An overview on glycation: molecular mechanisms, impact on proteins, pathogenesis, and inhibition.

The formation of a heterogeneous set of advanced glycation end products (AGEs) is the final outcome of a non-enzymatic process that occurs in vivo on long-life biomolecules. This process, known as glycation, starts with the reaction between reducing sugars, or their autoxidation products, with the amino groups of proteins, DNA, or lipids, thus gaining relevance under hyperglycemic conditions. Once AGEs are formed, they might affect the biological function of the biomacromolecule and, therefore, induce the development of pathophysiological events. In fact, the accumulation of AGEs has been pointed as a triggering factor of obesity, diabetes-related diseases, coronary artery disease, neurological disorders, or chronic renal failure, among others. Given the deleterious consequences of glycation, evolution has designed endogenous mechanisms to undo glycation or to prevent it. In addition, many exogenous molecules have also emerged as powerful glycation inhibitors. This review aims to provide an overview on what glycation is. It starts by explaining the similarities and differences between glycation and glycosylation. Then, it describes in detail the molecular mechanism underlying glycation reactions, and the bio-molecular targets with higher propensity to be glycated. Next, it discusses the precise effects of glycation on protein structure, function, and aggregation, and how computational chemistry has provided insights on these aspects. Finally, it reports the most prevalent diseases induced by glycation, and the endogenous mechanisms and the current therapeutic interventions against it.

Understanding the effect of the membrane-mimetic micelles on the interplay between α-synuclein and Cu(II)/Cu(I) cations.

α-Synuclein (αS) is a presynaptic protein whose aggregates are considered as a hallmark of Parkinson's disease (PD). Although its physiological function is still under debate, it is widely accepted that its functions are always mediated by its interaction with membranes. The association of αS with phospholipid membranes occurs concomitant to its folding from its monomeric, unfolded state towards an antiparallel amphipathic α-helix. Besides this, copper ions can also bind αS and modify its aggregation propensity. The effect of Cu(II) and Cu(I) on the lipid-αS affinity and on the structure of the membrane-bound αS have not yet been studied. This knowledge is relevant to understand the molecular pathogenesis of PD. Therefore, we have here studied the affinities between Cu(II) and Cu(I) and the micelle-bound αS, as well as the effect of these cations on the structure of micelle-bound αS. Cu(II) or Cu(I) did not affect the α-helical structure of the micelle-bound αS. However, while Cu(I) binds at the same sites of αS in the presence or in the absence of micelles, the micelle-bound αS displays different Cu(II) binding sites than unbound αS. In any case, sodium docecyl sulphate -micelles reduce the stability of the αS complexes with both Cu(II) and Cu(I). Finally, we have observed that the micelle-bound αS is still able to prevent the Cu(II)-catalysed oxidation of neuronal metabolites (e.g. ascorbic acid) and the formation of reactive oxygen species, thus this binding does not impair its biological function as part of the antioxidant machinery.

Tyrosine Nitroxidation Does Not Affect the Ability of α-Synuclein to Bind Anionic Micelles, but It Diminishes Its Ability to Bind and Assemble Synaptic-like Vesicles.

Parkinson's disease (PD) is characterized by dopaminergic neuron degeneration and the accumulation of neuronal inclusions known as Lewy bodies, which are formed by aggregated and post-translationally modified α-synuclein (αS). Oxidative modifications such as the formation of 3-nitrotyrosine (3-NT) or di-tyrosine are found in αS deposits, and they could be promoted by the oxidative stress typical of PD brains. Many studies have tried to elucidate the molecular mechanism correlating nitroxidation, αS aggregation, and PD. However, it is unclear how nitroxidation affects the physiological function of αS. To clarify this matter, we synthetized an αS with its Tyr residues replaced by 3-NT. Its study revealed that Tyr nitroxidation had no effect on either the affinity of αS towards anionic micelles nor the overall structure of the micelle-bound αS, which retained its α-helical folding. Nevertheless, we observed that nitroxidation of Y39 lengthened the disordered stretch bridging the two consecutive α-helices. Conversely, the affinity of αS towards synaptic-like vesicles diminished as a result of Tyr nitroxidation. Additionally, we also proved that nitroxidation precluded αS from performing its physiological function as a catalyst of the clustering and the fusion of synaptic vesicles. Our findings represent a step forward towards the completion of the puzzle that must explain the molecular mechanism behind the link between αS-nitroxidation and PD.

On the effect of methionine oxidation on the interplay between α-synuclein and synaptic-like vesicles.

