The Writers, Readers, and Erasers in Redox Regulation of GAPDH.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme, which is crucial for the breakdown of glucose to provide cellular energy. Over the past decade, GAPDH has been reported to be one of the most prominent cellular targets of post-translational modifications (PTMs), which divert GAPDH toward different non-glycolytic functions. Hence, it is termed a moonlighting protein. During metabolic and oxidative stress, GAPDH is a target of different oxidative PTMs (oxPTM), e.g., sulfenylation, S-thiolation, nitrosylation, and sulfhydration. These modifications alter the enzyme's conformation, subcellular localization, and regulatory interactions with downstream partners, which impact its glycolytic and non-glycolytic functions. In this review, we discuss the redox regulation of GAPDH by different redox writers, which introduce the oxPTM code on GAPDH to instruct a redox response; the GAPDH readers, which decipher the oxPTM code through regulatory interactions and coordinate cellular response via the formation of multi-enzyme signaling complexes; and the redox erasers, which are the reducing systems that regenerate the GAPDH catalytic activity. Human pathologies associated with the oxidation-induced dysregulation of GAPDH are also discussed, featuring the importance of the redox regulation of GAPDH in neurodegeneration and metabolic disorders

Kinetically Inhibited Order in a Diamond-Lattice Antiferromagnet

Frustrated magnetic systems exhibit highly degenerate ground states and strong fluctuations, often leading to new physics. An intriguing example of current interest is the antiferromagnet on a diamond lattice, realized physically in A-site spinel materials. This is a prototypical system in three dimensions where frustration arises from competing interactions rather than purely geometric constraints, and theory suggests the possibility of unusual magnetic order at low temperature. Here we present a comprehensive single-crystal neutron scattering study of CoAl2O4, a highly frustrated A-site spinel. We observe strong diffuse scattering that peaks at wavevectors associated with Neel ordering. Below the temperature T*=6.5 K, there is a dramatic change in the elastic scattering lineshape accompanied by the emergence of well-defined spin-wave excitations. T* had previously been associated with the onset of glassy behavior. Our new results suggest instead that T* signifies a first-order phase transition, but with true long-range order inhibited by the kinetic freezing of domain walls. This scenario might be expected to occur widely in frustrated systems containing first-order phase transitions and is a natural explanation for existing reports of anomalous glassy behavior in other materials.Comment: 40 pages, 9 figures, Introduction and discussion altered and expanded. Additional section and figure added to Supplementary Informatio

Regulation of the CoA Biosynthetic Complex Assembly in Mammalian Cells

Coenzyme A (CoA) is an essential cofactor present in all living cells. Under physiological conditions, CoA mainly functions to generate metabolically active CoA thioesters, which are indispensable for cellular metabolism, the regulation of gene expression, and the biosynthesis of neurotransmitters. When cells are exposed to oxidative or metabolic stress, CoA acts as an important cellular antioxidant that protects protein thiols from overoxidation, and this function is mediated by protein CoAlation. CoA and its derivatives are strictly maintained at levels controlled by nutrients, hormones, metabolites, and cellular stresses. Dysregulation of their biosynthesis and homeostasis has deleterious consequences and has been noted in a range of pathological conditions, including cancer, diabetes, Reye’s syndrome, cardiac hypertrophy, and neurodegeneration. The biochemistry of CoA biosynthesis, which involves five enzymatic steps, has been extensively studied. However, the existence of a CoA biosynthetic complex and the mode of its regulation in mammalian cells are unknown. In this study, we report the assembly of all five enzymes that drive CoA biosynthesis, in HEK293/Pank1β and A549 cells, using the in situ proximity ligation assay. Furthermore, we show that the association of CoA biosynthetic enzymes is strongly upregulated in response to serum starvation and oxidative stress, whereas insulin and growth factor signaling downregulate their assembly

Extensive Anti-CoA Immunostaining in Alzheimer’s Disease and Covalent Modification of Tau by a Key Cellular Metabolite Coenzyme A

