Exploiting preQ₁ Riboswitches To Regulate Ribosomal Frameshifting

Knowing the molecular details of the interaction between riboswitch aptamers and their corresponding metabolites is important to understand gene expression. Here we report on a novel <i>in vitro</i> assay to study preQ₁ riboswitch aptamers upon binding of 7-aminomethyl-7-deazaguanine (preQ₁). The assay is based on the ability of the preQ₁ aptamer to fold, upon ligand binding, into a pseudoknotted structure that is capable of stimulating −1 ribosomal frameshifting (−1 FS). Aptamers from three different species were found to induce between 7% and 20% of −1 FS in response to increasing preQ₁ levels, whereas preQ₁ analogues were 100–1000-fold less efficient. In depth mutational analysis of the <i>Fusobacterium nucleatum</i> aptamer recapitulates most of the structural details previously identified for preQ₁ aptamers from other bacteria by crystallography and/or NMR spectroscopy. In addition to providing insight into the role of individual nucleotides of the preQ₁ riboswitch aptamer in ligand binding, the presented system provides a valuable tool to screen small molecules against bacterial riboswitches in a eukaryotic background

Cellular Polyamines Promote Amyloid-Beta (Aβ) Peptide Fibrillation and Modulate the Aggregation Pathways

The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer’s disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (Aβ) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric Aβ(1–40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 μM spermine (normal intracellular concentration is ∼1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce Aβ(1–40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of Aβ peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer’s disease