scholarly journals Proteome-wide identification of S-sulfenylated cysteines in Brassica napus

Author(s):  
Sidra Iqbal ◽  
Liangqian Yu ◽  
Yuting Zhang ◽  
Guofang Zhang ◽  
Usman Ali ◽  
...  

Deregulation of reduction-oxidation (redox) metabolism under environmental stresses results in enhanced production of intracellular reactive oxygen species (ROS), which ultimately leads to posttranslational modifications (PTMs) in structure and molecular function of responsive proteins. Redox PTMs are important mediators of cellular signalling and regulation and several proteomic approaches attempted to quantify them under various stresses in plants. We aimed to generate large-scale redox proteomics data in response to short-term salt stress in Brassica napus by analyzing reversible cysteine modification. We employed iodoTMT approach to analyze the redox proteome of Brassica napus seedlings under control and salt stress conditions. We identified 2,010 peptides in 1,017 proteins, of which 1,821 sites in 912 proteins had oxidative modification. The redox homeostasis of biology processes in chloroplast and cytoplasm were mainly affected and the modification levels of proteins involved in photosynthesis and glycolysis pathways were significantly changed. Two oxidatively modified proteins were selected from the candidates and their in vitro activity under oxidative stress was assayed and validated the findings of this proteomics study. This targeted approach should contribute to the understanding of redox-based molecular changes prevailing in Brassica napus proteome subjected to salt stress and the mechanism adopted to cope with it.

2020 ◽  
Vol 477 (19) ◽  
pp. 3673-3693
Author(s):  
Aleksandra Liszka ◽  
Regina Schimpf ◽  
Krupskaya Ivannova Cartuche Zaruma ◽  
Annika Buhr ◽  
Thorsten Seidel ◽  
...  

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2–C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.


Author(s):  
Daniel N. El Kodsi ◽  
Jacqueline M. Tokarew ◽  
Rajib Sengupta ◽  
Nathalie A. Lengacher ◽  
Andy C. Ng ◽  
...  

SUMMARYWe recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H2O2) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H2O2 levels were unchanged in adult prkn-/- brain, a parkin-dependent reduction of cytosolic H2O2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn-/- mice. Nevertheless, this rise in oxidative stress led to changes in mitochondrial enzyme activities and the metabolism of glutathione in cells and mammalian brain. In parkin’s absence reduced glutathione concentrations were increased including in human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H2O2 reduction and glutathione regeneration. These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance.


2020 ◽  
Vol 21 (18) ◽  
pp. 6647 ◽  
Author(s):  
Eloise Keeling ◽  
Annabelle J. Culling ◽  
David A. Johnston ◽  
David S. Chatelet ◽  
Anton Page ◽  
...  

Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.


Author(s):  
Derin Keskin ◽  
Brendan Marshall ◽  
David Munn ◽  
Andrew Mellor ◽  
Debra Gearhart

AbstractThe activity of indoleamine 2, 3-dioxygenase (IDO; E.C. 1.13.11.42) catalyzes the oxidative cleavage of tryptophan to form kynurenine. IDO activity consumes superoxide anions; therefore, we postulated that over-expression of IDO might mitigate superoxide-anion dependent, oxidative modification of cellular proteins in vitro. We prepared and characterized RAW 264.7 macrophages that were stably transfected with either an IDO expression vector or the control (empty) vector. We detected IDO mRNA, protein, and enzyme activity in the IDO-transfected macrophages, but not in the macrophages transfected with the empty vector. To generate superoxide anions in situ, we treated the IDO-and control-transfected cultures with xanthine or hypoxanthine, and then used ELISA methods to quantitate the relative levels of oxidatively modified proteins in total cell lysates. The levels of protein carbonyls were similar in IDO-transfected and vector-transfected macrophages; however, protein nitration was significantly less in IDO-transfected cells compared to control transfectants. In addition, steady-state levels of superoxide anions were significantly lower in the IDO-transfected cultures compared with control transfectants. Our results are consistent with the concept that, besides degrading tryptophan, IDO activity may protect cells from oxidative damage.


2020 ◽  
Author(s):  
Szymon W. Kmiecik ◽  
Katarzyna Drzewicka ◽  
Frauke Melchior ◽  
Matthias P. Mayer

AbstractThe heat shock response (HSR) is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Posttranslational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by SUMO (small ubiquitin-like modifier) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low level SUMOylation at other sites. None of the tested E3 SUMO ligases increased SUMOylation efficacy as compared to the level in the presence of high concentrations of the E2 Ubc9. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA-bound, and SUMOylation of Hsf1 does neither alter DNA binding affinity nor does it affect Hsc70 and DnaJB1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the HSR.


2021 ◽  
Vol 118 (32) ◽  
pp. e2108391118
Author(s):  
Yu Chen ◽  
Jens Nielsen

Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.


Author(s):  
J. Metuzals

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.


1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.


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