Cellular Redox Processes Research Articles

Absence of mitochondrial CX9C-CX10C protein Cox12 generates oxidative and nitrosative stress in Saccharomyces cerevisiae: Implication on cellular redox homeostasis

Mitochondrial intermembrane space (IMS) harbors a series of small, evolutionarily conserved redox-active cysteine-rich proteins. These proteins are essential for the functioning of cytochrome c oxidase, but their role in maintaining cellular redox processes is unknown. Here, we find out that in the absence of two such cysteine-rich Cx9C-Cx10C proteins, cytochrome c oxidase subunit 12 (Cox12) or cytochrome c oxidase assembly factor 6 (Coa6), Saccharomyces cerevisiae cells become sensitive under the oxidative and nitrosative stress. Interestingly, knockout of COX12 generates a significant amount of endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) as evidenced by FACS analysis. Moreover, cellular redox status, redox-active enzymes glutathione reductase, catalase, S-nitroso glutathione reductase, and protein nitration were significantly affected in Cox12 null cells. Further, we found that an overexpression of COX12 partially protects mitochondrial respiratory subunit Sdh2 under oxidative and nitrosative stress. Taken together, we provide proof of evidence that cysteine-rich proteins in the IMS dynamically control the cellular redox milieu and actively prevent reactive nitrogen and oxygen species generation.

Selection and Gene Duplication Associated With High-Elevation Diversification in Pristimantis, the Largest Terrestrial Vertebrate Genus.

The genus Pristimantis diversified in the tropical Andes mountains and is the most speciose genus of terrestrial vertebrates. Pristimantis are notable among frogs in that they thrive at high elevations (>2,000 m) and are direct developers without a tadpole stage. Despite their ecological significance, little is known about the genetic and physiological traits enabling their success. We conducted transcriptomic analysis on seven Pristimantis species sampled across elevations in the Ecuadorean Andes to explore three hypotheses for their success: (i) unique genes are under selection relative to all other frogs, (ii) common selection occurs across all direct developers, or (iii) common selection occurs across all high-elevation frog clades. Comparative analysis with 34 frog species revealed unique positive selection in Pristimantis genes related to aerobic respiration, hemostasis, signaling, cellular transportation of proteins and ions, and immunity. Additionally, we detected positive selection across all direct developers for genes associated with oxygenase activity and metal ion binding. While many genes under selection in Pristimantis were not positively selected in other high-elevation frog species, we identified some shared genes and pathways linked to lipid metabolism, innate immunity, and cellular redox processes. We observed more positive selection in duplicated- versus single-copy genes, while relaxed purifying selection was prevalent in single-copy genes. Notably, copy number of an innate immunity complement gene was positively correlated with Pristimantis species elevation. Our findings contribute novel insights into the genetic basis of adaptation in Pristimantis and provide a foundation for future studies on the evolutionary mechanisms leading to direct development and coping with high elevations.

Асбест әсерінен туындаған өкпе аурулары жасушалық механизмдеріндегі митохондрияның рөлі

Mitochondria are one of the main sources of reactive oxygen species (ROS) and therefore they are actively involved in the regulation of cellular redox processes and ROS signalling. Mitochondrial dysfunction plays a critical role in bioenergy metabolism and the pathogenesis of many lung diseases. The processes of mitochondrial oxidative phosphorylation and their cellular functions are specific to different cells and tissues and can be heterogeneous even within a single cell due to the existence of subpopulations of mitochondria with distinct functional and structural properties. Mitochondrial function can change in response to changes in cellular metabolism. Damage to mitochondrial DNA (mtDNA) causes mitochondrial dysfunction, including disruption of the electron transport chain and loss of mitochondrial membrane potential. In addition, damaged mtDNA also acts as a damage-associated molecular pattern that triggers inflammatory and immune responses. Asbestos causes various lung diseases, the cellular and molecular mechanisms of which are not fully understood. Asbestos fibers can induce the production of mitochondrial ROS in lung epithelial cells and macrophages. This review article examines the molecular mechanisms of mitochondrial functioning when exposed to asbestos, as well as the formation and damage of mtDNA, reactive oxygen species, the effect of asbestos on ultrastructural changes in lung tissue mitochondria, and the role of microRNA in the functional activity of mitochondria.

