Dynamics of human acetylcholinesterase bound to non-covalent and covalent inhibitors shedding light on changes to the water network structure

© 2016 the Owner Societies.We investigated the effects of non-covalent reversible and covalent irreversible inhibitors on human acetylcholinesterase and human butyrylcholinesterase. Remarkably a non-covalent inhibitor, Huperzine A, has almost no effect on the molecular dynamics of the protein, whereas the covalently binding nerve agent soman renders the molecular structure stiffer in its aged form. The modified movements were studied by incoherent neutron scattering on different time scales and they indicate a stabilization and stiffening of aged human acetylcholinesterase. It is not straightforward to understand the forces leading to this strong effect. In addition to the specific interactions of the adduct within the protein, some indications point towards an extensive water structure change for the aged conjugate as water Bragg peaks appeared at cryogenic temperature despite an identical initial hydration state for all samples. Such a change associated to an apparent increase in free water volume upon aging suggests higher ordering of the hydration shell that leads to the stiffening of protein. Thus, several additive contributions seem responsible for the improved flexibility or stiffening effect of the inhibitors rather than a single interaction

Pressure-induced molten globule state of human acetylcholinesterase: Structural and dynamical changes monitored by neutron scattering

© the Owner Societies 2015. We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein

Interaction of prion protein with acetylcholinesterase: potential pathobiological implications in prion diseases

The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases. Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infected cell cultures. Furthermore, reduction of AChE levels in prion-infected heterozygous AChE knock-out mice leads to slightly but significantly prolonged incubation time. Finally, we found that AChE levels were altered in prion-infected cells and tissues, suggesting that AChE might be directly associated with abnormal PrP. Our results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target

Progress in the development of enzyme-based nerve agent bioscavengers

International audienceAcetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers. Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger. Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime. Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established

Potential of a Pycnoporus sanguineus laccase in bioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye

In the frame of the development of a bioprocess using the competences of White Rot Fungi to decolourise and detoxify dye contaminated effluents, laccases were produced by the strain Pycnoporus sanguineus MUCL 41582 in a malt extract medium. Two isoenzymes were detected among which LAC-1 was concentrated. A survey of the composition of industrial effluents showed that wastewaters from textile industry usually contain high concentrations of Na2SO4 or NaCl. Regarding the activity profile of the biocatalyst against pH, salts, temperature and target substrates, LAC-1 appears to be a good candidate for application in acid dye bath treatments. Studying the model anthraquinonic dye ABu62 decolourisation, we proved that this dye was a good substrate for LAC-1. Furthermore, unusual kinetic behaviour was observed suggesting that LAC- I was activated in presence of ABu62. On the contrary, a classical Michaelis-Menten behaviour was observed for the oxidation of ABTS and LAC- I showed a high affinity for this substrate as compared to data available for other laccases. To our knowledge, this is the first report showing this atypical behaviour of a laccase in the presence of dyes. (c) 2007 Elsevier Inc. All rights reserved

Activity and molecular dynamics relationship within the family of human cholinesterase

The temperature dependence of the dynamics of recombinant human acetylcholinesterase (hAChE) and plasma human butyrylcholinesterase (hBChE) is examined using elastic incoherent neutron scattering. These two enzymes belong to the same family and present 50% amino acid sequence identity. However, significantly higher flexibility and catalytic activity of hAChE when compared to the ones of hBChE are measured. At the same time, the average height of the potential barrier to the motions is increased in the hBChE, e.g. more thermal energy is needed to cross it in the latter case, which might be the origin of the increase in activation energy and the reduction in the catalytic rate of hBChE observed experimentally. These results suggest that the motions on the picosecond timescale may act as a lubricant for those associated with activity occurring on a slower millisecond timescale

Laccase chloride inhibition reduction by an anthraquinonic substrate

Due to their low substrate specificity, fungal laccases have a great potential in industrial applications, including the bioremediation of colored wastewaters from textile industry. However, the presence of halides in these effluents (up to 1M NaCl) which inhibit laccases is a drawback for bioremediation processes. In order to develop an efficient enzymatic remediation process for textile dye effluent, the possibility to reduce this halide inhibition is conditioned by a better understanding of the phenomenon. The present study gives a detailed account of the kinetics of chloride inhibition of both ABTS (a model substrate) and ABu62 (an anthraquinonic acid dye) oxidations catalyzed by Trametes versicolor laccase (LacIIIb). Chloride inhibition can be described by a mixed model for ABTS and a non-competitive model for ABu62 and both inhibitions are linear suggesting a single inhibitory site for chloride. Experiments were also conducted in presence of both substrates. An apparent activation of laccase was observed in the presence of ABu62 leading to an enhancement of the oxidation rate of ABTS. The extent of activation increased in the presence of chloride anions. Finally, for the first time to our knowledge, we evidenced that inhibition of ABTS oxidation by chloride can be reduced in the presence of ABu62

Pressure-induced molten globule state of human acetylcholinesterase: Structural and dynamical changes monitored by neutron scattering

© the Owner Societies 2015. We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein

Dynamics of human acetylcholinesterase bound to non-covalent and covalent inhibitors shedding light on changes to the water network structure

International audienc