Abstractγ‐Secretase inhibitors (GSIs) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSIs against a broader panel of substrates, we demonstrate that clinical GSIs are pharmacologically distinct. GSIs show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH1 cleavage is inhibited. Several GSIs are also potent inhibitors of select signal peptide peptidase (SPP/SPPL) family members. Extending these findings to mammosphere inhibition assays in triple‐negative breast cancer lines, we establish that these GSIs have different functional effects. We also demonstrate that the processive γ‐secretase cleavage pattern established for amyloid precursor protein (APP) occurs in multiple substrates and that potentiation of γ‐secretase cleavage is attributable to a direct action of low concentrations of GSIs on γ‐secretase. Such data definitively demonstrate that the clinical GSIs are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds.
Streptococcus salivarius is a lactose-and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3 end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.Streptococcus salivarius is an oral bacterium that is phylogenetically closely related to Streptococcus thermophilus, which is used in food fermentation (16,17,25,29). Both species were initially placed in the S. salivarius taxon as S. salivarius subsp. salivarius and S. salivarius subsp. thermophilus (7) but were regarded as distinct species by Schleifer et al. (28) on the basis of both genetic and phenetic criteria. These two species, together with Streptococcus vestibularis, form a distinct cluster within the streptococcal phylogenetic tree (13,16,25). Lactose, the principal energy source used by S. thermophilus for growth in milk, is transported into the cell by a permease (LacS) belonging to the glycoside-pentoside-hexuronide-cation symporter family (23). Lactose is hydrolyzed within the cell into glucose and galactose by -galactosidase. Glucose is metabolized to lactic acid via the glycolytic, Embden-Meyerhof-Parnas pathway, whereas in most strains galactose cannot be metabolized and is expelled into the external medium (11,14). The organization of the galactose operon coding for the Leloir pathway enzymes in S. thermophilus has recently been elucidated (5, 24, 36), indicating that the inability of S. thermophilus to metabolize galactose is not caused by the absence of the genetic information required for the synthesis of suitable metabolic pathways. Moreover, the activities of the enzymes involved in the Leloir pathway (galactokinase, galactose-1-P uridylyltransferase, and...
BackgroundRecent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer’s disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD.PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function.MethodsWe assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays.ResultsPLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function.ConclusionsThe PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.Electronic supplementary materialThe online version of this article (10.1186/s13195-019-0469-0) contains supplementary material, which is available to authorized users.
Rare coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with increased risk for Alzheimer's disease (AD), but how they confer this risk remains uncertain. We assessed binding of TREM2, AD‐associated TREM2 variants to various forms of Aβ and APOE in multiple assays. TREM2 interacts directly with various forms of Aβ, with highest affinity interactions observed between TREM2 and soluble Aβ42 oligomers. High‐affinity binding of TREM2 to Aβ oligomers is characterized by very slow dissociation. Pre‐incubation with Aβ is shown to block the interaction of APOE. In cellular assays, AD‐associated variants of TREM2 reduced the amount of Aβ42 internalized, and in NFAT assay, the R47H and R62H variants decreased NFAT signaling activity in response to Aβ42. These studies demonstrate i) a high‐affinity interaction between TREM2 and Aβ oligomers that can block interaction with another TREM2 ligand and ii) that AD‐associated TREM2 variants bind Aβ with equivalent affinity but show loss of function in terms of signaling and Aβ internalization.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.