A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

Pinnix, Inga; Musunuru, Usha; Tun, Han W.; Sridharan, Arati; Golde, Todd E.; Eckman, Christopher B.; Ziani-Cherif, Chewki; Onstead, Luisa; Sambamurti, Kumar

doi:10.1074/jbc.m005968200

Cited by 135 publications

(132 citation statements)

References 65 publications

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“…This was not entirely unexpected, because, as for other substrates ␥-secretase, it is extremely difficult to detect ICD, probably because of rapid intracellular turnover (31,32). Yet, several groups have developed cell-free assays, allowing them to demonstrate ICD generation from APP by ␥-secretase (28,(33)(34)(35)(36). Using a similar assay for syndecan, we detected a 7-kDa fragment that was continuously released over a period of at least 4 h into the supernatant of S3-MEF membranes (Fig.…”

Section: Resultsmentioning

confidence: 88%

Syndecan 3 Intramembrane Proteolysis Is Presenilin/γ-Secretase-dependent and Modulates Cytosolic Signaling

Schulz

Annaert

Vandekerckhove

et al. 2003

Journal of Biological Chemistry

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The syndecans play critical roles in several signal transduction pathways. The core proteins of these heparan sulfate proteoglycans are characterized by highly conserved transmembrane and intracellular domains which are required for signaling across the membrane and for interaction with cytosolic proteins. However, regulatory mechanisms controlling these functions remain largely unknown. Here we show that, upon ligandinduced primary proteolytic cleavage within the ectodomain, the intracellular domain of syndecan 3 is released by regulated intramembrane proteolysis. The cleavage is mediated by presenilin/␥-secretase complex and negatively regulates the plasma membrane targeting of the transcriptional cofactor CASK.Heparan sulfate proteoglycans (HSPG) 1 are present on the surface of all adherent cells. Binding to a large number of ligands depends upon highly charged heparan sulfate chains, linear polysaccharide chains that can be modified by sequential enzymatic modifications. These modifications lead to enormous structural diversity (for review see Ref. 1). Although numerous functions for the heparan sulfate moiety of proteoglycans have been shown in vivo and in vitro, the roles of the core proteins remain largely elusive (2, 3). The syndecan family of HSPG, as opposed to the GPI-anchored glypicans or matrix-associated HSPG, contains a transmembrane and cytosolic domain that is highly conserved across homologues and species, suggesting an important role for this part of the protein (4). Indeed, a role in FGF signaling, neurite outgrowth, dendritic spine morphogenesis, and left-right axis formation has been demonstrated (5-8), but the regulatory mechanisms are incompletely understood. The cytosolic domain can be subdivided into a membrane-proximal C1 and a C-terminal C2 conserved region that is nearly identical in all syndecans and has a more variable V region in the middle. The most conserved residues are serines and tyrosines that at least partially undergo phosphorylation, a basic motif that binds nonreceptor tyrosine kinases and cytoskeletal proteins, and the C terminus that is required for interaction with PDZ proteins such as syntenin, CASK, synectin, or synbindin (for review see Ref. 4).The heparan sulfate-bearing syndecan ectodomain has been shown to undergo ligand-activated or stress-induced proteolytic cleavage and shedding (9 -11), but the fate of the Cterminal fragment (CTF) that remains associated with the membrane is unknown. We surmised that the remaining CTF is subject to further proteolytic degradation, ectodomain-shedding being a first step in an intracellular signaling cascade. Because regulated intramembrane proteolysis, a novel mechanism of signal transduction across the membrane (12), has been demonstrated to depend upon prior ectodomain cleavage of type I transmembrane protein substrates (13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and is mediated by presenilins (PS) as part of a larger so-called ␥-secretase complex (23), we investigated whether syndecan 3 undergoes PS-dependent cleavage. EXPERI...

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Section: Resultsmentioning

confidence: 88%

Syndecan 3 Intramembrane Proteolysis Is Presenilin/γ-Secretase-dependent and Modulates Cytosolic Signaling

Schulz

Annaert

Vandekerckhove

et al. 2003

Journal of Biological Chemistry

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“…We also examined AICD production in H4 neuroglioma cells induced for FE65L1 with a cell-free AICD generation assay (27,34,37). Our data show that incubation of microsomal membranes over a 2-h period produces similar levels of AICD in cells uninduced and induced for FE65L1 overexpression (Fig.…”

Section: Fe65l1 Increases ␥-Secretase Processing Of App Ctfsmentioning

confidence: 88%

“…Cell-free AICD Generation-AICD generation was examined in vitro using an active membrane preparation as described by Pinnix et al (27). The 10,000 ϫ g active post-nuclear membrane fraction containing the same amount of protein (5 mg) isolated from cells uninduced or induced for FE65L1 was resuspended in 200 l of Buffer A (50 mM HEPES buffer, 150 mM NaCl, 5 mM EDTA, pH 7.4, containing 5 mM 1,10-phenanthroline) and incubated at 37°C or on ice for the indicated times.…”

