Unraveling the complexity of γ-secretase.
Ontology highlight
ABSTRACT:
SUBMITTER: Wolfe MS
PROVIDER: S-EPMC7371508 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Over two decades, γ-secretase has been the target for extensive therapeutic development due to its pivotal role in pathogenesis of Alzheimer's disease and cancer. However, it has proven to be a challenging task owing to its large set of substrates and our limited understanding of the enzyme's structural and mechanistic features. The scientific community is taking bigger strides towards solving this puzzle with recent advancement in techniques like cryogenic electron microscopy (cryo-EM) and photo-affinity labelling (PAL). This review highlights the significance of the PAL technique with multiple examples of photo-probes developed from γ-secretase inhibitors and modulators. The binding of these probes into active and/or allosteric sites of the enzyme has provided crucial information on the γ-secretase complex and improved our mechanistic understanding of this protease. Combining the knowledge of function and regulation of γ-secretase will be a decisive factor in developing novel γ-secretase modulators and biological therapeutics.
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Project description:Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.
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Project description:Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid beta-peptide (Abeta), the major component of senile plaques in the brains of Alzheimer disease patients. Therefore, gamma-secretase is a prime target for Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex composed of four different subunits, presenilin (PS), APH-1, nicastrin, and PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the catalytically active site, which is critically required for the aspartyl protease activity of gamma-secretase. Moreover, numerous familial Alzheimer disease-associated mutations within the PSs increase the production of the aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a substrate receptor, although this has recently been challenged. PEN-2 is required to stabilize PS within the gamma-secretase complex. No particular function has so far been assigned to APH-1. The four components are sufficient and required for gamma-secretase activity. At least six different gamma-secretase complexes exist that are composed of different variants of PS and APH-1. All gamma-secretase complexes can exert pathological Abeta production. Assembly of the gamma-secretase complex occurs within the endoplasmic reticulum, and only fully assembled and functional gamma-secretase complexes are transported to the plasma membrane. Structural analysis by electron microscopy and chemical cross-linking reveals a water-containing cavity, which allows intramembrane proteolysis. Specific and highly sensitive gamma-secretase inhibitors have been developed; however, they interfere with the physiological function of gamma-secretase in Notch signaling and thus cause rather significant side effects in human trials. Modulators of gamma-secretase, which selectively affect the production of the pathological 42-amino acid Abeta, do not inhibit Notch signaling.
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Project description:Protein ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as one of the most prevalent posttranslational modifications (PTMs), regulating nearly every cellular pathway. The diversity of signaling associated with this particular PTM stems from the myriad ways in which a target protein can be modified by ubiquitin, e.g., monoubiquitin, multi-monoubiquitin, and polyubiquitin linkages. In this Review, we focus on developments in both enzymatic and chemical methods that engender ubiquitin with new chemical and physical properties. Moreover, we highlight how these methods have enabled studies directed toward (i) characterizing enzymes responsible for reversing the ubiquitin modification, (ii) understanding the influence of ubiquitin on protein function and crosstalk with other PTMs, and (iii) uncovering the impact of polyubiquitin chain linkage and length on downstream signaling events.
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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.
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Project description:The numerous processes involved in the etiology of breast cancer such as cell survival, metabolism, proliferation, differentiation, and angiogenesis are currently being elucidated. However, underlying mechanisms that drive breast cancer progression and drug resistance are still poorly understood. As we discuss here in detail, the Notch signaling pathway is an important regulatory component of normal breast development, cell fate of normal breast stem cells, and proliferation and survival of breast cancer initiating cells. Notch exerts a wide range of critical effects through a canonical pathway where it is expressed as a type I membrane precursor heterodimer followed by at least two subsequent cleavages induced by ligand engagement to ultimately release an intracellular form to function as a transcriptional activator. Notch and its ligands are overexpressed in breast cancer, and one method of effectively blocking Notch activity is preventing its cleavage at the cell surface with ?-secretase inhibitors. In the context of Notch signaling, the application of clinically relevant anti-Notch drugs in treatment regimens may contribute to novel therapeutic interventions and promote more effective clinical response in women with breast cancer.
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Project description:The intramembrane protease gamma-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer’s disease. To identify naturally short substrates and non-substrates of gamma-secretase, we used four human cell lines of different tissue origins, breast cancer MCF7 cells, cervix carcinoma HeLa cells, T cell leukemia Jurkat cells and lymphoma U937 macrophage-like cells. The cell lines were treated overnight with the established gamma-secretase inhibitor DAPT or DMSO as a control. The proteomes of membrane fractions were determined by nano-liquid chromatography-tandem mass spectrometry and label-free quantitative proteomics. TNFRSF12A, PTPRCAP and C16orf54 were identified as potential naturally short gamma-secretase substrates, whereas other proteins with a short ectodomain including ‘pituitary tumor-transforming gene 1-interacting protein’ (PTTG1IP) did not show an increased abundance upon DAPT treatment.
2024-05-24 | PXD045701 | Pride
Project description:Fifteen human healthy volunteers, divided into three groups, received a single dose of placebo or either 140 mg or 280 mg of the γ-secretase inhibitor semagacestat (LY450139). Endogenous peptides in CSF, sampled prior to administration of the drug and at six subsequent time points, were analyzed by liquid chromatography coupled to mass spectrometry, using isobaric labeling based on the tandem mass tag approach for relative quantification.
2016-03-14 | PXD003075 | Pride
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.
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