Murburn posttranslational modifications of proteins: Cellular redox processes and murzyme-mediated metabolo-proteomics.

Murburn concept constitutes the thesis that diffusible reactive species or DRS are obligatorily involved in routine metabolic and physiological activities. Murzymes are defined as biomolecules/proteins that generate/modulate/sustain/utilize DRS. Murburn posttranslational modifications (PTMs) result because murburn/murzyme functionalism is integral to cellular existence. Cells must incorporate the inherently stochastic nature of operations mediated by DRS. Due to the earlier/inertial stigmatic perception that DRS are mere agents of chaos, several such outcomes were either understood as deterministic modulations sponsored by house-keeping enzymes or deemed as unregulated nonenzymatic events resulting out of "oxidative stress". In the current review, I dispel the myths around DRS-functions, and undertake systematic parsing and analyses of murburn modifications of proteins. Although it is impossible to demarcate all PTMs into the classical or murburn modalities, telltale signs of the latter are evident from the relative inaccessibility of the locus, non-specificities and mechanistic details. It is pointed out that while many murburn PTMs may be harmless, some others could have deleterious or beneficial physiological implications. Some details of reversible/irreversible modifications of amino acid residues and cofactors that may be subjected to phosphorylation, halogenation, glycosylation, alkylation/acetylation, hydroxylation/oxidation, etc. are listed, along with citations of select proteins where such modifications have been reported. The contexts of these modifications and their significance in (patho)physiology/aging and therapy are also presented. With more balanced explorations and statistically verified data, a definitive understanding of normal versus pathological contexts of murburn modifications would be obtainable in the future.

Adult patient diagnosed with NADSYN1 associated congenital NAD deficiency and analysis of NAD levels to be published in: European Journal of Medical Genetics

IntroductionNicotinamide adenine dinucleotide (NAD) is an essential cosubstrate/coenzyme in multiple cellular redox processes and a substrate in several non-redox reactions. NADSYN1 encodes NAD synthetase 1, an enzyme in the NAD de novo synthesis pathway and the Preiss-Handler pathway, and biallelic pathogenic variants causes NAD deficiency associated with vertebral, cardiac, renal and limb defects. Szot et al. and Kortbawi et al. have reported a total of seven patients with NADSYN1 associated congenital NAD deficiency disorder with the oldest patient being seven years old. Patient dataWe present a male patient age 30 with a height of 130 cm and numerous skeletal malformations including segmentation defects of the spine, rib anomalies and unequal leg length as well as bilateral ptosis, cleft palate and asymmetric dysmorphic facial features. The patient underwent surgery for an aortic stenosis due to a bicuspid valve. No malformations of the kidneys or urinary tract were identified. ResultsTrio exome sequencing revealed a homozygous missense variant in NADSYN1 c.1717G > A (p.Ala573Thr). Both parents were unaffected carriers of the variant. Analysis of NAD levels showed that the patient had a lower NAD pool compared to his unaffected siblings. The NAD pool rose approximately 25% after supplementation with nicotinamide, a NAD precursor for the salvage pathway. ConclusionThe variant was previously reported in four patients and functional analyses by Szot et al. support the pathogenicity of the variant. We report an adult patient with NADSYN1 associated congenital NAD deficiency disorder and expand the phenotypic spectrum. We also present analysis of the NAD levels before and after supplementation with nicotinamide.

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow−/− BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow−/− BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow−/− BMDMs. Furthermore, Selenow−/− BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Reducing Chemotherapy-Induced DNA Damage via nAChR-Mediated Redox Reprograming-A New Mechanism for SCLC Chemoresistance Boosted by Nicotine.

