Oxidative Post-translational Modifications Research Articles

Cysteine thiol-based post-translational modification: What do we know about transcription factors?

Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis inadopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Brachypodium distachyon Seedlings Display Accession-Specific Morphological and Transcriptomic Responses to the Microgravity Environment of the International Space Station.

Plants have been recognized as key components of bioregenerative life support systems for space exploration, and many experiments have been carried out to evaluate their adaptability to spaceflight. Unfortunately, few of these experiments have involved monocot plants, which constitute most of the crops used on Earth as sources of food, feed, and fiber. To better understand the ability of monocot plants to adapt to spaceflight, we germinated and grew Brachypodium distachyon seedlings of the Bd21, Bd21-3, and Gaz8 accessions in a customized growth unit on the International Space Station, along with 1-g ground controls. At the end of a 4-day growth period, seedling organ's growth and morphologies were quantified, and root and shoot transcriptomic profiles were investigated using RNA-seq. The roots of all three accessions grew more slowly and displayed longer root hairs under microgravity conditions relative to ground control. On the other hand, the shoots of Bd21-3 and Gaz-8 grew at similar rates between conditions, whereas those of Bd21 grew more slowly under microgravity. The three Brachypodium accessions displayed dramatically different transcriptomic responses to microgravity relative to ground controls, with the largest numbers of differentially expressed genes (DEGs) found in Gaz8 (4527), followed by Bd21 (1353) and Bd21-3 (570). Only 47 and six DEGs were shared between accessions for shoots and roots, respectively, including DEGs encoding wall-associated proteins and photosynthesis-related DEGs. Furthermore, DEGs associated with the "Oxidative Stress Response" GO group were up-regulated in the shoots and down-regulated in the roots of Bd21 and Gaz8, indicating that Brachypodium roots and shoots deploy distinct biological strategies to adapt to the microgravity environment. A comparative analysis of the Brachypodium oxidative-stress response DEGs with the Arabidopsis ROS wheel suggests a connection between retrograde signaling, light response, and decreased expression of photosynthesis-related genes in microgravity-exposed shoots. In Gaz8, DEGs were also found to preferentially associate with the "Plant Hormonal Signaling" and "MAP Kinase Signaling" KEGG pathways. Overall, these data indicate that Brachypodium distachyon seedlings exposed to the microgravity environment of ISS display accession- and organ-specific responses that involve oxidative stress response, wall remodeling, photosynthesis inhibition, expression regulation, ribosome biogenesis, and post-translational modifications. The general characteristics of these responses are similar to those displayed by microgravity-exposed Arabidopsis thaliana seedlings. However, organ- and accession-specific components of the response dramatically differ both within and between species. These results suggest a need to directly evaluate candidate-crop responses to microgravity to better understand their specific adaptability to this novel environment and develop cultivation strategies allowing them to strive during spaceflight.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) invitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Current State of Cold Atmospheric Plasma and Cancer-Immunity Cycle: Therapeutic Relevance and Overcoming Clinical Limitations Using Hydrogels.

Cold atmospheric plasma (CAP) is a partially ionized gas that gains attention as a well-tolerated cancer treatment that can enhance anti-tumor immune responses, which are important for durable therapeutic effects. This review offers a comprehensive and critical summary on the current understanding of mechanisms in which CAP can assist anti-tumor immunity: induction of immunogenic cell death, oxidative post-translational modifications of the tumor and its microenvironment, epigenetic regulation of aberrant gene expression, and enhancement of immune cell functions. This shouldprovide a rationale for the effective and meaningful clinical implementation of CAP. As discussed here, despite its potential, CAP faces different clinical limitations associated withthe current CAP treatment modalities: direct exposure of cancerous cells to plasma, and indirect treatment through injection of plasma-treated liquids in the tumor. To this end, a novel modality is proposed: plasma-treated hydrogels (PTHs) that can not only help overcome some of the clinical limitations but also offer a convenient platform for combining CAP with existing drugs to improve therapeutic responses and contribute to the clinical translation of CAP. Finally, by integrating expertise in biomaterials and plasma medicine, practical considerations and prospective for the development of PTHs are offered.

