Membrane inserted APP fragments containing the βA4 sequence of Alzheimer's disease do not aggregate

Abstract

Previously w¢ have shown that the COOH.terminal fragment (A4CT) of flu= Ahhcimcr amyloid protein precursor (APP), which at the N H_.-tcrminus ¢arricl the ~quencc of the amyloid .8A4 protein, forms highly inrmluble aggre~tes [EMBO .I. (1988) 7. 949-957]. Here ~ report thai aggregation is prevented if A4CT is ¢xpreucd in vitro with tt signal ~=quenee at the NH:.terminus (SPA4CT) under conditions which allow membrane immrtion.Aglirellates from gPA4CT are obtained alter removal of membranes h)' chlorofon~methunol extraction or heating.A4CT; In vitro translation; AIIllrqlation; Alxheimer's disca~The histopathological features of Alzheimer's disease (AD) and the mechanisms underlying amyloid deposition in AD have been intensively investigated [2][3][4][5][6][7]. The isolation and sequence analysis of#A4 [3-6] from intra~llular and extracellalar amyloid in AD and Dawn's syndrome brains has led to the isolation and characterization of the family of amyloid protein precursors (APP's) as glycosylated, tyrosine-sulfated transmem. brane proteins [8][9][10][11][12], The ~A4 protein is encoded within the transmembrane and extracellular domains of the APP's [8].The normal route of APP processing involves cleavage of APP within the,aA4 sequence [12][13][14]. This suggests that the NHa-terminus of'the amyloid/~A4 protein is not produced by the normal cleavage which leads to shedding of the extraceliular part of cell-surface transmembrane APP's.The exact amyloidogeni¢ mechanism by which pA4 is cleaved from APP and deposited in AD remains unknown, although our earlier [1] and reins studies [15] suggest that th¢ initial step of the abnormal processing of full-length APP in Alzheimer's disease may occur at the NH,.terminus of the 3A4 sequence and generate a COOH-terminal fragment of 100 residues (A4CT), which includes the amyloid pA4 sequence, and the transmembrane and cytoplasmic domains of all known transmembrane APP forms.To stud~/the amyloidogenic properties of this hypo- Here we =how that expression of this APP fragment in the rabbit reticulocyte ly~ate (RRL) resulted in an aggregating protein, Membrane insertion of A4C'T obtained by expression of" gPA4CT in the presence of dog pancreas membranes prevents aggregation. Post-translational removal of the membranes led to aggregation, EXPERIMENTAL 2,1, Clonhtg pmcedurr~"Preparation of plaimid DNA, restriction enzyme digestion, al~ros¢ 8el ¢lectrophor~is of DNA, DNA libation and bacterial transform;t. tion were carried out as deicri~d by Maniatis et al, [16]. PhtsmM ¢onstrurtionConstruct SP6$/A4CT [1] was obtained by cloning the 961 bp Dell II ttindlll fra~nem of the APP595 eDNA clone [S] into pSP65, The resulting plasmid includes metllionine ¢odon 596 of AlaPfi95 as initia. tion codon, the anaylold ,BA4 Jequence (radons 597-6391640 of APP 69S) and the entire COOH.tcrminal domain, For construction of SF651SPA4CT, plasmid SP6S/A4C'rl. which was obtained by cloninga NCOI oligolinker into SphllE¢oRl.diBested SPfS/A4CT, wa~ digested with NCOI, incubated with $1 to ereat, blunt ends and ...

Histone Deacetylase Inhibitor Valproic Acid Inhibits Cancer Cell Proliferation via Down-regulation of the Alzheimer Amyloid Precursor Protein

“…To determine the growth promoting activity of APP, we used the shorter SPA4CT construct, lacking the Nterminal domain of APP. SPA4CT-transfected cells (40) did not promote increased proliferation rate as compared with mock-transfected controls. Furthermore, we showed that addition of conditioned medium from APP-overproducing cells could stimulate proliferation of SPA4CT-and mock-transfected cells.…”

“…To test this hypothesis, we used the shorter construct lacking the N-terminal domain (SPA4CT). The SPA4CT construct contains the signal peptide of APP followed by leucine and glutamic acid and the C-terminal fragment (C99) of APP (40). In contrast, the SPA4CT construct that lacks the ectodomain of APP had no effect on cellular growth compared with mock-transfected neuroblastoma cells.…”

Independent Inhibition of Alzheimer Disease β- and γ-Secretase Cleavage by Lowered Cholesterol Levels

“…It was previously shown that directly expressed SP-C99 protein is processed to A␤ 40 and A␤ 42 in the same way as full-length APP (48). To facilitate efficient removal of the signal peptide by SP peptidase, the dipeptide LE was introduced directly between the original APP signal peptide and C99 (49).…”

The intramembrane cleavage site of the amyloid precursor protein depends on the length of its transmembrane domain

“…Plasmids pUC18͞SPC99-⌬NT, ⌬CT, and CTI were generated as described earlier for pUC͞C99-T43A (15), by using suited oligonucleotides and plasmid pSP65͞SPC99 (26) as template. The KpnI͞SpeI fragments of the pUC18͞ SPC99-⌬NT and CTI constructs were cloned into the pCEP4 vector digested with KpnI͞NheI to generate the mutated expression plasmids pCEP4͞SPC99 plasmids.…”

“…The KpnI͞SpeI fragments of these constructs were cloned into the pCEP4 vector that was digested with KpnI͞NheI. Plasmid pSP65͞SPC99-NTI was obtained by using the Quik Change Site-Directed Mutagenesis Kit (Stratagene), the corresponding sense and antisense primers, and pSP65͞SPC99 as DNA-template (26). The SmaI͞SpeI fragment of pSP65͞SPC99-NTI was cloned into the pCEP4 vector that was digested with NheI͞PvuII.…”