Simple SummaryTobacco smoking is the dominant risk factor for lung cancer, particularly for small cell lung cancer (SCLC). Smoking is also associated with worse clinical outcomes of SCLC. For SCLC patients, platinum-based chemotherapy (cisplatin or carboplatin), in combination with etoposide, has been the first-line therapy for decades, even with the recent introduction of immunotherapy. One key limitation of such chemotherapy is the quick acquisition of drug resistance. Here, we found that nicotine and its main metabolite, cotinine, reduced the efficacy of chemotherapies to SCLC cells and accelerated drug resistance, which could contribute to poorer survival rates in SCLC patients who continue to smoke.Up to 60% of patients with small cell lung cancer (SCLC) continue to smoke, which is associated with worse clinical outcomes. Platinum-based chemotherapies, in combination with topoisomerase inhibitors, are first-line therapies for SCLC, with rapid chemoresistance as a major barrier. We provided evidence in this study that nicotine and its major metabolite, cotinine, at physiologically relevant concentrations, reduced the efficacy of platinum-based chemotherapies and facilitated chemoresistance in SCLC cells. Mechanistically, nicotine or cotinine reduced chemotherapy-induced DNA damage by modulating cellular redox processes, with nAChRs as the upstream targets. Surprisingly, cisplatin treatment alone also increased the levels of nAChRs in SCLC cells, which served as a self-defense mechanism against platinum-based therapies. These discoveries were confirmed in long-term in vitro and in vivo studies. Collectively, our results depicted a novel and clinically important mechanism of chemoresistance in SCLC treatment: nicotine exposure significantly compromises the efficacy of platinum-based chemotherapies in SCLC treatment by reducing therapy-induced DNA damage and accelerating chemoresistance acquisition. The results also emphasized the urgent need for tobacco cessation and the control of NRT use for SCLC management.

Influence of complex treatment on the functional state of the hepatobiliary system in patients with diabetes mellitus

Liver damage in diabetes mellitus is of particular interest, since this factor significantly affects the course of the disease, the level of compensation and prognosis of the underlying disease.
 The aim. To study the effect of complex treatment using ursosan on the functional state of the hepatobiliary system in patients with diabetes mellitus.
 Materials and methods. The study included 30 patients with type 1 diabetes mellitus and 48 patients with type 2 diabetes. According to the duration of diabetes, patients were divided into three subgroups: a) up to five years; b) 5–10 years; c) more than 10 years. The number of patients was: 1a group – 13, 1b group – 10, 1c group – 7 people; Group 2a – 23, 2b group – 13, 2c group – 10 people. The control group consisted of 23 apparently healthy people. Ursosan was prescribed at a dose of 10-12 mg per 1 kg of body per day for 6 months. Clinical laboratory and instrumental research methods were used to study the functional state of the liver and gallbladder.
 Research results. After the course of treatment with Ursosan, patients with diabetes mellitus showed normalization of protein, pigment, enzymatic metabolism and, to a lesser extent, lipid metabolism in the liver, which led to an improvement in cellular metabolism and redox processes, providing a stable course of diabetes.
 Conclusions. A 6-month course of treatment with Ursosan in diabetic patients promotes long-term diabetes compensation. Patients during treatment have a decrease in cytolysis syndrome indicators (alanine aminotransferase (AlAT), aspartate aminotransferase (AsAT), lactate dehydrogenesis (LDH)) by about 1.5 times in all 3 indicators compared to the indicators before treatment. The same trend was observed in terms of gamma-glutamine transferase (GGT) and alkaline phosphatase (ALP)

Oxidative and anti-oxidative responses to metal toxicity in an extremophilic alga (Cyanidium caldarium) and a neutrophilic alga (Chlamydomonas reinhardtii)

