Maple syrup urine disease diagnosis in Brazilian patients by massive parallel sequencing.

Biallelic pathogenic variants cause maple syrup urine disease (MSUD) in one of the branched-chain α-keto acid dehydrogenase (BCKDH) complex genes (BCKDHA, BCKDHB, DBT, DLD, and PPM1K) leading to the accumulation of leucine, isoleucine, and valine. This study aimed to perform a molecular diagnosis of Brazilian patients with MSUD using gene panels and massive parallel sequencing. Eighteen Brazilian patients with a biochemical diagnosis of MSUD were analyzed by massive parallel sequencing in the Ion PGM Torrent Server using a gene panel with the BCKDHA, BCKDHB, and DBT genes. The American College of Medical Genetics and Genomics guidelines were used to determine variant pathogenicity. Thirteen patients had both variants found by massive parallel sequencing, whereas 3 patients had only one variant found. In 2 patients, the variants were not found by this analysis. These 5 patients required additional Sanger sequencing to confirm their genotype. Twenty-five pathogenic variants were identified in the 3 MSUD-related genes (BCKDHA, BCKDHB, and DBT). Most variants were present in the BCKDHB gene, and no common variants were found. Nine novel variants were observed: c.922 A > G, c.964C > A, and c.1237 T > C in the BCKDHA gene; and c.80_90dup, c.384delA, c.478 A > T, c.528C > G, c.977 T > C, and c.1039-2 A > G in the BCKDHB gene. All novel variants were classified as pathogenic. Molecular modeling of the novel variants indicated that the binding of monomers was affected in the BCKDH complex tetramer, which could lead to a change in the stability and activity of the enzyme. Massive parallel sequencing with targeted gene panels seems to be a cost-effective method that can provide a molecular diagnosis of MSUD.

Structure determination needs to go viral.

Viral diseases are expected to cause new epidemics in the future, therefore, it is essential to assess how viral diversity is represented in terms of deposited protein structures. Here, data were collected from the Protein Data Bank to screen the available structures of viruses of interest to WHO. Excluding SARS-CoV-2 and HIV-1, less than 50 structures were found per year, indicating a lack of diversity. Efforts to determine viral structures are needed to increase preparedness for future public health challenges.

Fungal heat shock proteins: molecular phylogenetic insights into the host takeover.

Heat shock proteins are constitutively expressed chaperones induced by cellular stress, such as changes in temperature, pH, and osmolarity. These proteins, present in all organisms, are highly conserved and are recruited for the assembly of protein complexes, transport, and compartmentalization of molecules. In fungi, these proteins are related to their adaptation to the environment, their evolutionary success in acquiring new hosts, and regulation of virulence and resistance factors. These characteristics are interesting for assessment of the host adaptability and ecological transitions, given the emergence of infections by these microorganisms. Based on phylogenetic inferences, we compared the sequences of HSP9, HSP12, HSP30, HSP40, HSP70, HSP90, and HSP110 to elucidate the evolutionary relationships of different fungal organisms to suggest evolutionary patterns employing the maximum likelihood method. By the different reconstructions, our inference supports the hypothesis that these classes of proteins are associated with pathogenic gains against endothermic hosts, as well as adaptations for phytopathogenic fungi.

Structural Characteristics of Glycocins: Unraveling the Role of S-Linked Carbohydrates and Other Structural Elements.

Glycocins are antimicrobial peptides with glycosylations, often an S-linked monosaccharide. Their recent structure elucidation has brought forth questions about their mechanisms of action as well as the impact of S-glycosylation on their structural behavior. Here, we investigated structural characteristics of glycocins using a computational approach. Depending on the peptide's class (sublancin- or glycocin F-like), the sugar changes the peptide's flexibility. Also, the presence of glycosylation is necessary for the lack of structure of Asm1. The C-terminal tail in glycocin F-like peptides influenced their structured regions, acting like a regulator. These findings corroborate the versatility of these post-translational modifications, pointing toward their potential use in molecular engineering.

Making it Rain: Cloud-Based Molecular Simulations for Everyone.

We present a user-friendly front-end for running molecular dynamics (MD) simulations using the OpenMM toolkit on the Google Colab framework. Our goals are (1) to highlight the usage of a cloud-computing scheme for educational purposes for a hands-on approach when learning MD simulations and (2) to exemplify how low-income research groups can perform MD simulations in the microsecond time scale. We hope this work facilitates teaching and learning of molecular simulation throughout the community.

