Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

The glyoxalase pathway: the first hundred years... and beyond.

The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time. Its association to detoxification and anti-glycation defence are its presently accepted roles, since methylglyoxal exerts irreversible effects on protein structure and function, associated with misfolding. This functional defence role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic target, since its inhibition might lead to an increased methylglyoxal concentration and cellular damage. However, metabolic pathway analysis showed that glyoxalase effects on methylglyoxal concentration are likely to be negligible and several organisms, from mammals to yeast and protozoan parasites, show no phenotype in the absence of one or both glyoxalase enzymes. The aim of the present review is to show the evolution of thought regarding the glyoxalase pathway since its discovery 100 years ago, the current knowledge on the glyoxalase enzymes and their recognized role in the control of glycation processes.

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains.

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⁻¹ for L. acidophilus, with a cell mass yield (Y (X/S)) of 0.37 g g⁻¹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⁻¹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis-Menten constant (K(m)) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⁻¹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K(m) for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.

The glyoxalase pathway in protozoan parasites.

The glyoxalase system is the main catabolic route for methylglyoxal, a non-enzymatic glycolytic byproduct with toxic and mutagenic effects. This pathway includes two enzymes, glyoxalase I and glyoxalase II, which convert methylglyoxal to d-lactate by using glutathione as a catalytic cofactor. In protozoan parasites the glyoxalase system shows marked deviations from this model. For example, the functional replacement of glutathione by trypanothione (a spermidine-glutathione conjugate) is a characteristic of trypanosomatids. Also interesting are the lack of glyoxalase I and the presence of two glyoxalase II enzymes in Trypanosoma brucei. In Plasmodium falciparum the glyoxalase pathway is glutathione-dependent, and glyoxalase I is an atypical monomeric enzyme with two active sites. Although it is tempting to exploit these differences for their potential therapeutic value, they provide invaluable clues regarding methylglyoxal metabolism and the evolution of protozoan parasites. Glyoxalase enzymes have been characterized in only a few protozoan parasites, namely Plasmodium falciparum and the trypanosomatids Leishmania and Trypanosoma. In this review, we will focus on the key features of the glyoxalase pathway in major human protozoan parasites, with particular emphasis on the characterized systems in Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. We will also search for genes encoding glyoxalase I and II in Toxoplasma gondii, Entamoeba histolytica, and Giardia lamblia.

Enlightening the molecular basis of trypanothione specificity in trypanosomatids: mutagenesis of Leishmania infantum glyoxalase II.

Leishmania infantum glyoxalase II shows absolute specificity towards its trypanothione thioester substrate. In the previous work, we performed a comparative analysis of glyoxalase II structures determined by X-ray crystallography which revealed that Tyr291 and Cys294, absent in the human homologue, are essential for substrate binding. To validate this trypanothione specificity hypothesis we produced a mutant L. infantum GLO2 enzyme by replacing Tyr291 and Cys294 by arginine and lysine, respectively. This new enzyme is capable to use the glutathione thioester substrate, with kinetic parameters similar to the ones from the human enzyme. Substrate specificity is likely to be mediated by spermidine moiety binding, providing a primer for understanding the molecular basis of trypanothione specificity.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum.

Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-D-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-shaped form exhibited diffraction to about 2.0 A resolution. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 A and three dimers of the enzyme per asymmetric unit.

Protein glycation in vivo: functional and structural effects on yeast enolase.

Protein glycation is involved in structure and stability changes that impair protein functionality, which is associated with several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and beta-fibre formation, numerous studies have been carried out in vitro. All of these previous experiments were conducted in non-physiological or pseudo-physiological conditions that bear little to no resemblance to what may happen in a living cell. In vivo, glycation occurs in a crowded and organized environment, where proteins are exposed to a steady-state of glycation agents, namely methylglyoxal, whereas in vitro, a bolus of a suitable glycation agent is added to diluted protein samples. In the present study, yeast was shown to be an ideal model to investigate glycation in vivo since it shows different glycation phenotypes and presents specific protein glycation targets. A comparison between in vivo glycated enolase and purified enolase glycated in vitro revealed marked differences. All effects regarding structure and stability changes were enhanced when the protein was glycated in vitro. The same applies to enzyme activity loss, dimer dissociation and unfolding. However, the major difference lies in the nature and location of specific advanced glycation end-products. In vivo, glycation appears to be a specific process, where the same residues are consistently modified in the same way, whereas in vitro several residues are modified with different advanced glycation end-products.

Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum.

In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222(1) (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 angstroms) and diffract beyond 2.15 angstroms using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.

Argpyrimidine, a methylglyoxal-derived advanced glycation end-product in familial amyloidotic polyneuropathy.

FAP (familial amyloidotic polyneuropathy) is a systemic amyloid disease characterized by the formation of extracellular deposits of transthyretin. More than 80 single point mutations are associated with amyloidogenic behaviour and the onset of this fatal disease. It is believed that mutant forms of transthyretin lead to a decreased stability of the tetramer, which dissociates into monomers that are prone to unfolding and aggregation, later forming beta-fibrils in amyloid deposits. This theory does not explain the formation of beta-fibrils nor why they are toxic to nearby cells. Age at disease onset may vary by decades for patients with the same mutation. Moreover, non-mutated transthyretin also forms the same deposits in SSA (senile systemic amyloidosis), suggesting that mutations may only accelerate this process, but are not the determinant factor in amyloid fibril formation and cell toxicity. We propose that glycation is involved in amyloidogenesis, since amyloid fibrils present several properties common to glycated proteins. It was shown recently that glycation causes the structural transition from the folded soluble form to beta-fibrils in serum albumin. We identified for the first time a methylglyoxal-derived advanced glycation end-product, argpyrimidine [N(delta)-(5-hydroxy-4,6-dimethylpyrimidin-2-yl)-L-ornithine] in amyloid fibrils from FAP patients. Unequivocal argpyrimidine identification was achieved chromatographically by amino acid analysis using dabsyl (4-dimethylaminoazobenzene-4'-sulphonyl) chloride. Argpyrimidine was found at a concentration of 162.40+/-9.05 pmol/mg of protein in FAP patients, and it was not detected in control subjects. The presence of argpyrimidine in amyloid deposits from FAP patients supports the view that protein glycation is an important factor in amyloid diseases.

Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation.

The glyoxalase pathway of Leishmania infantum was kinetically characterized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A K(m) of 0.253 mm and a V of 0.21 micromol.min(-1).mg(-1)for glyoxalase I, and a K(m) of 0.098 mm and a V of 0.18 micromol.min(-1).mg(-1) for glyoxalase II, were obtained. Modelling and computer simulation were used for evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in Leishmania infantum. A sensitivity analysis of the pathway was performed using experimentally validated kinetic models and experimentally determined metabolite concentrations and kinetic parameters. The measurement of metabolites in L. infantum involved the identification and quantification of methylglyoxal and intracellular thiols. Methylglyoxal formation in L. infantum is nonenzymatic. The sensitivity analysis revealed that the most critical parameters for controlling the intracellular concentration of methylglyoxal are its formation rate and the concentration of trypanothione. Glyoxalase I and II activities play only a minor role in maintaining a low intracellular methylglyoxal concentration. The importance of the glyoxalase pathway as a therapeutic target is very small, compared to the much greater effects caused by decreasing trypanothione concentration or increasing methylglyoxal concentration.

A quantitative model of the generation of N(epsilon)-(carboxymethyl)lysine in the Maillard reaction between collagen and glucose.

The Maillard reaction between reducing sugars and amino groups of biomolecules generates complex structures known as AGEs (advanced glycation endproducts). These have been linked to protein modifications found during aging, diabetes and various amyloidoses. To investigate the contribution of alternative routes to the formation of AGEs, we developed a mathematical model that describes the generation of CML [ N(epsilon)-(carboxymethyl)lysine] in the Maillard reaction between glucose and collagen. Parameter values were obtained by fitting published data from kinetic experiments of Amadori compound decomposition and glycoxidation of collagen by glucose. These raw parameter values were subsequently fine-tuned with adjustment factors that were deduced from dynamic experiments taking into account the glucose and phosphate buffer concentrations. The fine-tuned model was used to assess the relative contributions of the reaction between glyoxal and lysine, the Namiki pathway, and Amadori compound degradation to the generation of CML. The model suggests that the glyoxal route dominates, except at low phosphate and high glucose concentrations. The contribution of Amadori oxidation is generally the least significant at low glucose concentrations. Simulations of the inhibition of CML generation by aminoguanidine show that this compound effectively blocks the glyoxal route at low glucose concentrations (5 mM). Model results are compared with literature estimates of the contributions to CML generation by the three pathways. The significance of the dominance of the glyoxal route is discussed in the context of possible natural defensive mechanisms and pharmacological interventions with the goal of inhibiting the Maillard reaction in vivo.

Optimization of time-course experiments for kinetic model discrimination.

Systems biology relies heavily on the construction of quantitative models of biochemical networks. These models must have predictive power to help unveiling the underlying molecular mechanisms of cellular physiology, but it is also paramount that they are consistent with the data resulting from key experiments. Often, it is possible to find several models that describe the data equally well, but provide significantly different quantitative predictions regarding particular variables of the network. In those cases, one is faced with a problem of model discrimination, the procedure of rejecting inappropriate models from a set of candidates in order to elect one as the best model to use for prediction.In this work, a method is proposed to optimize the design of enzyme kinetic assays with the goal of selecting a model among a set of candidates. We focus on models with systems of ordinary differential equations as the underlying mathematical description. The method provides a design where an extension of the Kullback-Leibler distance, computed over the time courses predicted by the models, is maximized. Given the asymmetric nature this measure, a generalized differential evolution algorithm for multi-objective optimization problems was used.The kinetics of yeast glyoxalase I (EC 4.4.1.5) was chosen as a difficult test case to evaluate the method. Although a single-substrate kinetic model is usually considered, a two-substrate mechanism has also been proposed for this enzyme. We designed an experiment capable of discriminating between the two models by optimizing the initial substrate concentrations of glyoxalase I, in the presence of the subsequent pathway enzyme, glyoxalase II (EC 3.1.2.6). This discriminatory experiment was conducted in the laboratory and the results indicate a two-substrate mechanism for the kinetics of yeast glyoxalase I.

α-Synuclein aggregation in the saliva of familial transthyretin amyloidosis: a potential biomarker.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the formation of transthyretin (TTR) amyloid deposits. This crippling and fatal disease is associated with point mutations in TTR, a protein mainly produced in the liver. Hence, liver transplantation is the only treatment capable of halting disease progression. Ideally, liver transplantation should be performed as early as possible in the disease course before significant neurologic disability has been incurred. Early detection of disease before serious pathological lesions occur is crucial for the clinical management of patients and for morbidity delay. Unfortunately, the presence of TTR mutations by itself is not a predictor of disease onset or progression. In the present work, we observed an increased oligomerization of α-synuclein in the saliva of ATTR symptomatic individuals comparatively to asymptomatic carriers of the same TTR mutation and healthy control subjects. Based on this observation, we propose monitoring α-synuclein oligomers in saliva as a biomarker of ATTR progression. Since α-synuclein plays a major role in several neurodegenerative disorders, assessing its oligomerization state in this fluid provides a non-invasive approach to survey these pathologies.

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production.

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.