Glyoxalase 2: Towards a Broader View of the Second Player of the Glyoxalase System.

Glyoxalase 2 is a mitochondrial and cytoplasmic protein belonging to the metallo-ß-lactamase family encoded by the hydroxyacylglutathione hydrolase (HAGH) gene. This enzyme is the second enzyme of the glyoxalase system that is responsible for detoxification of the α-ketothaldehyde methylglyoxal in cells. The two enzymes glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) form the complete glyoxalase pathway, which utilizes glutathione as cofactor in eukaryotic cells. The importance of Glo2 is highlighted by its ubiquitous distribution in prokaryotic and eukaryotic organisms. Its function in the system has been well defined, but in recent years, additional roles are emerging, especially those related to oxidative stress. This review focuses on Glo2 by considering its genetics, molecular and structural properties, its involvement in post-translational modifications and its interaction with specific metabolic pathways. The purpose of this review is to focus attention on an enzyme that, from the most recent studies, appears to play a role in multiple regulatory pathways that may be important in certain diseases such as cancer or oxidative stress-related diseases.

The Role of Artificial Intelligence in the Diagnosis and Prognosis of Renal Cell Tumors.

The increasing availability of molecular data provided by next-generation sequencing (NGS) techniques is allowing improvement in the possibilities of diagnosis and prognosis in renal cancer. Reliable and accurate predictors based on selected gene panels are urgently needed for better stratification of renal cell carcinoma (RCC) patients in order to define a personalized treatment plan. Artificial intelligence (AI) algorithms are currently in development for this purpose. Here, we reviewed studies that developed predictors based on AI algorithms for diagnosis and prognosis in renal cancer and we compared them with non-AI-based predictors. Comparing study results, it emerges that the AI prediction performance is good and slightly better than non-AI-based ones. However, there have been only minor improvements in AI predictors in terms of accuracy and the area under the receiver operating curve (AUC) over the last decade and the number of genes used had little influence on these indices. Furthermore, we highlight that different studies having the same goal obtain similar performance despite the fact they use different discriminating genes. This is surprising because genes related to the diagnosis or prognosis are expected to be tumor-specific and independent of selection methods and algorithms. The performance of these predictors will be better with the improvement in the learning methods, as the number of cases increases and by using different types of input data (e.g., non-coding RNAs, proteomic and metabolic). This will allow for more precise identification, classification and staging of cancerous lesions which will be less affected by interpathologist variability.

Side Effects of Curcumin: Epigenetic and Antiproliferative Implications for Normal Dermal Fibroblast and Breast Cancer Cells.

BACKGROUND: Curcumin is a yellow-orange pigment obtained from the plant Curcuma longa, which is known to exert beneficial effects in several diseases, including cancer. However, at high doses, it may produce toxic and carcinogenic effects in normal cells. In this context, we studied the effects of curcumin on normal human dermal fibroblast (HDF) cells and breast cancer cells (MCF7). METHODS: We used cellular viability and growth assays to evaluate the antiproliferative action of curcumin, analyzed the endogenous glutathione levels, conducted cell cycle, apoptosis, and necrosis analyses, and performed immunodetection of glutathionylated and acetylated H3 histones. RESULTS: We found that HDFs are more sensitive to curcumin treatment than MCF7 cells, resulting in pronounced arrest of cell cycle progression and higher levels of cellular death. In both cell types, the homeostasis of the redox cellular environment did not change after curcumin treatment; however, significant differences were observed in glutathione (GSH) levels and in S-glutathionylation of H3 histones. CONCLUSION: Curcumin administration can potentially confer benefits, but high doses may be toxic. Thus, its use as a dietary supplement or in cancer therapies has a double edge.

A Spectroscopic Study on Secondary Structure and Thermal Unfolding of the Plant Toxin Gelonin Confirms Some Typical Structural Characteristics and Unravels the Sequence of Thermal Unfolding Events.

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.

Glutathione compartmentalization and its role in glutathionylation and other regulatory processes of cellular pathways.

Glutathione is considered the major non-protein low molecular weight modulator of redox processes and the most important thiol reducing agent of the cell. The biosynthesis of glutathione occurs in the cytosol from its constituent amino acids, but this tripeptide is also present in the most important cellular districts, such as mitochondria, nucleus, and endoplasmic reticulum, thus playing a central role in several metabolic pathways and cytoprotection mechanisms. Indeed, glutathione is involved in the modulation of various cellular processes and, not by chance, it is a ubiquitous determinant for redox signaling, xenobiotic detoxification, and regulation of cell cycle and death programs. The balance between its concentration and redox state is due to a complex series of interactions between biosynthesis, utilization, degradation, and transport. All these factors are of great importance to understand the significance of cellular redox balance and its relationship with physiological responses and pathological conditions. The purpose of this review is to give an overview on glutathione cellular compartmentalization. Information on its subcellular distribution provides a deeper understanding of glutathione-dependent processes and reflects the importance of compartmentalization in the regulation of specific cellular pathways. © 2018 BioFactors, 45(2):152-168, 2019.

Protein-protein interactions of human glyoxalase II: findings of a reliable docking protocol.

Glyoxalase II (GlxII) is an antioxidant glutathione-dependent enzyme, which catalyzes the hydrolysis of S-d-lactoylglutathione to form d-lactic acid and glutathione (GSH). The last product is the most important thiol reducing agent present in all eukaryotic cells that have mitochondria and chloroplasts. It is generally known that GSH plays a crucial role not only in the cellular redox state but also in various cellular processes. One of them is protein S-glutathionylation, a process that can occur through an oxidation reaction of proteins' thiol groups by GSH. Changes in protein S-glutathionylation have been associated with a range of human diseases such as diabetes, cardiovascular and pulmonary diseases, neurodegenerative diseases and cancer. Within a major project aimed at elucidating the role of GlxII in the mechanism of S-glutathionylation, a reliable computational protocol consisting of a protein-protein docking approach followed by atomistic Molecular Dynamics (MD) simulations was developed and it was applied to the prediction of molecular associations between human GlxII (in the presence and absence of GSH) and some proteins that are known to be S-glutathionylated in vitro, such as actin, malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The computational results show a high propensity of GlxII to interact with actin and MDH through its active site and a high stability of the GlxII-protein systems when GSH is present. Moreover, close proximities of GSH with actin and MDH cysteine residues have been found, suggesting that GlxII could be able to perform protein S-glutathionylation by using the GSH molecule present in its catalytic site.

A possible S-glutathionylation of specific proteins by glyoxalase II: An in vitro and in silico study.

Glyoxalase II, the second of 2 enzymes in the glyoxalase system, is a hydroxyacylglutathione hydrolase that catalyses the hydrolysis of S-d-lactoylglutathione to form d-lactic acid and glutathione, which is released from the active site. The tripeptide glutathione is the major sulfhydryl antioxidant and has been shown to control several functions, including S-glutathionylation of proteins. S-Glutathionylation is a way for the cells to store reduced glutathione during oxidative stress, or to protect protein thiol groups from irreversible oxidation, and few enzymes involved in protein S-glutathionylation have been found to date. In this work, the enzyme glyoxalase II and its substrate S-d-lactoylglutathione were incubated with malate dehydrogenase or with actin, resulting in a glutathionylation reaction. Glyoxalase II was also submitted to docking studies. Computational data presented a high propensity of the enzyme to interact with malate dehydrogenase or actin through its catalytic site and further in silico investigation showed a high folding stability of glyoxalase II toward its own reaction product glutathione both protonated and unprotonated. This study suggests that glyoxalase II, through a specific interaction of its catalytic site with target proteins, could be able to perform a rapid and specific protein S-glutathionylation using its natural substrate S-d-lactoylglutathione. SIGNIFICANCE: This article reports for the first time a possible additional role of Glo2 that, after interacting with a target protein, is able to promote S-glutathionylation using its natural substrate SLG, a glutathione derived compound. In this perspective, Glo2 can play a new important regulatory role inS-glutathionylation, acquiring further significance in cellular post-translational modifications of proteins.

The thermal unfolding of the ribosome-inactivating protein saporin-S6 characterized by infrared spectroscopy.

Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein.

Characterization of thymoquinone binding to human α1-acid glycoprotein.

Thymoquinone (TQ) is the main bioactive component isolated from Nigella sativa essential oil and seeds and has been used for the treatment of inflammations, liver disorders, arthritis, and is of great importance as a promising therapeutic drug for different diseases including cancer. This paper reports the first experimental evidence on binding of TQ to human α(1)-acid glycoprotein (AGP), an important drug-binding glycoprotein in human plasma, which affects pharmacokinetic properties of various therapeutic agents. The interaction of TQ with AGP has been characterized by Fourier transform infrared (FTIR) and fluorescence spectroscopy, as well as by molecular docking experiments. FTIR spectroscopy showed that the binding of TQ to AGP slightly increases its thermal stability and shifts the existence of a molten globule-like state observed in a previous study to higher temperature. The binding constants K(a); the number of binding sites n; and the corresponding thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated through fluorescence spectroscopy. Fluorescence quenching experiments indicated that TQ binding involves hydrophobic interactions and to a lower extent hydrogen bonds, in agreement with molecular docking experiments. The data on binding ability of TQ to AGP represent basic information for the TQ pharmacokinetics such as drug metabolism and distribution in the body.

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.

Mink growth hormone structural-functional relationships: effects of renaturing and storage conditions.

Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.

A strategic fluorescence labeling of D-galactose/D-glucose-binding protein from Escherichia coli helps to shed light on the protein structural stability and dynamics.

The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.

Temperature-, SDS-, and pH-induced conformational changes in protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a dynamic simulation and fourier transform infrared spectroscopic study.

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.

The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure.

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.

Structure-activity relationship on fungal laccase from Rigidoporus lignosus: a Fourier-transform infrared spectroscopic study.

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse.

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.