Alzheimer’s disease (AD) is a neurodegenerative disorder, accounting for at least two-thirds of dementia cases. A combination of genetic, epigenetic and environmental triggers is widely accepted to be responsible for the onset and development of AD. Accumulating evidence shows that oxidative stress and dysregulation of energy metabolism play an important role in AD pathogenesis, leading to neuronal dysfunction and death. Redox-induced protein modifications have been reported in the brain of AD patients, indicating excessive oxidative damage. Coenzyme A (CoA) is essential for diverse metabolic pathways, regulation of gene expression and biosynthesis of neurotransmitters. Dysregulation of CoA biosynthesis in animal models and inborn mutations in human genes involved in the CoA biosynthetic pathway have been associated with neurodegeneration. Recent studies have uncovered the antioxidant function of CoA, involving covalent protein modification by this cofactor (CoAlation) in cellular response to oxidative or metabolic stress. Protein CoAlation has been shown to both modulate the activity of modified proteins and protect cysteine residues from irreversible overoxidation. In this study, immunohistochemistry analysis with highly specific anti-CoA monoclonal antibody was used to reveal protein CoAlation across numerous neurodegenerative diseases, which appeared particularly frequent in AD. Furthermore, protein CoAlation consistently co-localized with tau-positive neurofibrillary tangles, underpinning one of the key pathological hallmarks of AD. Double immunihistochemical staining with tau and CoA antibodies in AD brain tissue revealed co-localization of the two immunoreactive signals. Further, recombinant 2N3R and 2N4R tau isoforms were found to be CoAlated in vitro and the site of CoAlation mapped by mass spectrometry to conserved cysteine 322, located in the microtubule binding region. We also report the reversible H_{2}O_{2}-induced dimerization of recombinant 2N3R, which is inhibited by CoAlation. Moreover, CoAlation of transiently expressed 2N4R tau was observed in diamide-treated HEK293/Pank1β cells. Taken together, this study demonstrates for the first time extensive anti-CoA immunoreactivity in AD brain samples, which occurs in structures resembling neurofibrillary tangles and neuropil threads. Covalent modification of recombinant tau at cysteine 322 suggests that CoAlation may play an important role in protecting redox-sensitive tau cysteine from irreversible overoxidation and may modulate its acetyltransferase activity and functional interactions

Диференціація генотипів сосни звичайної за поліморфізмом довжини інтронів генів дефензинів

The search for highly informative DNA markers to support the breeding programs aimed at increasing the productivity and biological stability of forest stands is an urgent task, especially in the context of global climate change. In this paper, we first investigate a new type of genetic markers based on the intron length polymorphism (ILP) of defensin genes to determine their informativeness and promising use for the estimation of the potential resistance of pine genotypes to biological damage. In the course of our study we applied the following methods: total DNA isolation by STAB method, PCR amplification with specific primers to pine defensin genes and Heterobasidion annosum s. s., electrophoretic separation of PCR fragments on a polyacrylamide gel under nondenaturing conditions, and also data analysis using software GenAlEx 6.053 and Statistica 10. Having conducted the research, we can present the following results. To investigate the defensin genes polymorphism in pine trees, we used ILP markers. These markers were developed based on the nucleotide sequences of the exons of PsDef1-4 genes. Analysis of PCR fragments obtained after amplification of each pair of primers with genomic DNA of pine showed that only one pair of primers, which is specific to defensin 2 (IPL-PsDef2), forms a wide range of amplified products, indicating their promising use for genetic characterization of genotypes of Scots pine. To clarify the use of IPL-PsDef2 markers for the study of genetic polymorphism we analyzed 196 trees and revealed that the average PIC was 0.287. IPL-PsDef2 markers were used to analyze the different genotypes of Scots pine on the areas affected by root rot disease. Based on the results of cluster analysis, the samples were divided into two groups, which differ in resistance to the root sponge. In addition, we identified PCR fragments that are specific to each of these groups and can serve as markers for the evaluation of the resistance of pine genotypes to Heterobasidion annosum. Thus, our conclusion is as follows: genotyping of defensin genes loci of pine breeding material is promising for the development of environmentally friendly technologies in order to enhance the sustainability of improved genetic material of pine trees to biotic stress.Наведено результати дослідження генотипів сосни звичайної (Pinus sylvestris L.) із використанням молекулярних маркерів на підстаі генетичного поліморфізму довжини інтронів генів дефензинів PsDef1-4. Виявлено високу інформативність маркерів на підстаі інтрону гена PsDef2 (IPL-PsDef2) для дослідження внутрішньовидового поліморфізму в сосни звичайної. Встановлено межі осередка інфекції кореневої губки (Heterobasidion annosum (Fr.) Bref.) в сосновому насадженні за допомогою полімеразно-ланцюгової реакції (ПЛР) із використанням видоспецифічних праймерів. Проведено ДНК-профілювання модельних дерев способом електрофоретичного розділення ампліконів IPL-PsDef2 маркерів. За результатами поліморфізму довжини інтронів генів дефензинів здійснено кластеризацію генотипів сосни звичайної із використанням алгоритму UPGMA. на підстаі дендрограми виділено два кластери генотипів зі 100 % бутстреп підтримкою, які відрізняються за стійкістю до кореневої губки. Методом К-середніх визначено значущість кожного з ампліконів для диференціації генотипів сосни на групи. Виявлено амплікони, які можуть бути генетичними маркерами для оцінки потенційної стійкості генотипів сосни звичайної до кореневої губки. Отримані дані щодо поліморфізму локусів генів дефензинів, а також результати кластерного аналізу вказують на перспективність використання IPL-PsDef2 маркерів для генотипування сосни звичайної

