Endoproteolytic Processing and Stabilization of Wild-type and Mutant Presenilin

Ratovitski, Tamara; Slunt, Hilda H.; Wang, Shuai; Price, Donald L.; Sisodia, Sangram S.; Borchelt, David R.

doi:10.1074/jbc.272.39.24536

Cited by 192 publications

(178 citation statements)

References 24 publications

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“…As a consequence, PEN-2-⌬C behaved as a highly unstable protein and was rapidly turned over by the proteasome. This is in contrast to correctly assembled ␥-secretase complex components, which have been shown to be very stable and have a long half-life time (9,17,44,48). The failure of PEN-2-⌬C to stably assemble with PS, NCT, and APH-1 was associated with a marked selective instability of the PS1 NTF and CTF, which also underwent proteasomal degradation, whereas NCT and APH-1 were stable, consistent with previous findings that these proteins can stabilize each other independently (20,24,25).…”

Section: Pen-2-dependent Stabilization Of Ps Ntf/ctf Heterodimermentioning

confidence: 90%

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

Prokop

Shirotani

Edbauer

et al. 2004

Journal of Biological Chemistry

111

128

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␥-Secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated ␤-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-⌬C mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N-and C-terminal fragments and to maturate NCT. PEN-2-⌬C was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the ␥-secretase complex. In addition, expression of PEN-2-⌬C caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 ⌬exon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for ␥-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the ␥-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for ␥-secretase complex maturation independent of its activity in PS1 endoproteolysis.The Alzheimer's disease (AD) 1 -associated ␥-secretase is a high molecular weight complex with an aspartyl protease activity that catalyzes the second of two subsequent proteolytic cleavages of the ␤-amyloid precursor protein (APP) implicated in AD (1). This unusual, intramembranous cleavage liberates the neurotoxic amyloid-␤ peptide from the membrane. Amyloid-␤ peptide aggregates and is deposited in the brain of AD patients in amyloid plaques, an invariant pathological hallmark of AD (2). The ␥-secretase complex is composed of either of the two presenilins (PS), PS1 and PS2, polytopic membrane proteins that are endoproteolytically cleaved into stable heterodimers consisting of an N-and C-terminal fragment (NTF and CTF) (3), the type I transmembrane glycoprotein nicastrin (NCT) (4, 5), and the polytopic membrane proteins PEN-2 (6), APH-1a, and/or APH-1b (6, 7). Expression of the ␥-secretase complex components is coordinately regulated. RNA interference (RNAi)-mediated knockdown studies in cultured Drosophila and mammalian cells as well as studies with cells derived from PS1, PS1/2, or NCT knockout mice have shown that ␥-secretase complex formation is coordinately regulated. Downregulation or genetic ablation of a given compone...

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Section: Pen-2-dependent Stabilization Of Ps Ntf/ctf Heterodimermentioning

confidence: 90%

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

Prokop

Shirotani

Edbauer

et al. 2004

Journal of Biological Chemistry

111

128

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“…Turnover of PS2 and Del3-We compared the stability of full-length PS2 and Del3, since PS1⌬E10 has been reported to show a longer half-life than full-length PS1 (25,27). PS2N141I and Del3N141I cells were labeled for 1 h and chased for up to 20 h, followed by immunoprecipitation with the PS2N53 antibody.…”

Section: Endoproteolytic Cleavage and Pathological Function Of Ps2mentioning

confidence: 99%

“…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 (21,24,25). However, the enzyme involved in the normal PS cleavage remains to be determined.…”

mentioning

confidence: 99%

“…Interestingly, PS1⌬E10 has a longer half-life than that of full-length PS1, although it does not undergo endoproteolytic processing because of the lack of the cleavage region. In addition, the overexpression of PS1⌬E10 causes a compensatory decrease of endogenous presenilins and it is incorporated into the high molecular weight complex (17,21,25,27,28). In contrast, exogenous expression of NTF or CTF alone leads to neither diminished accumulation of endogenous presenilin fragments nor increased production of A␤42, even though they have FAD mutations (31-35).…”

mentioning

confidence: 99%

“…However, the enzyme involved in the normal PS cleavage remains to be determined. The processed fragments form stable heterodimers and occur as a high molecular weight complex, while the remaining full-length proteins are rapidly degraded (20,23,(25)(26)(27)(28)(29)(30). Therefore, it is proposed that these heterodimers consisting of NTF and CTF are functional units of PSs.…”

mentioning

confidence: 99%

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

Shirotani¹,

Takahashi²,

Araki³

et al. 2000

Journal of Biological Chemistry

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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...

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Identity and function of γ‐secretase

Kimberly

Wolfe

2003

J of Neuroscience Research

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gamma-Secretase catalyzes intramembrane proteolysis of various type I membrane proteins, including the amyloid-beta precursor protein and the Notch receptor. Despite its importance in the pathogenesis of Alzheimer's disease and to normal development, this protease has eluded identification until only very recently. Four membrane proteins are now known to be members of the protease complex: presenilin, nicastrin, aph-1, and pen-2. Recent findings suggest that these four proteins are sufficient to reconstitute the active gamma-secretase complex and that together they mediate the cell surface signaling of a variety of receptors via intramembrane proteolysis.

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Endoproteolytic Processing and Stabilization of Wild-type and Mutant Presenilin

Cited by 192 publications

References 24 publications

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

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

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

Identity and function of γ‐secretase

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