ABSTRACT Redox metabolism is integral to the functioning of photosynthetic organisms. The highly oxidative reactive oxygen species (ROS) are products of cellular redox processes and can cause major cellular damage. ROS are also produced in the face of environmental and cellular stress, including metal-induced oxidative stress. The degree of ROS production and the subsequent anti-oxidative response will vary according to the tolerance level of the species and the degree and type of stress it experiences. The extremophilic alga Cyanidium caldarium and neutrophilic alga Chlamydomonas reinhardtii have contrasting Zn tolerances, with the EC50 of the extremophile 128 times higher than that of the neutrophile. We examined the oxidative stress responses of these algae to zinc stress, using ROS production to establish the metal-induced oxidative state of the organisms. The anti-oxidative responses measured included total free-sulphydryl groups and total protein content as well as a heat shock protein 70 (HSP70) response assay to explore the role the stress protein HSP70 might play in the contrasting metal tolerance mechanisms in these two species. Results showed that the oxidative response in Cy. caldarium was dose-dependent, while in Ch. reinhardtii the oxidative response was more dependent on the time of exposure to Zn. Cyanidium caldarium managed to regain its base level of protein (i.e. that seen in naïve cells) upon acclimation (long-term exposure) to sub-toxic Zn levels. On the other hand, Ch. reinhardtii showed reduced total protein content upon acclimation. This raises questions about the different roles that ROS signalling might play in metal stress tolerance.

Redox Homeostasis and Prospects for Therapeutic Targeting in Neurodegenerative Disorders.

Reactive species, such as those of oxygen, nitrogen, and sulfur, are considered part of normal cellular metabolism and play significant roles that can impact several signaling processes in ways that lead to either cellular sustenance, protection, or damage. Cellular redox processes involve a balance in the production of reactive species (RS) and their removal because redox imbalance may facilitate oxidative damage. Physiologically, redox homeostasis is essential for the maintenance of many cellular processes. RS may serve as signaling molecules or cause oxidative cellular damage depending on the delicate equilibrium between RS production and their efficient removal through the use of enzymatic or nonenzymatic cellular mechanisms. Moreover, accumulating evidence suggests that redox imbalance plays a significant role in the progression of several neurodegenerative diseases. For example, studies have shown that redox imbalance in the brain mediates neurodegeneration and alters normal cytoprotective responses to stress. Therefore, this review describes redox homeostasis in neurodegenerative diseases with a focus on Alzheimer's and Parkinson's disease. A clearer understanding of the redox-regulated processes in neurodegenerative disorders may afford opportunities for newer therapeutic strategies.

Can a Nonorganometallic Ruthenium(II) Polypyridylamine Complex Catalyze Hydride Transfer? Mechanistic Insight from Solution Kinetics on the Reduction of Coenzyme NAD+ by Formate.

Application of organometallic ruthenium(II) arene complexes has been successful for the modulation of cellular redox processes via their interaction with species such as formate to control the NAD+/NADH balance in cells. Here we present the first evidence that similar effects can be reached with the application of a nonorganometallic ruthenium(II) polypyridyl complex. Kinetic studies performed demonstrate the ability of [RuII(terpy)(en)(H2O/EtOH)]2+ in water/ethanol (1:9, v/v) solution, where terpy = 2,2':6',2″-terpyridine and en = ethylenediamine, to catalyze the reduction of the NAD+ coenzyme to NADH in the presence of formate as hydride transfer source. In this case, terpy instead of arene is responsible for the labilization of coordinated solvent. The suggested catalytic cycle begins with the fast anation of the [RuII(terpy)(en)(H2O/EtOH)]2+ complex by formate. This is followed by the rate-determining formate-catalyzed decarboxylation of the generated ruthenium(II) formato complex to form [RuII(terpy)(en)H]+. Rapid hydride transfer to NAD+ from [RuII(terpy)(en)H]+ to form NADH and to regenerate the starting ruthenium(II) solvato complex, closes the overall catalytic cycle.

Nanoscale Dynamic Readout of a Chemical Redox Process Using Radicals Coupled with Nitrogen-Vacancy Centers in Nanodiamonds.

Biocompatible nanoscale probes for sensitive detection of paramagnetic species and molecules associated with their (bio)chemical transformations would provide a desirable tool for a better understanding of cellular redox processes. Here, we describe an analytical tool based on quantum sensing techniques. We magnetically coupled negatively charged nitrogen-vacancy (NV) centers in nanodiamonds (NDs) with nitroxide radicals present in a bioinert polymer coating of the NDs. We demonstrated that the T1 spin relaxation time of the NV centers is very sensitive to the number of nitroxide radicals, with a resolution down to ∼10 spins per ND (detection of approximately 10-23 mol in a localized volume). The detection is based on T1 shortening upon the radical attachment, and we propose a theoretical model describing this phenomenon. We further show that this colloidally stable, water-soluble system can be used dynamically for spatiotemporal readout of a redox chemical process (oxidation of ascorbic acid) occurring near the ND surface in an aqueous environment under ambient conditions.