Nerve excitability changes related to muscle weakness in chronic progressive external ophthalmoplegia

Objective: To explore potential spreading to peripheral nerves of the mitochondrial dysfunction in chronic progressive external ophthalmoplegia (CPEO) by assessing axonal excitability. Methods: CPEO patients (n = 13) with large size deletion of mitochondrial DNA and matching healthy controls (n = 22) were included in a case-control study. Muscle strength was quantified using MRC sum-score and used to define two groups of patients: CPEO-weak and CPEO-normal (normal strength). Nerve excitability properties of median motor axons were assessed with the TROND protocol and changes interpreted with the aid of a model. Results: Alterations of nerve excitability strongly correlated with scores of muscle strength. CPEO-weak displayed abnormal nerve excitability compared to CPEO-normal and healthy controls, with increased superexcitability and responses to hyperpolarizing current. Modeling indicated that the CPEO-weak recordings were best explained by an increase in the ‘Barrett-Barrett’ conductance across the myelin sheath. Conclusion: CPEO patients with skeletal weakness presented sub-clinical nerve excitability changes, which were not consistent with axonal membrane depolarization, but suggested Schwann cell involvement. Significance: This study provides new insights into the spreading of large size deletion of mitochondrial DNA to Schwann cells in CPEO patients

Using small molecules to facilitate exchange of bicarbonate and chloride anions across liposomal membranes

Bicarbonate is involved in a wide range of biological processes, which include respiration, regulation of intracellular pH and fertilization. In this study we use a combination of NMR spectroscopy and ion-selective electrode techniques to show that the natural product prodigiosin, a tripyrrolic molecule produced by microorganisms such as Streptomyces and Serratia, facilitates chloride/bicarbonate exchange (antiport) across liposomal membranes. Higher concentrations of simple synthetic molecules based on a 4,6-dihydroxyisophthalamide core are also shown to facilitate this antiport process. Although it is well known that proteins regulate Cl-/HCO3- exchange in cells, these results suggest that small molecules may also be able to regulate the concentration of these anions in biological systems

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

International audienceABSTRACT: BACKGROUND: Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. METHODS: Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. RESULTS: Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain. CONCLUSION: Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

The SECURE project – Stem canker of oilseed rape: : molecular methods and mathematical modelling to deploy durable resistance

N Evans et al, "The SECURE Project - Stem Canker of oilseed rape: Molecular methods and mathematical modeling to deploy durable resistance", in Vol 4 of the Proceedings of the 12th International Rapeseed Congress : Sustainable Development in Cruciferous Oilseed Crops Production, Wuhan, China, March 26 - 30, 2007. The proceedings are available online at: http://gcirc.org/intranet/irc-proceedings/12th-irc-wuhan-china-2007-vol-4.htmlModelling done during the SECURE project has demonstrated the dynamic nature of the interaction between phoma stem canker (Leptosphaeria maculans), the oilseed rape host (Brassica napus) and the environment. Experiments done with near-isogenic lines of L. maculans to investigate pathogen fitness support field data that suggest a positive effect of the avirulence allele AvrLm4 on pathogen fitness, and that the loss of this allele renders isolates less competitive under field conditions on cultivars without the resistance gene Rlm4. The highlight of molecular work was the cloning of AvrLm1 and AvrLm6. L. maculans is now one of the few fungal species for which two avirulence loci have been cloned. Subsequent research focused on understanding the function of AvrLm1 and AvrLm6 and on the analysis of sequences of virulent isolates to understand molecular evolution towards virulence. Isolates of L. maculans transformed with GFP and/or DsRed were used to follow growth of the fungus in B. napus near-isogenic-lines (NIL) with or without MX (Rlm6) resistance under different temperature and wetness conditions. The results greatly enhanced our knowledge of the infection process and the rate and extent of in planta growth on different cultivars. Conclusions from work to model durability of resistance have been tested under field conditions through a series of experiments to compare durability of resistance conferred by the major resistance gene Rlm6 alone in a susceptible background (EurolMX) or in a resistant background (DarmorMX) under recurrent selection over 4 growing seasons. A major priority of the project was knowledge transfer of results and recommendations to target audiences such as plant breeding companies and extension services. CETIOM developed a “diversification scheme” that encourages French growers to make an informed choice about the cultivars that are grown within the rotation based on the resistance genes carried by the individual cultivars. Use of such schemes, in association with survey data on the population structure of L. maculans at both national and European scales will provide opportunities for breeders and the industry to manage available B. napus resistance more effectively.Non peer reviewe