Genome Mining for Antimicrobial Compounds in Wild Marine Animals-Associated Enterococci.

New ecosystems are being actively mined for new bioactive compounds. Because of the large amount of unexplored biodiversity, bacteria from marine environments are especially promising. Further, host-associated microbes are of special interest because of their low toxicity and compatibility with host health. Here, we identified and characterized biosynthetic gene clusters encoding antimicrobial compounds in host-associated enterococci recovered from fecal samples of wild marine animals remote from human-affected ecosystems. Putative biosynthetic gene clusters in the genomes of 22 Enterococcus strains of marine origin were predicted using antiSMASH5 and Bagel4 bioinformatic software. At least one gene cluster encoding a putative bioactive compound precursor was identified in each genome. Collectively, 73 putative antimicrobial compounds were identified, including 61 bacteriocins (83.56%), 10 terpenes (13.70%), and 2 (2.74%) related to putative nonribosomal peptides (NRPs). Two of the species studied, Enterococcus avium and Enterococcus mundtti, are rare causes of human disease and were found to lack any known pathogenic determinants but yet possessed bacteriocin biosynthetic genes, suggesting possible additional utility as probiotics. Wild marine animal-associated enterococci from human-remote ecosystems provide a potentially rich source for new antimicrobial compounds of therapeutic and industrial value and potential probiotic application.

Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

The fructose-1,6-bisphosphatase deficiency and the p.(Lys204ArgfsTer72) variant.

Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inborn error of fructose metabolism caused by pathogenic variants in the FBP1 gene. As gluconeogenesis is affected, catabolic episodes can induce ketotic hypoglycemia in patients. FBP1 analysis is the most commonly used approach for the diagnosis of this disorder. Herein, a Brazilian patient is reported. The proband, a girl born to a consanguineous couple, presented with severe hypoglycemia crisis in the neonatal period. At the age 17 months, presented a new crisis accompanied by metabolic acidosis associated with a feverish episode. Genetic analysis was performed by next-generation sequencing (NGS), identifying the NM_000507.3:c.611_614del variant in homozygosis in the FBP1 gene. In silico analysis and 3D modeling were performed, suggesting that this variant is associated with a loss of sites for substrate and Mg2+ binding and for posttranslational modifications of FBPase. The c.611_614del variant is located in a repetitive region of the FBP1 gene that appears to be a hotspot for mutational events. This frameshift creates a premature termination codon in the last coding exon which escapes the nonsense-mediated decay mechanism, according to in silico analysis. This variant results in an intrinsically disordered protein with loss of substrate recognition and post-translational modification sites.

Guarana supplementation attenuated obesity, insulin resistance, and adipokines dysregulation induced by a standardized human Western diet via brown adipose tissue activation.

Obesity is a metabolic disorder associated with adverse health consequences that has increased worldwide at an epidemic rate. This has encouraged many people to utilize nonprescription herbal supplements for weight loss without knowledge of their safety or efficacy. However, mounting evidence has shown that some herbal supplements used for weight loss are associated with adverse effects. Guarana seed powder is a popular nonprescription dietary herb supplement marketed for weight loss, but no study has demonstrated its efficacy or safety when administered alone. Wistar rats were fed four different diets (low-fat diet and Western diet with or without guarana supplementation) for 18 weeks. Metabolic parameters, gut microbiota changes, and toxicity were then characterized. Guarana seed powder supplementation prevented weight gain, insulin resistance, and adipokine dysregulation induced by Western diet compared with the control diet. Guarana induced brown adipose tissue expansion, mitochondrial biogenesis, uncoupling protein-1 overexpression, AMPK activation, and minor changes in gut microbiota. Molecular docking suggested a direct activation of AMPK by four guarana compounds tested here. We propose that brown adipose tissue activation is one of the action mechanisms involved in guarana supplementation-induced weight loss and that direct AMPK activation may underlie this mechanism. In summary, guarana is an attractive potential therapeutic agent to treat obesity.

Modulation of Disordered Proteins with a Focus on Neurodegenerative Diseases and Other Pathologies.