Section: Methodsmentioning

confidence: 99%

Generation of the β-Amyloid Peptide and the Amyloid Precursor Protein C-terminal Fragment γ Are Potentiated by FE65L1

Chang

Tesco

Jeong

et al. 2003

Journal of Biological Chemistry

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Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of ␣-secretase-derived APP (APPs␣). Increased ␤-amyloid (A␤) generation was also observed in cells showing the FE65-dependent increase in APPs␣. To understand the mechanism for the observed increase in both A␤ and APPs␣ given that ␣-secretase cleavage of a single APP molecule precludes A␤ generation, we examined the effects of FE65L1 overexpression on APP Cterminal fragments (APP CTFs). Our data show that FE65L1 potentiates ␥-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP Cterminal domain and A␤40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on ␥-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.Processing of the amyloid precursor protein (APP) 1 results in the generation of the amyloidogenic peptide, A␤, which plays a central role in the pathogenesis of Alzheimer's disease. Cleavage of C99, the APP C-terminal fragment derived from ␤-secretase processing of APP, by ␥-secretase generates the A␤ peptide. Furthermore, ␥-secretase cleavage of C99 and C83, the ␣-secretase derived APP C-terminal fragment (APP CTF), releases the APP C-terminal domain (AICD), a 6-kDa peptide also called CTF␥ or AID, that regulates transcription after translocation to the nucleus (1-4).The majority of proteins reported to bind the 47-amino acid intracellular region of APP (5-7), including the FE65 protein family members FE65, FE65L1, and FE65L2, bind the YENPTY sorting motif of APP via a phosphotyrosine interaction domain (PID/PTB). YENP is a clathrin-coated pit internalization domain required for trafficking of APP into the endocytic pathway (8, 9). Previous studies have shown that FE65 protein family members can alter the processing of APP by influencing APP trafficking. Increased maturation of APP and increased ␣-secretase-cleaved APP (APPs␣) secretion was observed in H4 neuroglioma cells induced for FE65L1 overexpression (10). Furthermore, enhanced secretion of APPs␣ and A␤ was reported in Madin-Darby canine kidney APP695 cells stably overexpressing FE65 (11). Cell surface APP levels were elevated in these cells, and increased routing of APP into the endocytic pathway from the plasma membrane was suggested to account for the observed increase in A␤ (11).The FE65 proteins are adaptor proteins that have three protein-protein interaction dom...

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“…(H) In DS, the percentage of CTF-beta to alpha increased progressively with age. To detect CTF, the antibody CT20 was used and the specificity has been described previously [48]. Steady state levels of APP show no direct relationship with age but are higher overall in the DS cases.…”

Section: Discussionmentioning

confidence: 99%

“…CT20 (also called O443) detects CTF-α, CTF-β and CTF-γ and full length APP [48]. After a series of washes in TTBS, membranes were incubated for an hour at room temperature in horseradish peroxidase conjugated goat anti-mouse or anti-rabbit respectively (Biorad).…”

Section: Total App and C-terminal Fragments Western Blots And Quantifmentioning

confidence: 99%

Alpha- and beta-secretase activity as a function of age and beta-amyloid in Down syndrome and normal brain

Nistor

Don

Parekh

et al. 2007

Neurobiology of Aging

107

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Aged individuals with Down syndrome (DS) develop Alzheimer's disease (AD) neuropathology by the age of 40 years. The purpose of the current study was to measure age-associated changes in APP processing in 36 individuals with DS (5 months-69 years) and in 26 controls (5 months-100 years). Alpha-secretase significantly decreased with age in DS, particularly in cases over the age of 40 years and was stable in controls. The levels of C-terminal fragments of APP reflecting alphasecretase processing (CTF-alpha) decreased with age in both groups. In both groups, there was significant increase in beta-secretase activity with age. CTF-beta remained constant with age in controls suggesting compensatory increases in turnover/clearance mechanisms. In DS, young individuals had the lowest CTF-beta levels that may reflect rapid conversion of beta-amyloid (Aβ) to soluble pools or efficient CTF-beta clearance mechanisms. Treatments to slow or prevent AD in the general population targeting secretase activity may be more efficacious in adults with DS if combined with approaches that enhance Aβ degradation and clearance.

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A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

Cited by 135 publications

References 65 publications

Syndecan 3 Intramembrane Proteolysis Is Presenilin/γ-Secretase-dependent and Modulates Cytosolic Signaling

Syndecan 3 Intramembrane Proteolysis Is Presenilin/γ-Secretase-dependent and Modulates Cytosolic Signaling

Generation of the β-Amyloid Peptide and the Amyloid Precursor Protein C-terminal Fragment γ Are Potentiated by FE65L1

Alpha- and beta-secretase activity as a function of age and beta-amyloid in Down syndrome and normal brain

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