Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization.
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ABSTRACT:
SUBMITTER: Perrin F
PROVIDER: S-EPMC7749410 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:TMP21 has been shown to be associated with the gamma-secretase complex and can specifically regulate gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of gamma-secretase activity was investigated independent of epsilon-cleavage using an amyloid-beta precursor proteinepsilon (APPepsilon) construct which lacks the amyloid intracellular domain domain. The APPepsilon construct behaves similarly to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments, consistent with an inhibition of gamma-secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the gamma-secretase complex and could decrease gamma-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased gamma-cleavage activity.
| S-EPMC2781407 | biostudies-literature
Project description:The amyloid ?-peptide (A?) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid ? protein-precursor (APP CTF?) by the ?-secretase complex. This processing produces A? ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of A? peptides is the result of successive ?-secretase cleavages, with endoproteolysis first occurring at the ? sites to generate A?48 or A?49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of A?: the primary A?49-46-43-40 line and a minor A?48-45-42-38 line. The major secreted A? species are A?40 and A?42, and an increased proportion of the longer, aggregation-prone A?42 compared to A?40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ? site endoproteolysis and carboxypeptidase trimming of CTF? by ?-secretase. We determined that the C-terminal negative charge of the intermediate A?49 does not play a role in its trimming by ?-secretase. Peptidomimetic probes suggest that ?-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ? sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ? cleavage of CTF?. We also show that instability of the CTF? TMD helix near the ? site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by ?-secretase. In addition, we found that CTF? dimers are not endoproteolyzed by ?-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ? cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.
| S-EPMC5539764 | biostudies-literature
Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.
| S-EPMC7236921 | biostudies-literature
Project description:Intramembrane cleavage of the ?-amyloid precursor protein C99 substrate by ?-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for ?-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired ?-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ?-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.
| S-EPMC6554489 | biostudies-literature
Project description:γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aβ products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aβ peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aβ peptides.
| S-EPMC7961089 | biostudies-literature
Project description:Extracellular deposition of ?-amyloid (A?) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon ?-secretase-mediated cleavage of the ? C-terminal fragment (?-CTF) from the A? precursor protein, the ?-secretase complex produces the A? peptides associated with AD. The familial T43I mutation within the transmembrane domain of the ?-CTF (also referred to as C99) increases the ratio between the A?42 and A?40 peptides largely due to a decrease in A?40 formation. A?42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the A?42/A?40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for ?-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted A?40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases A?40 formation. We discuss how the observed structural changes in the T43I mutant may decrease A?40 formation and increase the A?42/A?40 ratio.
| S-EPMC6463720 | biostudies-literature
Project description:The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP) in Alzheimer's Disease (AD) is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cholesterol resides, and the endoplasmic reticulum (ER), where most cholesterol regulatory enzymes localize. The transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs), also called ER-regulatory pools of cholesterol. When formed, these ER-DRMs induce cholesterol esterification to maintain homeostasis, while attenuating cholesterol synthesis. Here, we show that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER, acts as a lipid-sensing peptide that forms ER-DRMs called mitochondria-associated ER membranes (MAM). In cellular AD models, increased levels of C99 in the ER upregulate the formation of MAMs by inducing the internalization of cholesterol and its trafficking from the PM to the ER. These results suggest a role for C99 as a mediator of cholesterol disturbances in AD.
| S-SCDT-EMBOJ-2019-103791 | biostudies-other
Project description:Six new pimprinine alkaloids (1-6), including four dimers, dipimprinines A-D (1-4), and two monomers, (±)-Pimprinol D (5), and pimprinone A (6), along with six known congeners (7-12), were isolated from a soil-derived actinomycete Streptomyces sp. NEAU-C99. Structures of the new compounds were elucidated by extensive spectroscopic analyses, single-crystal X-ray diffractions, and ECD calculations. Dipimprinines A-D (1-4) showed weak cytotoxic activities against five tumor cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW-480, with IC50 values ranging from 12.7 to 30.7 μM.
| S-EPMC7040024 | biostudies-literature
Project description:Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by β and γ secretases leads to the generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aβ42:Aβ40 ratio and the accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.
| S-EPMC8709358 | biostudies-literature
Project description:Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer’s disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down Syndrome (Trisomy 21) requires the extra gene copy of amyloid precursor protein (APP) and is mediated by the beta cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with Down Syndrome (DS), lysosomal degradation of autophagic and endocytic substrates is selectively impaired causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) associated with slowed LC3 turnover and the inactivation of cathepsin D (CTSD) and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, implicating pH elevation as their primary basis. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one BACE1 allele from adult Ts2 mice had similar rescue effects in vivo. The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD.
2019-05-30 | GSE127880 | GEO