Mutational Analysis of Intrinsic Regions of Presenilin 2 That Determine Its Endoproteolytic Cleavage and Pathological Function

Abstract

To investigate the significance of endoproteolytic processing of presenilin 2 (PS2) on its pathological function, we constructed PS2 cDNAs causing amino acid substitutions or deletions around the cleavage site. We found that a PS2 mutant (Del3) with a 20-amino acid deletion was not endoproteolytically processed, while other PS2s with amino acid substitutions and short deletions were cleaved. Overproduction of all the mutant proteins led to a compensatory decrease of endogenous PS1 fragments, but did not affect the amyloid ␤ peptide X-42/A␤ X-40 ratio without the familial Alzheimer's disease mutation. The Del3 mutant did not exhibit significant deficits in ␥-secretase activity. The turnover rate of the Del3 holoprotein was the same as that of full-length PS2. These data suggest that the determinants of the PS2 cleavage site reside within a large region and that the pathological function of PS2 is exerted by familial Alzheimer's disease mutations not related to the cleavage of holoproteins. We also found that PS2 with an 18-amino acid deletion at the C-terminal end was not processed. Its overexpression led neither to diminished accumulation of endogenous PS1 fragments nor to increased production of amyloid ␤ peptide X-42. The Cterminal end of PS2 seems to possess the signal for entry into the processing pathway.Mutations in homologous presenilin 1 and 2 (PS1 1 and PS2) genes are associated with early-onset autosomal dominant familial Alzheimer's disease (FAD) (1-3). To date, more than 50 different pathogenic missense mutations and one splice site mutation (loss of exon 10, designated as PS1⌬E10) have been found in PS1, and two missense mutations have been identified in PS2. The gene products of PS1 and PS2 are thought to contain six or eight transmembrane (TM) domains, and the N and C termini and a large hydrophilic loop region following the sixth TM domain are located within the cytoplasm (4 -6). PS1 proteins are involved in cell fate decisions by facilitating Notch signaling pathway (7-9). Recently, PS1 proteins have been shown to have a role in the endoproteolytic processing of ␤-amyloid precursor protein (APP) (10, 11) and , and trafficking of proteins including APP (15) and Notch (12, 13), while the physiological function of PS2 proteins is poorly understood. The amount of highly amyloidogenic A␤42 (amyloid ␤ peptide 42) increases in cells and transgenic mice with the PS1 and PS2 mutant genes (16 -20), supporting a hypothesis that mutations in PS lead to Alzheimer's disease by increasing the extracellular levels of A␤42.The PS proteins are proteolytically cleaved at the hydrophilic loop region and detected predominantly as N-terminal fragments (NTF) and C-terminal fragments (CTF) in culture cells and tissues (19,(21)(22)(23). The formation of PS fragments is highly regulated in a saturable and stoichiometric manner, since overproduction of full-length PS in transfected cells and transgenic mice does not yield a linear increase of fragments (21) and causes a compensatory decrease of the endogenous PS fragments...

Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

“…Nevertheless, in most cases, except mutants harboring mutations at the cleavage site (e.g. M292D-PS1) (40,44,45), the previously reported cleavagedefective mutations simultaneously confer definitive modification of ␥-secretase activity and/or specificity (12,16,17,25,46). This is the case for PS1 LA mutants, which are distinctive in mediating exclusively ␥-43 cleavage of APP, but not self-endoproteolysis, ␥-40 cleavage, and ⑀-cleavage.…”

A Loss of Function Mutant of the Presenilin Homologue SEL-12 Undergoes Aberrant Endoproteolysis in Caenorhabditis elegans and Increases Aβ42 Generation in Human Cells

“…Cleavage of human PS1 and PS2 is heterogeneous and appears to occur at three different sites. Arrows denote reported cleavage sites (69,74,75,77). Boldface letters indicate conserved amino acids.…”

Calsenilin Is a Substrate for Caspase-3 That Preferentially Interacts with the Familial Alzheimer's Disease-associated C-terminal Fragment of Presenilin 2

“…Recent data demonstrate that while expression of full-length presenilin constructs that contain FAD mutations is associated with an increase in the production of A␤, expression of constructs that contain an FAD mutation but lack the C-terminal domain of the protein do not alter A␤ production (14). Moreover, the C-terminal domain but not the large hydrophilic loop (which is where the majority of presenilin interactors bind) is critical for the processing pathway of holoprotein (63,64). Therefore, because calsenilin interacts with the C-terminal domain of the presenilins and because calsenilin interacts preferentially with the 20-kDa PS2 CTF, whose generation is linked to the presenilin mutations, calsenilin may also be involved in presenilin mutation-associated increases in A␤.…”