Partial Purification and Characterization of γ-Secretase from Post-mortem Human Brain

Farmery, Mark R.; Tjernberg, Lars O.; Pursglove, Sharon E.; Bergman, Anna; Winblad, Bengt; Näslund, Jan

doi:10.1074/jbc.m211992200

Cited by 143 publications

(141 citation statements)

References 59 publications

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“…Other recent studies have also utilized BN/PAGE for analysis of PS complexes. DDM at 0.5% has been recently reported independently to preserve PS complex integrity in BN/PAGE with reports of complex size from 440 to 600 kDa [12,13,42,55,56]. Digitonin (1%) and BN/ PAGE produced PS1 complex mobility of 250-270 kDa in cells over-expressing the four main constituents of the c-secretase complex [40,41].…”

Section: Discussionmentioning

confidence: 99%

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

Culvenor

Ilaya

Ryan

et al. 2003

European Journal of Biochemistry

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The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N‐ and C‐terminal fragments in association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1. Solubilization of mouse or human brain membranes with dodecyl‐d‐maltoside produced a 360‐kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X‐100 and SDS, but stable to reducing agent. Addition of 5 m urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer‐2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second‐dimension analysis. Anterior pharynx defective‐1 was detected as an 18‐kDa form and 9‐kDa C‐terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36‐kDa band after presenilin 1 complex second‐dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1‐reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.

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Section: Discussionmentioning

confidence: 99%

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

Culvenor

Ilaya

Ryan

et al. 2003

European Journal of Biochemistry

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“…We monitored the activity of ␥-and ␤-secretase as reported previously (40,41). Solubilized membranes were incubated overnight at 37°C in 200 l of 50 mM Tris-HCl (pH 6.8) buffer containing 2 mM EDTA, 0.25% CHAPSO (w/v), and 10 M fluorescent substrate of ␥-secretase or were incubated for 1 h at 37°C in 200 l of 50 mM acetate buffer (pH 4.1) containing 100 mM sodium chloride, 0.025% bovine serum albumin, and 10 M fluorescent ␤-secretase substrate.…”

Section: Methodsmentioning

confidence: 99%

Prostaglandin E2 Stimulates the Production of Amyloid-β Peptides through Internalization of the EP4 Receptor

Hoshino

Namba

Takehara

et al. 2009

Journal of Biological Chemistry

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Amyloid-␤ (A␤) peptides, generated by the proteolysis of ␤-amyloid precursor protein by ␤-and ␥-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E 2 (PGE 2 ), a strong inducer of inflammation, stimulates the production of A␤ through EP 2 and EP 4 receptors, and here we have examined the molecular mechanism. Activation of EP 2 and EP 4 receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP 2 , but not EP 4 , receptor-mediated stimulation of the A␤ production. In contrast, inhibitors of endocytosis suppressed EP 4 , but not EP 2 , receptor-mediated stimulation. Activation of ␥-secretase was observed with the activation of EP 4 receptors but not EP 2 receptors. PGE 2 -dependent internalization of the EP 4 receptor was observed, and cells expressing a mutant EP 4 receptor lacking the internalization activity did not exhibit PGE 2 -stimulated production of A␤. A physical interaction between the EP 4 receptor and PS-1, a catalytic subunit of ␥-secretases, was revealed by immunoprecipitation assays. PGE 2 -induced internalization of PS-1 and co-localization of EP 4 , PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP 4 receptor null mice. These results suggest that PGE 2 -stimulated production of A␤ involves EP 4 receptor-mediated endocytosis of PS-1 followed by activation of the ␥-secretase, as well as EP 2 receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease. Alzheimer disease (AD)2 is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-␤ (A␤) peptides A␤40 and A␤42 (1, 2). To generate A␤, ␤-amyloid precursor protein (APP) is first cleaved by ␤-secretase and then by ␥-secretase. Cleavage of APP by ␣-secretase produces non-amyloidogenic peptides (3, 4). The ␥-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5). Early onset familial AD is linked to three genes, APP, PS1, and PS2 (5, 6), strongly suggesting that ␥-secretasedependent production of A␤ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the ␥-secretase-dependent production of A␤ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the transGolgi network (TGN). Then, they are internalized again and traf...

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“…The activity of g-secretase was measured using a specific fluorogenic substrate assay 53 . Cells were collected in cell lysis buffer containing Tumorigenicity assay.…”

Section: Methodsmentioning

confidence: 99%

Sphingosine-1-phosphate promotes expansion of cancer stem cells via S1PR3 by a ligand-independent Notch activation

et al. 2014

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Many tumours originate from cancer stem cells (CSCs), which is a small population of cells that display stem cell properties. However, the molecular mechanisms that regulate CSC frequency remain poorly understood. Here, using microarray screening in aldehyde dehydrogenase (ALDH)-positive CSC model, we identify a fundamental role for a lipid mediator sphingosine-1-phosphate (S1P) in CSC expansion. Stimulation with S1P enhances ALDH-positive CSCs via S1P receptor 3 (S1PR3) and subsequent Notch activation. CSCs overexpressing sphingosine kinase 1 (SphK1), an S1P-producing enzyme, show increased ability to develop tumours in nude mice, compared with parent cells or CSCs. Tumorigenicity of CSCs overexpressing SphK1 is inhibited by S1PR3 knockdown or S1PR3 antagonist. Breast cancer patient-derived mammospheres contain SphK1 þ /ALDH1 þ cells or S1PR3 þ /ALDH1 þ cells. Our findings provide new insights into the lipid-mediated regulation of CSCs via Notch signalling, and rationale for targeting S1PR3 in cancer.

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Partial Purification and Characterization of γ-Secretase from Post-mortem Human Brain

Cited by 143 publications

References 59 publications

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

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

Prostaglandin E2 Stimulates the Production of Amyloid-β Peptides through Internalization of the EP4 Receptor

Sphingosine-1-phosphate promotes expansion of cancer stem cells via S1PR3 by a ligand-independent Notch activation

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