Ricardo A. Gomes

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.

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

BackgroundInsulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimers disease, Parkinsons disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.ResultsMethylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.ConclusionsWe propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.

Yeast protein glycation in vivo by methylglyoxal Molecular modification of glycolytic enzymes and heat shock proteins

Protein glycation by methylglyoxal is a nonenzymatic post‐translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end‐products. Methylglyoxal‐derived advanced glycation end‐products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d‐glyceraldehyde 3‐phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI‐TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolases activity loss, in a glycation‐dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end‐products were determined by MALDI‐TOF. Yeast cells experienced selective pressure towards efficient use of d‐glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).

Protein glycation in Saccharomyces cerevisiae Argpyrimidine formation and methylglyoxal catabolism

Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal‐derived advanced glycation end‐products are involved in neurodegenerative disorders (Alzheimers, Parkinsons and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d‐glucose (100 mm) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d‐glucose concentration (250 mm) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady‐state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae.

Protein Adducts As Prospective Biomarkers of Nevirapine Toxicity

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.

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 (Alzheimers disease, Parkinsons disease and Andrades 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.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen's Chaperone Activity

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimers and Parkinsons. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Insights into the molecular mechanism of protein native-like aggregation upon glycation

Several human neurodegenerative diseases such as Alzheimers disease, Parkinsons disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c.

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.

Bin1 and CD2AP polarise the endocytic generation of beta‐amyloid

The mechanisms driving pathological beta‐amyloid (Aβ) generation in late‐onset Alzheimers disease (AD) are unclear. Two late‐onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aβ generation in neurons. We identify a novel neuron‐specific polarisation of Aβ generation controlled by Bin1 and CD2AP. We discover that Bin1 and CD2AP control Aβ generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aβ generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aβ in neurons increasing the risk for late‐onset AD.