PEN-2 and APH-1 Coordinately Regulate Proteolytic Processing of Presenilin 1

Abstract

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous ␥-secretase cleavage of the ␤-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH 2 -and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-␥-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the ␥-secretase activity. However, their precise function in regulating the PS complex and ␥-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.Alzheimer's disease is a progressive neurodegenerative disorder that is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides, termed ␤-amyloid (A␤) 1 (1). A␤ peptides are proteolytically derived from amyloid precursor protein (APP), a type 1 transmembrane glycoprotein that predominantly resides in the trans-Golgi network (TGN), by two distinct enzymatic activities known as ␤-secretase (or BACE) and ␥-secretase (2). Mutations of presenilins (presenilin 1 (PS1) and presenilin 2 (PS2)) are responsible for the majority of early-onset familial Alzheimer's disease. Several lines of evidence suggest that PSs play a crucial role in intramembranous ␥-secretase cleavage of select type I membrane proteins including APP and the signaling receptor, Notch-1 (3-9).PS1 encodes a hydrophobic protein with 467 amino acids spanning the membrane six to eight times (10, 11). Shortly after being synthesized, PS1 undergoes endoproteolytic cleavage by unknown proteases (named presenilinases) between transmembrane domains 6 and 7 to generate a 27-kDa NH 2 -terminal fragment (NTF) and a 17-kDa COOH-terminal fragment (CTF) (12). Cell biological studies indicated that fulllength PS1 resides almost exclusively in the endoplasmic reticulum (ER), while the NTF and CTF localize predominantly in the Golgi and, to a lesser extent, in the plasma membrane and endocytic compartments (13-15). Endoproteolytic cleavage of PS1 is suggested to take place in the ER and the cleaved fragments are subsequently transported to the Golgi compartments (16). The subcellular location of the ␥-...

Requirement of PEN-2 for Stabilization of the Presenilin N-/C-terminal Fragment Heterodimer within the γ-Secretase Complex

“…We first investigated the maturation of PS. Consistent with previous results (11,14,24), down-regulation of PEN-2 was associated with reduced levels of PS fragments and an accumulation of the PS1 holoprotein (Fig. 1D).…”

“…The observation by others (14,17,24) and us (Fig. 1C) that a PEN-2 knockdown causes an accumulation of the PS holoprotein suggests that PEN-2 is required for PS endoproteolysis.…”

“…Besides their requirement for ␥-secretase complex assembly and maturation, the precise functional role of the other ␥-secretase complex components is unclear. In particular, apart from its role in facilitation of PS endoproteolysis (14,17), functional information about the smallest subunit, the ϳ10-kDa PEN-2 protein, is scarce. In order to gain further insight into its functional role, we thus initiated a structure-function analysis of this protein.…”

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

“…This finding supports previous evidence, that NCT and APH-1 may associate independently of PS1 complexes [40,41,44] and that NCT may form a subcomplex in the absence of PS1 [13]. Low or no detection of APH-1 in the partially purified endogenous PS complex by second-dimension analysis, and minimal effect on APH-1 expression level with PS1 deficiency supports the proposal that APH-1 may be important for early assembly and stabilization of the complex [50][51][52].…”

Prostaglandin I 2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

“…Indeed, APH‐1 often combines with PEN‐2, nicastrin, and PS to generate an active form of γ‐secretase complex, which is responsible for the cleavage of β‐APP and for the deposition of Aβ (De Strooper, 2003). Once APH‐1 was found in C. elegans (Francis et al ., 2002), the APH‐1 complex was confirmed in several experimental models (Gu et al ., 2003; Luo et al ., 2003; Hansson et al ., 2004). Along these lines, APH‐1α/1β might also be involved in regulating the deposition of Aβ 1–42 in response to PGI 2 stimulation.…”