Intrinsically disordered proteins (IDPs) do not have rigid 3D structures, showing changes in their folding depending on the environment or ligands. Intrinsically disordered proteins are widely spread in eukaryotic genomes, and these proteins participate in many cell regulatory metabolism processes. Some IDPs, when aberrantly folded, can be the cause of some diseases such as Alzheimer's, Parkinson's, and prionic, among others. In these diseases, there are modifications in parts of the protein or in its entirety. A common conformational variation of these IDPs is misfolding and aggregation, forming, for instance, neurotoxic amyloid plaques. In this review, we discuss some IDPs that are involved in neurodegenerative diseases (such as beta amyloid, alpha synuclein, tau, and the "IDP-like" PrP), cancer (p53, c-Myc), and diabetes (amylin), focusing on the structural changes of these IDPs that are linked to such pathologies. We also present the IDP modulation mechanisms that can be explored in new strategies for drug design. Lastly, we show some candidate drugs that can be used in the future for the treatment of diseases caused by misfolded IDPs, considering that cancer therapy has more advanced research in comparison to other diseases, while also discussing recent and future developments in this area of research. Therefore, we aim to provide support to the study of IDPs and their modulation mechanisms as promising approaches to combat such severe diseases.

Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide.

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

Determining the pathogenicity of CFTR missense variants: Multiple comparisons of in silico predictors and variant annotation databases.

Pathogenic variants in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for cystic fibrosis (CF), the commonest monogenic autosomal recessive disease, and CFTR-related disorders in infants and youth. Diagnosis of such diseases relies on clinical, functional, and molecular studies. To date, over 2,000 variants have been described on CFTR (~40% missense). Since few of them have confirmed pathogenicity, in silico analysis could help molecular diagnosis and genetic counseling. Here, the pathogenicity of 779 CFTR missense variants was predicted by consensus predictor PredictSNP and compared to annotations on CFTR2 and ClinVar. Sensitivity and specificity analysis was divided into modeling and validation phases using just variants annotated on CFTR2 and/or ClinVar that were not in the validation datasets of the analyzed predictors. After validation phase, MAPP and PhDSNP achieved maximum specificity but low sensitivity. Otherwise, SNAP had maximum sensitivity but null specificity. PredictSNP, PolyPhen-1, PolyPhen-2, SIFT, nsSNPAnalyzer had either low sensitivity or specificity, or both. Results showed that most predictors were not reliable when analyzing CFTR missense variants, ratifying the importance of clinical information when asserting the pathogenicity of CFTR missense variants. Our results should contribute to clarify decision making when classifying the pathogenicity of CFTR missense variants.

All-Hydrocarbon Staples and Their Effect over Peptide Conformation under Different Force Fields.

Olefinic staples enhance α-helical content and conformational stability in peptides, maintaining a structural scaffold that allows the emulation of specific regions of protein surfaces for therapeutical purposes. The ability to anticipate the efficacy of adding a staple to a peptide through computational simulations may contribute to lowering the costs associated with rational drug design. We evaluated the capabilities of different force fields to reproduce the effect of all-hydrocarbon staples in molecular dynamics simulations. Using the AMBER99SB-ILDN, CHARMM36, and GROMOS54A7 force fields and two distinct initial conformations, we compared our results to experimentally obtained circular dichroism data. The GROMOS54A7 united-atom force field seems to be more accurate compared with all-atom force fields, despite being unable to reproduce the effect of the staple in some of the simulated systems. With further force field enhancements, MD simulations may be used to anticipate conformational effects of all-hydrocarbon staples in peptides.

Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease.

BACKGROUND: Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent ß-hairpin motif consistent with either neurotoxicity or pore formation. METHODS: Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the ß-hairpin motif (residues 61-74), JbtxΔ-ß; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter. RESULTS: 1) JbtxΔ-ß disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the ß-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis. MAJOR CONCLUSIONS: The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the ß-hairpin motif. Although the ß-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. GENERAL SIGNIFICANCE: Jbtx represents a new type of insecticidal and membrane-active peptide.

Structural glycobiology of human α1-acid glycoprotein and its implications for pharmacokinetics and inflammation.

