Dataset Information

Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin.

ABSTRACT: Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a growing list of protein substrates, including the Notch receptors and the amyloid precursor protein. The four components of gamma-secretase complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be essential for activity. The catalytic domain resides within PS proteins, NCT has been suggested to be critical for substrate recognition, and the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, which had been thought to be essential for gamma-secretase activity, is instead involved in complex maturation. Here, we report that NCT is dispensable for gamma-secretase activity. NCT-independent gamma-secretase activity can be detected in two independent NCT-deficient mouse embryonic fibroblast lines and blocked by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate and can cleave ligand-activated endogenous Notch receptors, indicating presence of this activity at the plasma membrane. Small interfering RNA knockdown experiments demonstrated that NCT-independent gamma-secretase activity requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying biochemical properties similar to those of gamma-secretase and roughly 50% of its activity when normalized to PS1 N-terminal fragment levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize gamma-secretase but is not required for substrate recognition.

SUBMITTER: Zhao G

PROVIDER: S-EPMC2853942 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin.

Zhao Guojun G Liu Zhenyi Z Ilagan Ma Xenia G MX Kopan Raphael R

The Journal of neuroscience : the official journal of the Society for Neuroscience 20100201 5

Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a growing list of protein substrates, including the Notch receptors and the amyloid precursor protein. The four components of gamma-secretase complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be essential for activity. The catalytic domain resides within PS proteins, NCT has been suggested to be critical for substrate recognition, and the contributions of Pen2 and Aph1 remain unclear. The role of ...[more]

PMID: 20130175

Similar Datasets

Project description:INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering A? production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP ?-site cleavage (equivalent to A?40), and Notch ?-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of A? production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain A? was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain A? reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting A? production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain A? in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain A? was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

Dataset Information

Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin.

Publications

Gamma-secretase composed of PS1/Pen2/Aph1a can cleave notch and amyloid precursor protein in the absence of nicastrin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets