Amyloid-b peptide ending at the 42nd residue (Ab42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Ab42 production are termed c-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the c-secretase, thereby decreasing production of Ab42, possibly by enhancing its conversion to Ab38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.
Drp35 is a protein induced by cell wall-affecting antibiotics or detergents; it possesses calcium-dependent lactonase activity. To determine the molecular basis of the lactonase activity, we first solved the crystal structures of Drp35 with and without Ca 2؉ ; these showed that the molecule has a six-bladed -propeller structure with two calcium ions bound at the center of the -propeller and surface region. Mutational analyses of evolutionarily conserved residues revealed that the central calcium-binding site is essential for the enzymatic activity of Drp35. Substitution of some other amino acid residues for the calcium-binding residues demonstrated the critical contributions of Glu 48 , Asp 138 , and Asp 236 to the enzymatic activity. Differential scanning calorimetric analysis revealed that the loss of activity of E48Q and D236N, but not D138N, was attributed to their inability to hold the calcium ion. Further structural analysis of the D138N mutant indicates that it lacks a water molecule bound to the calcium ion rather than the calcium ion itself. Based on these observations and structural information, a possible catalytic mechanism in which the calcium ion and its binding residues play direct roles was proposed for the lactonase activity of Drp35.Staphylococcus aureus is a major cause of hospital-and community-acquired infections. S. aureus causes serious and fatal diseases, such as toxic shock syndrome or septicemia (1). Moreover, S. aureus has the remarkable and unfortunate feature that it can become readily resistant to antibiotics. Indeed, it has acquired resistance to almost all antibiotics so far, resulting in an increase in incidence of acute hospital-acquired infections (2).Extensive studies have focused on how S. aureus acquires resistance to antibiotics, and genome sequencing analysis confirmed the existence of many resistance genes acquired by horizontal transfer from other species (3). In addition, S. aureus can cope with antibiotic stresses in an adaptive manner through regulation of the expression of many genes (4).Drp35 (a 35-kDa drug-responsive protein) is a cytoplasmic protein originally found to be markedly induced upon exposure of S. aureus to cell wall-affecting antibiotics (5). Antibiotic susceptibility experiments using a drp35 defective strain and overexpressing strain of S. aureus revealed that Drp35 is correlated with bacitracin resistance, although it did not show significant changes in minimal inhibitory concentration for -lactams, glycopeptides, or fosfomycin (6). Drp35 can also be induced by a variety of detergents, including Nonidet P-40, Triton X-100, SDS, and CHAPS 2 (6). These findings suggest that a broad range of stresses that perturb membrane integrity are responsible for the induction of Drp35 and that Drp35 may be a factor responsible for such general stresses rather than specific antibiotic stress.Interestingly, Drp35 possesses calcium-dependent lactonase activity, although it has not been clarified how this activity contributes to the ability of the S. aureus cell t...
BackgroundThe most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo.MethodWe generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence.ResultsZebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP.ConclusionThese novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0146-8) contains supplementary material, which is available to authorized users.
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