Human α1-acid glycoprotein (AGP) is an abundant human plasma glycoprotein that may be N-glycosylated at five positions. AGP plays important roles on pharmacokinetics and can rise up to 5-fold in inflammatory events. In such events, the glycan chains attached to Asn54, Asn75 and Asn85 may become fucosylated, originating a sialyl-Lewis X epitope. This epitope, in turn, can bind selectin proteins. Such interplay is important for immunomodulation. While the X-ray structure of unglycosylated AGP has been reported, the absence of the glycan chains hampered the further insights into its structural biology and, ultimately, into its biological function. Thus, the current work intends to contribute in the characterization of the structural glycobiology and function of AGP by building a structural model of its fully glycosylated form, taking into account the different glycoforms that are found in vivo. The obtained data points to the absence of a major influence of glycosylation on AGP's secondary structure, in agreement with crystallography observations. However, the glycan chains seem able to interfere with the protein dynamics, mainly at the AGP-ligand-binding site, indicating a possible role in its complexation to drugs and other bioactive compounds. By examining the influence of fucosylation on AGP structure and binding to selectins, it is proposed that the latter may bind to glycan chains linked to Asn54 and Asn75, and that this binding may involve other glycans, such as the one attached to Asn15. These results point to an increased participation of carbohydrates on the observed AGP roles in pharmacokinetics and inflammation.

Canavalia ensiformis urease, Jaburetox and derived peptides form ion channels in planar lipid bilayers.

Ureases catalyze the hydrolysis of urea into NH3 and CO2. They are synthesized by plants, fungi and bacteria but not by animals. Ureases display biological activities unrelated to their enzymatic activity, i.e., platelet and neutrophil activation, fungus inhibition and insecticidal effect. Urease from Canavalia ensiformis (jack bean) is toxic to several hemipteran and coleopteran insects. Jaburetox is an insecticidal fragment derived from jack bean urease. Among other effects, Jaburetox has been shown to interact with lipid vesicles. In this work, the ion channel activity of C. ensiformis urease, Jaburetox and three deletion mutants of Jaburetox (one lacking the N-terminal region, one lacking the C-terminal region and one missing the central ß-hairpin) were tested on planar lipid bilayers. All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states whose conductance ranges were 7-18pS and 32-79pS, respectively. Urease and the N-terminal mutant of Jaburetox were more active at negative potentials, while the channels of the other peptides did not display voltage-dependence. This is the first direct demonstration of the capacity of C. ensiformis urease and Jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be a crucial step of their diverse biological activities, including host defense.

Differential effect of solution conditions on the conformation of the actinoporins Sticholysin II and Equinatoxin II.

Actinoporins are a family of pore-forming proteins with hemolytic activity. The structural basis for such activity appears to depend on their correct folding. Such folding encompasses a phosphocholine binding site, a tryptophan-rich region and the activity-related N-terminus segment. Additionally, different solution conditions are known to be able to influence the pore formation by actinoporins, as for Sticholysin II (StnII) and Equinatoxin II (EqtxII). In this context, the current work intends to characterize the influence of distinct solution conditions in the conformational behavior of these proteins through molecular dynamics (MD) simulations. The obtained data offer structural insights into actinoporins dynamics in solution, characterizing its conformational behavior at the atomic level, in accordance with previous experimental data on StnII and EqtxII hemolytic activities.

3-to-1: unraveling structural transitions in ureases.

Ureases are nickel-dependent enzymes which catalyze the hydrolysis of urea to ammonia and carbamate. Despite the apparent wealth of data on ureases, many crucial aspects regarding these enzymes are still unknown, or constitute matter for ongoing debates. One of these is most certainly their structural organization: ureases from plants and fungi have a single unit, while bacterial and archaean ones have three-chained structures. However, the primitive state of these proteins--single- or three-chained--is yet unknown, despite many efforts in the field. Through phylogenetic inference using three different datasets and two different algorithms, we were able to observe chain number transitions displayed in a 3-to-1 fashion. Our results imply that the ancestral state for ureases is the three-chained organization, with single-chained ureases deriving from them. The two-chained variants are not evolutionary intermediates. A fusion process, different from those already studied, may explain this structural transition.

Slowly Making Sense: A Review of the Two-Step Venom System within Slow (Nycticebus spp.) and Pygmy Lorises (Xanthonycticebus spp.).