Redox proteomics and structural analyses provide insightful implications for additional non-catalytic thiol-disulfide motifs in PDIs

Protein disulfide isomerases (PDIs) catalyze redox reactions that reduce, oxidize, or isomerize disulfide bonds and act as chaperones of proteins as they fold. The characteristic features of PDIs are the presence of one or more catalytic thioredoxin (TRX)-like domains harboring typical CXXC catalytic motifs responsible for redox reactions, as well as non-catalytic TRX-like domain. As increasing attention is paid to oxidative post-translational modifications of cysteines (Cys ox-PTMs) with the recognition that they control cellular signaling, strategies to identify sites of Cys ox-PTM by redox proteomics have been optimized. Exploration of an available Cys redoxome dataset supported by modeled structure provided arguments for the existence of an additional non-catalytic thiol-disulfide motif, distinct from those contained in the TRX type patterns, typical of PDIAs. Further structural analysis of PDIA3 and 6 allows us to consider the possibility that this hypothesis could be extended to other members of PDI. These elements invite future studies to decipher the exact role of these non-catalytic thiol-disulfide motifs in the functions of PDIs. Strategies that would allow to validate this hypothesis are discussed.

Impact of Genetic and Post-Translational Modifications on Structure and Function of Fibrinogen

Introduction: Fibrinogen is a highly heterogeneous protein that is a result of several types of alteration, including genetic polymorphisms, alternative mRNA processing, posttranslational modifications, proteolytic cleavage and environmental factors. The different combinations of these alternations cause a formation of abnormal variants of fibrinogen that may affect the molecule. Fibrinogen is more vulnerable to oxidative stress than other plasmatic proteins. The oxidative post-translational modifications can be caused by different reactive oxygen species (ROS) and change the spatial structure, thus, impaired protein function. Here, we characterized 48 patients, belonging to 30 unrelated families, with fibrinogen mutation. This study aims to understand the behavior of these alternated fibrinogens in blood coagulation. Methods: We investigated these fibrinogens using a wide range of biochemical procedures, including fibrin polymerization, fibrinolysis, fibrinopeptides (Fps) release measurement, tandem mass spectrometry, thromboelastography, protein modeling, and scanning electron microscopy (SEM). Results: Dysfibrinogenemia was diagnosed in 35 cases, mostly in exon 2 of FGA gene, around thrombin cleavage site in N-termini of Aα chain. Hypofibrinogenemia was found in 13 remaining cases. Novel mutations were identified in 7 cases - heterozygous missense BβY416C and BβA68S, homozygous nonsense BβY345*, heterozygous nonsense BβW403*, heterozygous missense FGG c.8G>A, heterozygous missense γT34A, and heterozygous missense γG284E mutations. All patients had decreased levels of functional and/or plasmatic fibrinogen. Functional tests showed abnormal fibrin development in all patients with lower maximal absorbance. Moreover, fibrinopeptide cleavage measurements revealed differences between the release of Fps in patients with mutation in position close or in the binding site od thrombin. The results obtained from SEM were significantly different in fiber thicknesses or average number of fibers per 1µm2. Homology modeling showed the impaired protein structure of BβA68S and γG284E. Mass spectrometry detected several post-translational oxidations of fibrinogen. Conclusion: The results indicate that detected post-translational modifications, induced very likely by ROS, may affect the overall clottability of fibrinogen and clot morphology. Consequently, our study demonstrates that the identification, characterization, and modeling of novel fibrinogen variants brings new knowledge to fibrinogen domain structures and interactions that influence the assembly, secretion and function of this molecule. This work was supported by the project of the Ministry of Health, Czech Republic (00023736).

Abstract 12273: Crispr-Mutation of the Na + -k + Pump β1-Subunit Protects Against Cardiac Fibrosis and Heart Failure in Mice