Iron‐Catalysed Radical Polymerisation by Living Bacteria

AbstractThe ability to harness cellular redox processes for abiotic synthesis might allow the preparation of engineered hybrid living systems. Towards this goal we describe a new bacteria‐mediated iron‐catalysed reversible deactivation radical polymerisation (RDRP), with a range of metal‐chelating agents and monomers that can be used under ambient conditions with a bacterial redox initiation step to generate polymers. Cupriavidus metallidurans, Escherichia coli, and Clostridium sporogenes species were chosen for their redox enzyme systems and evaluated for their ability to induce polymer formation. Parameters including cell and catalyst concentration, initiator species, and monomer type were investigated. Water‐soluble synthetic polymers were produced in the presence of the bacteria with full preservation of cell viability. This method provides a means by which bacterial redox systems can be exploited to generate “unnatural” polymers in the presence of “host” cells, thus setting up the possibility of making natural–synthetic hybrid structures and conjugates.

Iron-Catalysed Radical Polymerisation by Living Bacteria.

The ability to harness cellular redox processes for abiotic synthesis might allow the preparation of engineered hybrid living systems. Towards this goal we describe a new bacteria-mediated iron-catalysed reversible deactivation radical polymerisation (RDRP), with a range of metal-chelating agents and monomers that can be used under ambient conditions with a bacterial redox initiation step to generate polymers. Cupriavidus metallidurans, Escherichia coli, and Clostridium sporogenes species were chosen for their redox enzyme systems and evaluated for their ability to induce polymer formation. Parameters including cell and catalyst concentration, initiator species, and monomer type were investigated. Water-soluble synthetic polymers were produced in the presence of the bacteria with full preservation of cell viability. This method provides a means by which bacterial redox systems can be exploited to generate "unnatural" polymers in the presence of "host" cells, thus setting up the possibility of making natural-synthetic hybrid structures and conjugates.

Mechanistic Insight of Na/K-ATPase Signaling and HO-1 into Models of Obesity and Nonalcoholic Steatohepatitis.

Obesity is a multifaceted pathophysiological condition that has been associated with lipid accumulation, adipocyte dysfunction, impaired mitochondrial biogenesis and an altered metabolic profile. Redox imbalance and excessive release of inflammatory mediators have been intricately linked in obesity-associated phenotypes. Hence, understanding the mechanisms of redox signaling pathways and molecular targets exacerbating oxidative stress is crucial in improving health outcomes. The activation of Na/K-ATPase/Src signaling, and its downstream pathways, by reactive oxygen species (ROS) has been recently implicated in obesity and subsequent nonalcoholic steatohepatitis (NASH), which causes further production of ROS creating an oxidant amplification loop. Apart from that, numerous studies have also characterized antioxidant properties of heme oxygenase 1 (HO-1), which is suppressed in an obese state. The induction of HO-1 restores cellular redox processes, which contributes to inhibition of the toxic milieu. The novelty of these independent mechanisms presents a unique opportunity to unravel their potential as molecular targets for redox regulation in obesity and NASH. The attenuation of oxidative stress, by understanding the underlying molecular mechanisms and associated mediators, with a targeted treatment modality may provide for improved therapeutic options to combat clinical disorders.

Redox Systems Biology: Harnessing the Sentinels of the Cysteine Redoxome.

