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.
Monoamine oxidase (MAO), which exists in two isozymic forms, MAO A and MAO B, is an important flavoenzyme responsible for the metabolism of amine neurotransmitters such as dopamine, serotonin and norepinephrine. Despite extensive research effort, neither the catalytic nor the inhibition mechanisms of MAO have been completely understood. There has also been dispute with regard to the protonation state of the substrate upon entering the active site, as well as the identity of residues that are important for the initial deprotonation of irreversible acetylenic inhibitors, in accordance with the recently proposed mechanism. Therefore, in order to investigate features essential for the modes of action of MAO, we have calculated pK a values of three relevant tyrosine residues in the MAO B active site, with and without dopamine bound as the substrate (as well as the pK a of the dopamine itself in the active site). The calculated pK a values for Tyr188, Tyr398 and Tyr435 in the complex are found to be shifted upwards to 13.0, 13.7 and 14.7, respectively, relative to 10.1 in aqueous solution, ruling out the likelihood that they are viable proton acceptors. The altered tyrosine pK a values could be rationalized as an interplay of two opposing effects: insertion of positively charged bulky dopamine that lowers tyrosine pK a values, and subsequent removal of water molecules from the active site that elevates tyrosine pK a values, in which the latter prevails. Additionally, the pK a value of the bound dopamine (8.8) is practically unchanged compared to the corresponding value in aqueous solution (8.9), as would be expected from a charged amine placed in a hydrophobic active site consisting of aromatic moieties. We also observed potentially favorable cation-π interactions between -NH 3 + group on dopamine and aromatic moieties, which provide stabilizing effect to the charged fragment. Thus, we offer here theoretical evidence that the amine is most likely to be present in the active site in its protonated form, which is similar to the conclusion from experimental studies of MAO A (Jones et al. J. Neural Trans. 2007, 114, 707-712). However, the free energy cost of 3 transferring the proton from the substrate to the bulk solvent is only 1.9 kcal mol -1 , leaving open the possibility that the amine enters the chemical step in its neutral form. In conjunction with additional experimental and computational work, the data presented here should lead towards a deeper understanding of mechanisms of the catalytic activity and irreversible inhibition of MAO B, which can allow for the design of novel and improved MAO B inhibitors.
We used quantum-chemical methods to study seven possible mechanisms of monoamine oxidase (MAO) inhibition by acetylenic inhibitors, considering neutral, cationic, anionic and radical mechanisms. MAO is a flavoenzyme responsible for the metabolism of the important neurotransmitters noradrenaline, serotonin and dopamine. It exists in two isoforms: MAO A and MAO B. Selective MAO A inhibitors are used in the treatment of depression, whereas selective MAO B inhibitors such as rasagiline and selegiline are used to relieve symptoms of Parkinson disease. Rasagiline and selegiline are irreversible MAO B inhibitors, each forming a covalent bond with the enzyme's flavin adenine dinucleotide (FAD) cofactor upon inhibition. Although widely used, they both exhibit numerous adverse effects. Our calculations, performed at the B3LYP/6-311++G(2d,2p)//B3LYP/6-31+G(d) level of theory,
Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies.
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.