Introduction: The Na+-K+ pump controls translocation of Na+ and K+ across the cell membrane which indirectly affects heart muscle contraction. Glutathionylation of the Na+-K+ pump’s β1 subunit (NKβ1) is a reversible post-translational oxidative modification, where a glutathione adduct forms a disulphide bond with the cysteine residue at site 45, leading to a reduction in pump activity. Using CRISPR/Cas9 technology, we created a transgenic mouse line (CRISPR-β1) that has a mutation in cysteine to serine at site 45 on NKβ1, causing insensitivity to β1-GSS. Hypothesis: We hypothesize that a mutation of the β1-GSS can inhibit transverse aortic constriction (TAC)-induced cardiac fibrosis. Methods and Results: TAC was induced in both the wild-type (WT) and CRISPR-NKβ1 mutated mice (CRISPR-β1) over a period of four weeks. Gene sequencing showed that cysteine 45 was replaced by serine, and immunoprecipitation of the free cysteine demonstrated that cysteine 45 was mutated in the β1 subunit of the CRISPR-β1 mice hearts. TAC significantly reduced cardiac function in the WT but not the CRISPR-β1 mice (ejection fraction: WT TAC: 36.24% ± 3.49%; CRISPR-β1 TAC: 57.5% ± 3.41%). Similarly, we found that the CRISPR-β1 mice were protected from cardiac hypertrophy, as evidenced by a significant reduction in cardiomyocytes size (WT TAC: 452.1 μm2 ± 30.74 μm2 ; CRISPR-β1 TAC: 271.1 μm 2 ± 11.0 μm 2 ). TAC significantly increased interstitial and perivascular fibrosis in the left ventricles of WT but not the CRISPR-β1 mice (interstitial: WT TAC: 18.4% ± 1.96%; CRISPR-β1 TAC: 5.2% ± 1.62%, perivascular: WT TAC: 42.6% ± 4.5%; CRISPR-β1 TAC: 22.01% ± 1.6%). Cardioprotective effects observed in the CRISPR-β1 mice were at least in part attributed to a significant reduction in the expression of ANP, BNP, Tgfb1, Fn1 and Ctgf genes. Conclusions: Our data indicate that CRISPR mutation of the Na+-K+ pump’s β1 subunit prevents the development of cardiac fibrosis and heart failure in a TAC-induced mouse model.

Photo-tuneable protein nitration by sensitiser tris(bipyridine)-Ruthenium(II) chloride complex

Post-translational modifications (PTMs) of proteins are a diverse source of variability that impacts on their functions, localisation, regulation, and lifetime. However, one of the main pitfalls in their study is that they appear in rather low frequencies and/or are only transiently observed. To overcome this issue and ease the study in vitro of stress-related protein PTMs, several methods have been proposed to model stress conditions and chemically introduce them. These techniques employ the combination of peroxides with transition metal ions or haem-containing proteins, as well as other possibilities such as peroxy radicals or UV radiation. However, their control, reproducibility and undesired secondary reactions that reduce the process yield are often a matter of concern. Here we introduce a photo-tuneable method that selectively targets nitration of aromatic residues. We initially present the adaptation of an oxidation method based on the photosensitiser tris(2,2′-bipyridine)-Ruthenium(II) chloride complex and ammonium persulfate, in which we employ an alternative radical neutralisation/trapping pathway that uses nitrite ions for the nitration of free l-Tyrosine and L-Tryptophan amino acids. After analysing the effect of several factors, we report the application of the photo-tuneable protein nitration (PTPN) method to four different model proteins in which we evaluate the nitration and oxidation of residues in each case. A mass spectrometry label-free quantitation of Tyr and Trp nitration is also described in order to compare the degree of modification and the accessibility of these residues. The method described could be employed to scale up the production of proteins with a selected range of oxidative PTMs for their characterisation, the assessment of their pathophysiological roles, and the development of detection and quantification methods to validate these PTMs as novel biomarkers associated with oxidative stress-related pathologies, such as in cardiovascular or neurodegenerative diseases.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes

Aims/hypothesisAntibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides.MethodsoxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs.ResultsWe observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12–21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11–30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21–30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies.Conclusions/interpretationOur findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.Graphical abstract

Prominent role of cysteine residues C49 and C343 in regulating Plasmodium falciparum pyruvate kinase activity

The protozoan parasite Plasmodium falciparum causes the most severe form of malaria and is highly dependent on glycolysis. Glycolytic enzymes were shown to be massively redox regulated, inter alia via oxidative post-translational modifications (oxPTMs) of their cysteine residues. In this study, we identified P. falciparum pyruvate kinase (PfPK) C49 and C343 as amino acid residues essentially involved in maintaining structural and functional integrity of the enzyme. The mutation of these cysteines resulted in an altered substrate affinity, lower enzymatic activities, and, as studied by X-ray crystallography, conformational changes within the A-domain where the substrate binding site is located. Although the loss of a cysteine evoked an impaired catalysis in both mutants, the effects observed for mutant C49A were more severe: multiple conformational changes, caused by the loss of two hydrogen bonds, impeded proper substrate binding and thus the transfer of phosphate upon catalysis.

Crosstalk between neuroinflammation and oxidative stress in epilepsy.

The roles of both neuroinflammation and oxidative stress in the pathophysiology of epilepsy have begun to receive considerable attention in recent years. However, these concepts are predominantly studied as separate entities despite the evidence that neuroinflammatory and redox-based signaling cascades have significant crosstalk. Oxidative post-translational modifications have been demonstrated to directly influence the function of key neuroinflammatory mediators. Neuroinflammation can further be controlled on the transcriptional level as the transcriptional regulators NF-KB and nrf2 are activated by reactive oxygen species. Further, neuroinflammation can induce the increased expression and activity of NADPH oxidase, leading to a highly oxidative environment. These factors additionally influence mitochondria function and the metabolic status of neurons and glia, which are already metabolically stressed in epilepsy. Given the implication of this relationship to disease pathology, this review explores the numerous mechanisms by which neuroinflammation and oxidative stress influence one another in the context of epilepsy. We further examine the efficacy of treatments targeting oxidative stress and redox regulation in animal and human epilepsies in the literature that warrant further investigation. Treatment approaches aimed at rectifying oxidative stress and aberrant redox signaling may enable control of neuroinflammation and improve patient outcomes.

Mitochondrial protein expression during sweet pepper (Capsicum annuum L.) fruit ripening: iTRAQ-based proteomic analysis and role of cytochrome c oxidase

The physiological process of fruit ripening is associated with the late developmental stages of plants in which mitochondrial organelles play an important role in the final success of this whole process. Thus, an isobaric tag for relative and absolute quantification (iTRAQ)-based analysis was used to quantify the mitochondrial proteome in pepper fruits in this study. Analysis of both green and red pepper fruits identified a total of 2284 proteins, of which 692 were found to be significantly more abundant in unripe green fruits as compared to red fruits, while 497 showed lower levels as the ripening process proceeded. Of the total number of proteins identified, 2253 (98,6%) were found to share orthologs with Arabidopsis thaliana. Proteomic analysis identified 163 proteins which were categorized as cell components, the major part assigned to cellular, intracellular space and other subcellular locations such as cytosol, plastids and, to a lesser extent, to mitochondria. Of the 224 mitochondrial proteins detected in pepper fruits, 78 and 48 were more abundant in green and red fruits, respectively. The majority of these proteins which displayed differential abundance in both fruit types were involved in the mitochondrial electron transport chain (mETC) and the tricarboxylic acid (TCA) cycle. The abundance levels of the proteins from both pathways were higher in green fruits, except for cytochrome c (CYC2), whose abundance was significantly higher in red fruits. We also investigated cytochrome c oxidase (COX) activity during pepper fruit ripening, as well as in the presence of molecules such as nitric oxide (NO) and hydrogen peroxide (H2O2), which promote thiol-based oxidative post-translational modifications (oxiPTMs). Thus, with the aid of in vitro assays, cytochrome c oxidase (COX) activity was found to be potentially inhibited by the PTMs nitration, S-nitrosation and carbonylation. According to protein abundance data, the final segment of the mETC appears to be a crucial locus with regard to fruit ripening, but also because in this location the biosynthesis of ascorbate, an antioxidant which plays a major role in the metabolism of pepper fruits, occurs.