Since the early 2000s, studies of the evolution of venom within animals have rapidly expanded, offering new revelations on the origins and development of venom within various species. The venomous mammals represent excellent opportunities to study venom evolution due to the varying functional usages, the unusual distribution of venom across unrelated mammals and the diverse variety of delivery systems. A group of mammals that excellently represents a combination of these traits are the slow (Nycticebus spp.) and pygmy lorises (Xanthonycticebus spp.) of south-east Asia, which possess the only confirmed two-step venom system. These taxa also present one of the most intriguing mixes of toxic symptoms (cytotoxicity and immunotoxicity) and functional usages (intraspecific competition and ectoparasitic defence) seen in extant animals. We still lack many pieces of the puzzle in understanding how this venom system works, why it evolved what is involved in the venom system and what triggers the toxic components to work. Here, we review available data building upon a decade of research on this topic, focusing especially on why and how this venom system may have evolved. We discuss that research now suggests that venom in slow lorises has a sophisticated set of multiple uses in both intraspecific competition and the potential to disrupt the immune system of targets; we suggest that an exudate diet reveals several toxic plants consumed by slow and pygmy lorises that could be sequestered into their venom and which may help heal venomous bite wounds; we provide the most up-to-date visual model of the brachial gland exudate secretion protein (BGEsp); and we discuss research on a complement component 1r (C1R) protein in saliva that may solve the mystery of what activates the toxicity of slow and pygmy loris venom. We conclude that the slow and pygmy lorises possess amongst the most complex venom system in extant animals, and while we have still a lot more to understand about their venom system, we are close to a breakthrough, particularly with current technological advances.

A Mechanism of Action of Metformin in the Brain: Prevention of Methylglyoxal-Induced Glutamatergic Impairment in Acute Hippocampal Slices.

Metformin, a biguanide compound (N-1,1-dimethylbiguanide), is widely prescribed for diabetes mellitus type 2 (T2D) treatment. It also presents a plethora of properties, such as anti-oxidant, anti-inflammatory, anti-apoptosis, anti-tumorigenic, and anti-AGE formation activity. However, the precise mechanism of action of metformin in the central nervous system (CNS) needs to be clarified. Herein, we investigated the neuroprotective role of metformin in acute hippocampal slices exposed to methylglyoxal (MG), a highly reactive dicarbonyl compound and a key molecule in T2D developmental pathophysiology. Metformin protected acute hippocampal slices from MG-induced glutamatergic neurotoxicity and neuroinflammation by reducing IL-1ß synthesis and secretion and RAGE protein expression. The drug also improved astrocyte function, particularly with regard to the glutamatergic system, increasing glutamate uptake. Moreover, we observed a direct effect of metformin on glutamate transporters, where the compound prevented glycation, by facilitating enzymatic phosphorylation close to Lys residues, suggesting a new neuroprotective role of metformin via PKC ζ in preventing dysfunction in glutamatergic system induced by MG. Proposed neuroprotection role of metformin in acute hippocampal slices against impairment in glutamatergic system induced in a model of methylglyoxal glycotoxicity. Metformin reversed methylglyoxal (MG)-induced neuroinflammation by reducing pro-inflammatory IL-1ß synthesis and secretion and RAGE protein expression. Metformin did not alter the effect of MG on S100B secretion (1). Both MG and metformin also influenced astrocyte function in hippocampal slices. Metformin did not reverse the elevation in GLO1 activity induced by glycotoxicity; however, it abrogated the high GSH level and the expression of the co-factor of GLO1 (2). Both treatments per se changed bioenergetic metabolism and increased glucose uptake, extracellular lactate content, and pyruvate kinase (PK) activity. The increment in glucose uptake and lactate levels ceased during the co-incubation of MG with metformin. Metformin reversed the elevation of hexokinase activity by MG (3). We suggest a new role of metformin in the glutamate system, whereby it protects the hippocampus against the derangements of the glutamatergic system induced by MG, possibly by phosphorylation via PKC ζ (4). The neuroprotective action of metformin may be mediated by the phosphorylation of specific amino acid residues (Lysine) of the glutamate transporters (GLAST and GLT-1), since metformin activated the PKC ζ signaling and promoted cascades of phosphorylation in p38 MAPK and Akt proteins. The transporter protein phosphorylation prevented the Lys-glycation and the impairment of glutamate uptake induced by MG (5).