requires tight control of muscle cell fusion by tetraspanins CD9 and CD81

“…S4e,f). The recent demonstration that CD9P-1, CD9 and CD81 associate with the g-secretase complex 40 raised the hypothesis that these molecules could increase the fusion by regulating the Notch pathway, which has a key role in the balance between self-renewal of satellite cells and their engagement towards differentiation 41 . Indeed, g-secretasemediated cleavage of Notch is an essential step of this pathway 42 .…”

A conserved tetraspanin subfamily promotes Notch signaling in Caenorhabditis elegans and in human cells

“…In view of the proposed role of tetraspanins in establishing or maintaining membrane microdomains, Wakabayashi et al (14) suggested that CD9 and CD81 modulate the proteolytic activity of γ-secretase or its interaction with β-APP. We favor a similar model in which members of the RDS subfamily of tetraspanins promote γ-secretase activity through promoting physical association between γ-secretase and Notch in membrane microdomains.…”

“…Wakabayashi et al (14) reported that two tetraspanins of the CD subfamily, CD9 and CD81, physically associate with γ-secretase and also showed that CD81 RNAi reduces γ-secretase processing of β-APP. We assessed the effect of depleting the activity of CD9 or CD81 individually in HeLa cells by RNAi and measured the effect on the activity of different leukemic mutant forms of NOTCH1 as above in two separate experiments.…”

“…7). More recently, the tetraspanins CD81 and CD9 were found to associate in a complex with γ-secretase and promote cleavage of β-amyloid precursor protein (β-APP) (14), and various tetraspanins were found to associate with ADAM10 and also to promote cleavage of β-APP (15). However, until now, there has been no biochemical or functional evidence implicating tetraspanins in Notch signaling.…”