Human alpha-synuclein (αS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, which are considered a hallmark of Parkinson's disease (PD). αS has four different Met, which are particularly sensitive to oxidation, as most of them are found as Met sulfoxide (MetO) in the αS deposits. Consequently, researchers have invested mounting efforts trying to elucidate the molecular mechanisms underlying the links between oxidative stress, αS aggregation and PD. However, it has not been described yet the effect of Met oxidation on the physiological function of αS. Trying to shed light on this aspect, we have here studied a synthetic αS that displayed all its Met replaced by MetO moieties (αS-MetO). Our study has allowed to prove that MetO diminishes the affinity of αS towards anionic micelles (SDS), although the micelle-bound fraction of αS-MetO still adopts an α-helical folding resembling that of the lipid-bound αS. MetO also diminishes the affinity of αS towards synaptic-like vesicles, and its hindering effect is much more pronounced than that displayed on the αS-micelle affinity. Additionally, we have also demonstrated that MetO impairs the physiological function of αS as a catalyst of the clustering and the fusion of synaptic vesicles (SVs). Our findings provide a new understanding on how Met oxidation affects one of the most relevant biological functions attributed to αS that is to bind and cluster SVs along the neurotransmission.

Glycation of α-synuclein hampers its binding to synaptic-like vesicles and its driving effect on their fusion.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders affecting the worldwide population. One of its hallmarks is the intraneuronal accumulation of insoluble Lewy bodies (LBs), which cause the death of dopaminergic neurons. α-Synuclein (αS) is the main component of these LBs and in them, it commonly contains non-enzymatic post-translational modifications, such as those resulting from its reaction with reactive carbonyl species arising as side products of the intraneuronal glycolysis (mainly methylglyoxal). Consequently, lysines of the αS found in LBs of diabetic individuals are usually carboxyethylated. A precise comprehension of the effect of Nε-(carboxyethyl)lysine (CEL) on the aggregation of αS and on its physiological function becomes crucial to fully understand the molecular mechanisms underlying the development of diabetes-induced PD. Consequently, we have here used a synthetic αS where all its Lys have been replaced by CEL moieties (αS-CEL), and we have studied how these modifications could impact on the neurotransmission mechanism. This study allows us to describe how the non-enzymatic glycosylation (glycation) affects the function of a protein like αS, involved in the pathogenesis of PD. CEL decreases the ability of αS to bind micelles, although the micelle-bound fraction of αS-CEL still displays an α-helical fold resembling that of the lipid-bound αS. However, CEL completely abolishes the affinity of αS towards synaptic-like vesicles and, consequently, it hampers its physiological function as a catalyst of the clustering and the fusion of the synaptic vesicles.

Frataxins Emerge as New Players of the Intracellular Antioxidant Machinery.

Frataxin is a mitochondrial protein which deficiency causes Friedreich's ataxia, a cardio-neurodegenerative disease. The lack of frataxin induces the dysregulation of mitochondrial iron homeostasis and oxidative stress, which finally causes the neuronal death. The mechanism through which frataxin regulates the oxidative stress balance is rather complex and poorly understood. While the absence of human (Hfra) and yeast (Yfh1) frataxins turn out cells sensitive to oxidative stress, this does not occur when the frataxin gene is knocked-out in E. coli. To better understand the biological roles of Hfra and Yfh1 as endogenous antioxidants, we have studied their ability to inhibit the formation of reactive oxygen species (ROS) from Cu2+- and Fe3+-catalyzed degradation of ascorbic acid. Both proteins drastically reduce the formation of ROS, and during this process they are not oxidized. In addition, we have also demonstrated that merely the presence of Yfh1 or Hfra is enough to protect a highly oxidation-prone protein such as α-synuclein. This unspecific intervention (without a direct binding) suggests that frataxins could act as a shield to prevent the oxidation of a broad set of intracellular proteins, and reinforces that idea that frataxin can be used to prevent neurological pathologies linked to an enhanced oxidative stress.

Nitration and Glycation Diminish the α-Synuclein Role in the Formation and Scavenging of Cu2+-Catalyzed Reactive Oxygen Species.

Human α-synuclein is a small monomeric protein (140 residues) essential to maintain the function of the dopaminergic neurons and the neuronal redox balance. However, it holds a dark side since it is able to clump inside the neurons forming insoluble aggregates known as Lewy bodies, which are considered the hallmark of Parkinson's disease. Sporadic mutations and nonenzymatic post-translational modifications are well-known to stimulate the formation of Lewy bodies. Yet, the effect of nonenzymatic post-translational modifications on the function of α-synuclein has been studied less intense. Therefore, here we study how nitration and glycation mediated by methylglyoxal affect the redox features of α-synuclein. Both diminish the ability of α-synuclein to chelate Cu2+, except when Nε-(carboxyethyl)lysine or Nε-(carboxymethyl)lysine (two advanced glycation end products highly prevalent in vivo) are formed. This results in a lower capacity to prevent the Cu-catalyzed ascorbic acid degradation and to delay the formation of H2O2. However, only methylglyoxal was able to abolish the ability of α-synuclein to inhibit the free radical release. Both nitration and glycation enhanced the α-synuclein availability to be damaged by O2•-, although glycation made α-synuclein less reactive toward HO•. Our data represent the first report describing how nonenzymatic post-translational modifications might affect the redox function of α-synuclein, thus contributing to a better understanding of its pathological implications.