NADPH oxidase 2 activity in Parkinson's disease

Mitochondrial dysfunction and oxidative stress are strongly implicated in Parkinson's disease (PD) pathogenesis and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2). Although NOX2 has been examined in the context of PD, most attention has focused on glial NOX2, and the role of neuronal NOX2 in PD remains to be defined. Additionally, pharmacological NOX2 inhibitors have typically lacked specificity. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and two animal models thereof, both neuronal and microglial NOX2 are highly active in substantia nigra under chronic conditions. However, in acute and sub-acute PD models, we observed neuronal, but not microglial NOX2 activation, suggesting that neuronal NOX2 may play a primary role in the early stages of the disease. Aberrant NOX2 activity is responsible for the formation of oxidative stress-related post-translational modifications of α-synuclein, and impaired mitochondrial protein import in vitro in primary ventral midbrain neuronal cultures and in vivo in nigrostriatal neurons in rats. In a rat model, administration of a brain-penetrant, highly specific NOX2 inhibitor prevented NOX2 activation in nigrostriatal neurons and its downstream effects in vivo, such as activation of leucine-rich repeat kinase 2 (LRRK2). We conclude that NOX2 is an important enzyme that contributes to progressive oxidative damage which in turn can lead to α-synuclein accumulation, mitochondrial protein import impairment, and LRRK2 activation. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.

A Computational Method for the Visualization of Nitrated Hsp90 Distribution in 3D Culture Models

Protein tyrosine (Y) nitration is an oxidative post‐translational modification that occurs in pathological conditions but is absent in normal tissue. We previously showed that nitrated proteins support tumor cell survival and migration, and recently identified the chaperone Heat Shock Protein 90 (Hsp90) as the first protein that, when nitrated, acquires a novel tumorigenic activity in glioblastoma multiforme (GBM). While Hsp90 is expressed in all cells, we showed that nitration of Hsp90 on Y33 and/or Y56 induces a pathological gain‐of‐function. Nitration on Y33 (Hsp90NY33) decreases mitochondrial metabolism by inhibiting complex IV activity, while nitration on Y56 (Hsp90NY56) activates the P2X7 receptor (P2X7R), which is linked to increased glycolysis in tumors. Together, these metabolic changes are characteristics of the Warburg effect, a metabolic phenotype common amongst tumors, suggesting that nitrated Hsp90 may play a role as a metabolic switch driving tumor progression. Here, we developed a computational approach to visualize the subcellular and spatial distribution of differentially nitrated Hsp90 in 3D cell culture models of GBM (tumoroids) using confocal Z‐Stack images. Our automated, high‐throughput method takes Z‐stack outputs and generates a 3D heatmap of a differentially localized protein of interest, normalized to a housekeeping fluorescence signal. These normalized heatmaps can be generated from any fluorescence image and serve as an unbiased visualization tool to use in conjunction with fluorescence microscopy images. GBM tumoroids were fluorescently labelled for Hsp90NY33or Hsp90NY56using specific and selective in‐house monoclonal antibodies, and for mitochondrial complex IV and P2X7R. DAPI was used as nuclear stain and tumoroids were imaged using confocal laser scanning microscopy. We observed a ~50% loss in fluorescence signal across the depth of tumoroid Z‐stacks, a common problem related to tissue penetration of laser light that leads to an underrepresented fluorescent signal at the core and bottom of imaged tumoroids. To overcome this problem, Python was used to analyze each focal plane within a confocal Z‐stack, normalizing the signal corresponding to the protein of interest by the mean DNA content (DAPI signal) at each focal plane. This normalized signal was used to generate a 3D heatmap of the protein of interest within the tumoroids. Method‐generated heatmaps confirmed the intracellular localization of Hsp90NY33in mitochondria and cytosol, and a distinct localization in the tumoroid periphery. In contrast, Hsp90NY56 was also localized in the nucleus, and was homogeneously distributed throughout the tumoroid. P2X7R showed a clear cell membrane localization, with increased expression in the tumoroid periphery. This suggests that Hsp90NY33may reduce aerobic respiration in the periphery of the tumoroid, allowing oxygen diffusion into the core, while Hsp90NY56 supports glycolysis throughout the tumoroid. Our results provide a novel fluorescence image analysis tool with potential to be expanded for several other 2D and 3D imaging modalities.

Effect of a Ketogenic Diet on Oxidative Posttranslational Protein Modifications and Brain Homogenate Denaturation in the Kindling Model of Epilepsy in Mice.