Significance: Cellular redox processes are highly interconnected, yet not in equilibrium, and governed by a wide range of biochemical parameters. Technological advances continue refining how specific redox processes are regulated, but broad understanding of the dynamic interconnectivity between cellular redox modules remains limited. Systems biology investigates multiple components in complex environments and can provide integrative insights into the multifaceted cellular redox state. This review describes the state of the art in redox systems biology as well as provides an updated perspective and practical guide for harnessing thousands of cysteine sensors in the redoxome for multiparameter characterization of cellular redox networks. Recent Advances: Redox systems biology has been applied to genome-scale models and large public datasets, challenged common conceptions, and provided new insights that complement reductionist approaches. Advances in public knowledge and user-friendly tools for proteome-wide annotation of cysteine sentinels can now leverage cysteine redox proteomics datasets to provide spatial, functional, and protein structural information. Critical Issues: Careful consideration of available analytical approaches is needed to broadly characterize the systems-level properties of redox signaling networks and be experimentally feasible. The cysteine redoxome is an informative focal point since it integrates many aspects of redox biology. The mechanisms and redox modules governing cysteine redox regulation, cysteine oxidation assays, proteome-wide annotation of the biophysical and biochemical properties of individual cysteines, and their clinical application are discussed. Future Directions: Investigating the cysteine redoxome at a systems level will uncover new insights into the mechanisms of selectivity and context dependence of redox signaling networks.

Metaproteomics reveal that rapid perturbations in organic matter prioritize functional restructuring over taxonomy in western Arctic Ocean microbiomes

We examined metaproteome profiles from two Arctic microbiomes during 10-day shipboard incubations to directly track early functional and taxonomic responses to a simulated algal bloom and an oligotrophic control. Using a novel peptide-based enrichment analysis, significant changes (p-value < 0.01) in biological and molecular functions associated with carbon and nitrogen recycling were observed. Within the first day under both organic matter conditions, Bering Strait surface microbiomes increased protein synthesis, carbohydrate degradation, and cellular redox processes while decreasing C1 metabolism. Taxonomic assignments revealed that the core microbiome collectively responded to algal substrates by assimilating carbon before select taxa utilize and metabolize nitrogen intracellularly. Incubations of Chukchi Sea bottom water microbiomes showed similar, but delayed functional responses to identical treatments. Although 24 functional terms were shared between experimental treatments, the timing, and degree of the remaining responses were highly variable, showing that organic matter perturbation directs community functionality prior to alterations to the taxonomic distribution at the microbiome class level. The dynamic responses of these two oceanic microbial communities have important implications for timing and magnitude of responses to organic perturbations within the Arctic Ocean and how community-level functions may forecast biogeochemical gradients in oceans.

Diffusion and Transport of Reactive Species Across Cell Membranes.

This chapter includes an overview of the structure of cell membranes and a review of the permeability of membranes to biologically relevant oxygen and nitrogen reactive species, namely oxygen, singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical, nitric oxide, nitrogen dioxide, peroxynitrite and also hydrogen sulfide. Physical interactions of these species with cellular membranesare discussed extensively, but also their relevance to chemical reactions such as lipid peroxidation. Most of these species are involved in different cellular redox processes ranging from physiological pathways to damaging reactions against biomolecules. Cell membranes separate and compartmentalize different processes, inside or outside cells, and in different organelles within cells. The permeability of these membranes to reactive species varies according to the physicochemical properties of each molecule. Some of them, such as nitric oxide and oxygen, are small and hydrophobic and can traverse cellular membranes virtually unhindered. Nitrogen dioxide and hydrogen sulfide find a slightly higher barrier to permeation, but still their diffusion is largely unimpeded by cellular membranes. In contrast, the permeability of cellular membranes to the more polar hydrogen peroxide, is up to five orders of magnitude lower, allowing the formation of concentration gradients, directionality and effective compartmentalization of its actions which can be further regulated by specific aquaporins that facilitate its diffusion through membranes. The compartmentalizing effect on anionic species such as superoxide and peroxynitrite is even more accentuated because of the large energetic barrier that the hydrophobic interior of membranes presents to ions that may be overcome by protonation or the use of anion channels. The large difference in cell membrane permeability for different reactive species indicates that compartmentalization is possible for some but not all of them.

In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified ( n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD generating superoxide and a flavin radical that oxidize the isoalloxazine ring C7α methyl group and a nearby tyrosine residue. This tyrosine and key residues surrounding the oxygen pocket are conserved in enzymes from related bacteria, including pathogens such as Staphylococcus aureus. Photo-sensitivity may thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria.