Understanding APP Metabolism♦ : The Transcriptionally Active Amyloid Precursor Protein (APP) Intracellular Domain Is Preferentially Produced from the 695 Isoform of APP in a β-Secretase-dependent Pathway
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PROVIDER: S-EPMC3009941 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Altered production of ?-amyloid (A?) from the amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of ?-secretase derived APP carboxyl terminal fragment (CTF?, C99) impairs processive cleavage by ?-secretase increasing production of long A?s (e.g., A?1-42, 43). Other studies report that APP CTF? dimers are not ?-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long A?, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long A?. To further evaluate how multimerization of substrate affects both initial ?-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ?-cleavage site utilization and A? production. These show that multimerization significantly impedes ?-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long A? production without altering the initial ?-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient ?-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter ?-secretase processivity.
| S-EPMC4211736 | biostudies-literature
Project description:Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-? (A?), released by sequential proteolytic processing of the amyloid precursor protein (APP) by ? - and ?-secretase. A? peptides can aggregate, leading to toxic A? oligomers and amyloid plaque formation. A? accumulation is not only dependent on de novo synthesis but also on A? degradation. Neprilysin (NEP) is one of the major enzymes involved in A? degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the ?-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APP?CT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1?exon9, and APP?CT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the A?-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD.
| S-EPMC4443740 | biostudies-literature
Project description:p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau.
| S-EPMC4562799 | biostudies-literature
Project description:sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.
| S-EPMC6674411 | biostudies-literature
Project description:Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.
| S-EPMC4489158 | biostudies-literature
Project description:Cleavage of amyloid precursor protein (APP) by ?- and ?-secretase generates amyloid-? (A?) and APP intracellular domain (AICD) peptides. Presenilin (PS) 1 or 2 is the catalytic component of the ?-secretase complex. Mitochondrial dysfunction is an established phenomenon in Alzheimer's disease (AD), but the causes and role of PS1, APP, and APP's cleavage products in this process are largely unknown. We studied the effect of these AD-associated molecules on mitochondrial features. Using cells deficient in PSs expression, expressing human wild-type PS1, or PS1 familial AD (FAD) mutants, we found that PS1 affects mitochondrial energy metabolism (ATP levels and oxygen consumption) and expression of mitochondrial proteins. These effects were associated with enhanced expression of the mitochondrial master transcriptional coactivator PGC-1? and its target genes. Importantly, PS1-FAD mutations decreased PS1's ability to enhance PGC-1? mRNA levels. Analyzing the effect of APP and its ?-secretase-derived cleavage products A? and AICD on PGC-1? expression showed that APP and AICD increase PGC-1? expression. Accordingly, PGC-1? mRNA levels in cells deficient in APP/APLP2 or expressing APP lacking its last 15 amino acids were lower than in control cells, and treatment with AICD, but not with A?, enhanced PGC-1? mRNA levels in these and PSs-deficient cells. In addition, knockdown of the AICD-binding partner Fe65 reduced PGC-1? mRNA levels. Importantly, APP/AICD increases PGC-1? expression also in the mice brain. Our results therefore suggest that APP processing regulates mitochondrial function and that impairments in the newly discovered PS1/APP/AICD/PGC-1? pathway may lead to mitochondrial dysfunction and neurodegeneration.
| S-EPMC4331788 | biostudies-literature
Project description:BackgroundAmyloid ? (A?), a causative peptide of Alzheimer's disease, is generated by intracellular metabolism of amyloid ?-protein precursor (APP). In general, mature APP (mAPP, N- and O-glycosylated form) is subject to successive cleavages by ?- or ?-, and ?-secretases in the late protein secretory pathway and/or at plasma membrane, while immature APP (imAPP, N-glycosylated form) locates in the early secretory pathway such as endoplasmic reticulum or cis-Golgi, in which imAPP is not subject to metabolic cleavages. X11-like (X11L) is a neural adaptor protein composed of a phosphotyrosine-binding (PTB) and two C-terminal PDZ domains. X11L suppresses amyloidogenic cleavage of mAPP by direct binding of X11L through its PTB domain, thereby generation of A? lowers. X11L expresses another function in the regulation of intracellular APP trafficking.MethodologyIn order to analyze novel function of X11L in intracellular trafficking of APP, we performed a functional dissection of X11L. Using cells expressing various domain-deleted X11L mutants, intracellular APP trafficking was examined along with analysis of APP metabolism including maturation (O-glycosylation), processing and localization of APP.ConclusionsX11L accumulates imAPP into the early secretory pathway by mediation of its C-terminal PDZ domains, without being bound to imAPP directly. With this novel function, X11L suppresses overall APP metabolism and results in further suppression of A? generation. Interestingly some of the accumulated imAPP in the early secretory pathway are likely to appear on plasma membrane by unidentified mechanism. Trafficking of imAPP to plasma membrane is observed in other X11 family proteins, X11 and X11L2, but not in other APP-binding partners such as FE65 and JIP1. It is herein clear that respective functional domains of X11L regulate APP metabolism at multiple steps in intracellular protein secretory pathways.
| S-EPMC3139598 | biostudies-literature
Project description:The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.
| S-EPMC2913973 | biostudies-literature
Project description:?-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ? precursor protein (APP) to release the amyloid ? peptide (A?). A? is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. ?-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing A? and the APP intracellular domain (AICD), referred to as ? and ?, respectively. The heterogeneous nature of the ? cleavage that produces various A? peptides is highly relevant to AD, as increased production of A? 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to A?) that plays a critical role in determining the final length of A? peptides released by ?-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of ?-secretase cleavage from 1-40 to 1-33 without significant changes to ? cleavage. These results further support a model where ? cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of ?-secretase.
| S-EPMC3220543 | biostudies-literature
Project description:Estrone, a steroidal estrogen that is persistently contaminating the surface water has been classified as an endocrine disruptor and as Group-1 carcinogen by the World Health Organization. Long-term exposure to estrone-contaminated water disrupt physiology, behaviour and sexual development of living organisms that lead to many disorders. So, it has to be eliminated from our surrounding. Its biological degradation is a cost effective and eco-friendly approach. The present study targets to predict the degradation pathway and understand the role of cyanobacterial enzymes: oxidoreductases (laccase, peroxidase) and esterase in estrone degradation. Poly-?-hydroxy butyrate (PHB) was also quantified as a by-product of estrone biodegradation. The estrone degradation pathway was predicted using EAWAG-BBD/PPS database. Spirulina CPCC-695 was grown in different concentration of estrone (20 mg/l, 50 mg/l, 100 mg/l and 200 mg/l). The culture without estrone was considered as control. The culture supernatant was used for testing laccase and esterase activity whereas the biomass was used to test peroxidase activity and quantify by-product (PHB). The enzymes showed concentration-dependent activities. Maximum enzyme activities were seen at 20 mg/l estrone. Spirulina CPCC-695 utilizes estrone as a carbon source and degrades it to produce pyruvate which forms acetyl CoA that undergo condensation, reduction and polymerization to form PHB. Maximum PHB (169 ?g) was also produced at 20 mg/l as a by-product during degradation.
| S-EPMC7215111 | biostudies-literature