This study focused on the ketogenic diet (KD) effects on oxidative posttranslational protein modification (PPM) as presumptive factors implicated in epileptogenesis. A 28-day of KD treatment was performed. The corneal kindling model of epileptogenesis was used. Four groups of adult male ICR mice (25-30g) were randomized in standard rodent chow (SRC) group, KD-treatment group; SRC + kindling group; KD + kindling group (n = 10 each). Advanced oxidation protein products (AOPP) and protein carbonyl contents of brain homogenates together with differential scanning calorimetry (DSC) were evaluated. Two exothermic transitions (Exo1 and Exo2) were explored after deconvolution of the thermograms. Factor analysis was applied. The protective effect of KD in the kindling model was demonstrated with both decreased seizure score and increased seizure latency. KD significantly decreased glucose and increased ketone bodies (KB) in blood. Despite its antiseizure effect, the KD increased the AOPP level and the brain proteome's exothermic transitions, suggestive for qualitative modifications. The ratio of the two exothermic peaks (Exo2/Exo1) of the thermograms from the KD vs. SRC treated group differed more than twice (3.7 vs. 1.6). Kindling introduced the opposite effect, changing this ratio to 2.7 for the KD + kindling group. Kindling significantly increased glucose and KB in the blood whereas decreased the BW under the SRC treatment. Kindling decreased carbonyl proteins in the brain irrespectively of the diet. Further evaluations are needed to assess the nature of correspondence of calorimetric images of the brain homogenates with PPM.

Thiol-based Oxidative Posttranslational Modifications (OxiPTMs) of Plant Proteins.

The thiol group of cysteine (Cys) residues, often present in the active center of the protein, is of particular importance to protein function, which is significantly determined by the redox state of a protein’s environment. Our knowledge of different thiol-based oxidative posttranslational modifications (oxiPTMs), which compete for specific protein thiol groups, has increased over the last 10 years. The principal oxiPTMs include S-sulfenylation, S-glutathionylation, S-nitrosation, persulfidation, S-cyanylation and S-acylation. The role of each oxiPTM depends on the redox cellular state, which in turn depends on cellular homeostasis under either optimal or stressful conditions. Under such conditions, the metabolism of molecules such as glutathione, NADPH (reduced nicotinamide adenine dinucleotide phosphate), nitric oxide, hydrogen sulfide and hydrogen peroxide can be altered, exacerbated and, consequently, outside the cell’s control. This review provides a broad overview of these oxiPTMs under physiological and unfavorable conditions, which can regulate the function of target proteins.

Oxidation, Glycation, and Carbamylation of Salivary Biomolecules in Healthy Children, Adults, and the Elderly: Can Saliva Be Used in the Assessment of Aging?

BackgroundAging is inextricably linked to oxidative stress, inflammation, and posttranslational protein modifications. However, no studies evaluate oxidation, glycation, and carbamylation of salivary biomolecules as biomarkers of aging. Saliva collection is non-invasive, painless, and inexpensive, which are advantages over other biofluids.MethodsThe study enrolled 180 healthy subjects divided into six groups according to age: 6–13, 14–19, 20–39, 40–59, 60–79, and 80–100 years. The number of individuals was determined a priori based on our previous experiment (power of the test = 0.8; α = 0.05). Non-stimulated saliva and plasma were collected from participants, in which biomarkers of aging were determined by colorimetric, fluorometric, and ELISA methods.ResultsThe study have demonstrated that modifications of salivary proteins increase with age, as manifested by decreased total thiol levels and increased carbonyl groups, glycation (Nε-(carboxymethyl) lysine, advanced glycation end products (AGE)) and carbamylation (carbamyl-lysine) protein products in the saliva of old individuals. Oxidative modifications of lipids (4-hydroxynonenal) and nucleic acids (8-hydroxy-2’-deoxyguanosine (8-OHdG)) also increase with age. Salivary redox biomarkers correlate poorly with their plasma levels; however, salivary AGE and 8-OHdG generally reflect their blood concentrations. In the multivariate regression model, they are a predictor of aging and, in the receiver operating characteristic (ROC) analysis, significantly differentiate children and adolescents (under 15 years old) from the working-age population (15–64 years) and the older people (65 years and older).ConclusionSalivary AGE and 8-OHdG have the most excellent diagnostic utility in assessing the aging process. Saliva can be used to evaluate the aging of the body.

Oxidative post-translational modifications following valproic acid treatment are dependent upon cellular differentiative state

Oxidative post-translational modifications following valproic acid treatment are dependent upon cellular differentiative state