Dataset Information

To Explore the Binding Affinity of Human γ-Secretase Activating Protein (GSAP) Isoform 4 with APP-C99 Peptides.

ABSTRACT: γ-Secretase activating protein (GSAP) is known to play an important role in the β-amyloid pathway. It acts as a modulator and accentuates the truncation of the amyloid precursor protein C-99 fragment through the γ-secretase complex. GSAP has four isoforms, out of which canonical isoform 1, a 16 kDa C-terminal portion, has been extensively studied, whereas the function of other three isoforms remains unknown. Here, we explore the GSAP isoform 4 (GSAP_I4) expression and purification from inclusion bodies followed by the refolding of the protein. The secondary structure of GSAP_I4 is predicted using circular dichroism. The protein is further characterized by western blotting and mass spectroscopy analysis. Additionally, biochemical assays and in silico molecular docking and molecular simulation are performed to investigate the binding of GSAP_I4 and APP-C99 peptide fragments. The results reflect that although GSAP_I1 and GSAP_I4 share high sequence similarity, the isoform 4 does not show any affinity toward APP-C99 peptide fragments. This hints toward the fact that GSAP_I4 might have a different role in the living system that is yet unexplored.

SUBMITTER: Angira D

PROVIDER: S-EPMC10099435 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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To Explore the Binding Affinity of Human γ-Secretase Activating Protein (GSAP) Isoform 4 with APP-C99 Peptides.

Angira Deekshi D Chaudhary Sonali S Abiramasundari Arumugam A Thiruvenkatam Vijay V

ACS omega 20230327 14

γ-Secretase activating protein (GSAP) is known to play an important role in the β-amyloid pathway. It acts as a modulator and accentuates the truncation of the amyloid precursor protein C-99 fragment through the γ-secretase complex. GSAP has four isoforms, out of which canonical isoform 1, a 16 kDa C-terminal portion, has been extensively studied, whereas the function of other three isoforms remains unknown. Here, we explore the GSAP isoform 4 (GSAP_I4) expression and purification from inclusion ...[more]

PMID: 37065030

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Project description:Backgroundγ-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.MethodsFor 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.ResultThese 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.ConclusionCollectively, 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.

Project description:The amyloid β-peptide (Aβ) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-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 ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Dataset Information

To Explore the Binding Affinity of Human γ-Secretase Activating Protein (GSAP) Isoform 4 with APP-C99 Peptides.

Publications

To Explore the Binding Affinity of Human γ-Secretase Activating Protein (GSAP) Isoform 4 with APP-C99 Peptides.

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