Single-molecule Force Spectroscopy Approach to Enzyme Catalysis

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.     

��-Amyloid Causes Depletion of Synaptic Vesicles Leading to Neurotransmission Failure

Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid β-peptide (Aβ), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, Aβ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the Aβ-induced pore prevented the delayed failure, indicating that Aβ blocks neurotransmission by causing vesicular depletion. This new mechanism for Aβ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of Aβ.     

Canonical Wnt3a Modulates Intracellular Calcium and Enhances Excitatory Neurotransmission in Hippocampal Neurons

A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca²⁺ transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest cross-talk between canonical and Wnt/Ca²⁺ signaling in central neurons.     

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants,Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-β (Aβ) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(1–16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.     

Flux regulation of cardiac ryanodine receptor channels

The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca²⁺-induced Ca²⁺ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca²⁺ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca²⁺ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca²⁺ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca²⁺ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel.     

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.Thermophilic and hyperthermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. In fact, many of the (hyper)thermophiles known were isolated from geothermal areas venting seawater (36). However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts and some, namely, di-myo-inositol-phosphate (DIP), mannosyl-di-myo-inositol-phosphate (MDIP), diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures (19, 22, 34, 38). Mannosylglycerate (2-α-d-mannosylglycerate; MG), for example, is a common compatible solute of thermophiles and hyperhermophiles (23, 27, 38) but has also been found in mesophilic organisms, such as red algae, where it was first identified (6). It should also be noted that there is a growing awareness that compatible solutes are involved in other types of stress; trehalose, for example, plays a role in osmotic stress, heat stress, desiccation, and freezing (9). Some compatible solutes of thermophilic organisms are extremely rare and have been encountered in only one or two, generally closely related, species. Among them are mannosylglyceramide in Rhodothermus marinus, diglycerol phosphate in Archaeoglobus fulgidus, and, more recently, mannosylglucosylglycerate (α-d-1→2-mannopyranosyl-α-d-1→2-glucopyranosylglycerate; MGG) identified in Petrotoga miotherma (16, 19, 38).The species of the genus Petrotoga represent slightly thermophilic members of the generally hyperthermophilic and deep-branching bacteria of the order Thermotogales (2, 3, 31). Organisms of this genus have all been isolated from hot oilfield water (21, 25), and have an optimum temperature for growth of 55 to 60°C in medium containing NaCl in the range of 0.5 to 10% (16). In Ptg. miotherma, the levels of MGG increased during low-level osmotic adaptation, whereas glutamate and proline were used for protection against hyperosmotic stress (16). The hyperthermophilic Thermotoga spp. accumulate primarily di-myo-inositol-phosphate and mannosyl-di-myo-inositol-phosphate during osmotic adjustment or during growth at temperatures above the optimum for growth (37).The novel compatible solute MGG is a derivative of glucosylglycerate (2-α-d-glucosylglycerate; GG) identified in the free form in Erwinia chrysanthemi, in the marine cyanobacteria Prochlorococcus marinus and Synechococcus sp. PCC7002, and in the thermophilic bacterium Persephonella marina, the latter of which possesses two alternative pathways for its synthesis (8, 13, 14, 18, 37). Glucosylglycerate has also been detected in trace amounts in Mycobacterium smegmatis, where it probably is the precursor of a polysaccharide involved in the regulation of fatty acid synthesis, as well as in the polar head group of a glycolipid from Nocardia otitidiscaviarum (17, 30).Two alternative pathways for the synthesis of GG have been identified and characterized. In the two-step reaction scheme, the synthesis of GG involves the condensation of nucleoside diphosphate (NDP)-glucose and d-3-phosphoglycerate (3-PGA) into glucosyl-3-phosphoglycerate (GPG), which in turn is dephosphorylated to yield GG. Yet, in a single-step pathway, the synthesis of GG occurs via the condensation of ADP-glucose with d-glycerate (13). Similar routes to those described above also lead to the synthesis of mannosylglycerate in Rhodothermus marinus (4).Two functionally connected genes encoding an “actinobacterial”-type glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase were detected in the genome of Petrotoga mobilis (12). In this study, we examine the synthesis of MGG through a phosphorylating pathway (with a phosphorylated intermediate) from 3-phosphoglycerate and UDP-glucose to the final compatible solute, in cell extracts and by functional characterization of recombinant enzymes. We also examine a second nonphosphorylating pathway (no phosphorylated intermediates) that could represent an alternative route for the synthesis of MGG in Ptg. mobilis that could lead to the direct conversion of GG and GDP-mannose to MGG. Pathway multiplicity likely reflects a crucial role for MGG in the physiology of Ptg. mobilis during stress adaptation.     

Asymmetric alkene epoxidation by Mn(III)salen catalyst in ionic liquids

The enantioselective epoxidation of 6-cyano-2,2-dimethylchromene (Chrom) catalysed by the Jacobsen catalyst, using sodium hypochlorite (NaOCl) as oxygen source, at room temperature, was performed in a series of 1,3-dialkylimidazolium and tetra-alkyl-dimethylguanidium based ionic liquids. All the room temperature ionic liquids (RTILs) could be used as reaction media for the enantioselective epoxidation of the alkene giving, generally, moderate to good epoxide yields and enantiomeric excesses (ee%).For the series of ionic liquids derived from the 1,3-dialkylimidazolium cation, it was observed some relationship between the RTILs physical properties and the catalytic reaction parameters, exemplified by linear correlations between (i) the ee% and the α Kamlet-Taft parameter (hydrogen bond acidity of the solvent) for CH₂Cl₂ and [C₄m_nim][BF₄] ionic liquids (n = 1 or 2), and (ii) the ee% and the β Kamlet-Taft parameter (hydrogen bond basicity of the solvent) for CH₂Cl₂ and [C₄mim][X] ionic liquids (X = PF₆, NTf₂ or BF₄).All the RTILs could be reused in further catalytic cycles, with the exception of [C₈mim][PF₆]. The reutilisation of the Jacobsen catalyst for four times generally led to a decrease in the epoxide yield and to a slight decrease in the enantioselectivity. The recycling of the catalyst could be improved by imparting an ionic character to the complex through abstraction of the axially coordinated chloride anion (Cat 2). Other oxygen sources, such as iodosylbenzene, hydrogen peroxide and urea-hydrogen peroxide adduct, were also tested coupled with Jacobsen catalyst, but the best results were achieved with NaOCl.