SFRR-E OP 4.1 [YIA]:
Expression signatures associated with oxidative stress sensitivity in 30 human cancer cell lines
Dr. Sander Bekeschus | Leibniz Institute for Plasma Science and Technology (INP) | Germany
Oxidative stress is described to have physiological, pathological, and therapeutic consequences in nearly all types of cancer. Yet, the expression signatures associated with oxidative stress sensitivity in cancer cells are understudied. To this end, we cultured more than 30 tumor cell lines and analyzed their basal expression on several levels: 1. Flow cytometry screening of 33 receptors and redox-related enzymes (e.g., catalase, NOS, NOX, MPO, aquaporins 1-10); 2. whole-genome gene expression transcriptomic analysis; 3. mass spectrometry and proteomics, including oxidative post-translational modifications (oxPTMs). In parallel to cell pellet collection, we exposed the cultured cell lines to oxidative stress induced via hydrogen peroxide, hypochlorous acid, or multi-ROS/RNS generating gas plasma technology at different concentrations. Subsequently, the metabolic activity was assessed, and for each cell line and oxidative stress-inducing agent, the IC25 was calculated to reflect oxidative eustress and distress elements. Next, these IC25 values were correlated with expression levels determined in flow cytometry, transcriptomic, and proteomic data to identify expression objects that correlate with oxidative stress sensitivity and resistance across the 30 cancer cell lines. For gas plasma treatment, NOX3 and AQP1 cell surface expression as well as cholesterol content and baseline metabolic activity correlated with exposure resistance. For hydrogen peroxide exposure, cell cycle-related genes but not antioxidant genes were associated with sensitivity to the treatment in the tumor cell lines. For hypochlorous acid exposure, proteins involved in cell migration and cytoskeleton correlated positively with resistance. Overall, gas plasma exposure with the atmospheric pressure argon plasma jet kINPen and hydrogen peroxide treatment correlated well, while hypochlorous acid did not. Expression assays and oxidative stress treatments were crossed analyzed to identify targets and pathways expressed in resting cells that predict their sensitivity (but not regulation) to subsequent oxidative stress induced by different agents.
SFRR-E OP 4.2:
The role of microRNA and oxidised microRNA-133 in muscle wasting during ageing and cachexia
Dr. Raúl Gonzalez Ojeda | National University of Ireland Galway, Galway | Ireland
miRNAs are short non-coding RNAs that play a central role in post-transcriptional gene regulation. microRNAs have been shown to play important roles in muscle development and homeostasis in adulthood. We hypothesized that microRNAs are oxidised and become pathological in muscle during ageing and cachexia, and this oxidation can alter their function. The aim of this study was to evaluate the therapeutic potential miR-133 overexpression and/or inhibition of oxidised miR-133 for muscle wasting through establishing changes in grip strength, muscle mass and fiber size in mice treated with miR-133 and/or antagomir to oxidised miR-133. Our data show that miR-133 and oxidised miR-133 inhibitor restore grip strength and lead to increase in fibre diameter in aged mice. Moreover, in adult mice, oxidised miR-133 led to increased abundance of mitochondrial complexes I and II in adult mice, mitochondrial biogenesis- and ER stress-related genes. In old mice, miR-133 and oxidised miR-133 inhibitor led to decrease in abundance of mitochondrial complexes I and V and modified ER stress-associated genes. Oxidised miRNAs were associated with an increase of senescence markers mainly in adult mice. Downregulation of muscle atrophy protein markers was detected with following oxo-miR-133 inhibitor treatment in aged mice. In cancer cachexia animal model, inhibitor to oxidised miR-133 led to a decrease in tumor weight in male and female mice. Cachectic male and female mice treated with inhibitor to oxidised miR-133 showed changes in p21 senescence marker, mitochondrial porin VDAC and p62 autophagy marker and mitochondrial complex I. Together, these data for the first time show functional consequences of microRNA oxidation in muscle and warrant further investigation in miR-133 overexpression and oxidised miR-133 inhibition therapeutic potential for caxechia and sarcopenia
SFRR-E OP 4.3 [YIA]:
Insights into the mitochondrial functions of glutaredoxin 2 in cellular and mouse models
Dr. Valeria Scalcon | University of Padova | Italy
Glutaredoxin 2 (Grx2) is a redox enzyme endowed with glutathionylation/de-glutathionylation activity, which can coordinate a [2Fe-2S] cluster in an enzymatically inactive holo-dimer. Different Grx2 splicing variants are present: the mitochondrial Grx2a, the nuclear Grx2b and the cytosolic Grx2c. Little is known about the different roles of these isoforms hence we studied the functions of Grx2a exploiting both cellular and animal models.
In HeLa cells, we observed that, following oxidative stress, mitochondrial Grx2 acts as a sensor of the cellular redox state and releases the [2Fe-2S] cluster promoting the consequent activation of cell death pathways. 
Basing on this result and to further explore the role of Grx2 in cancer, in collaboration with Dr. Marcus Conrad (MCD, Helmholtz Zentrum München) we generated Glrx2 knock out cell lines. The consequences of Grx2 deprivation on the cellular redox state, mitochondrial functions and the overall metabolism of cancer cells has been dissected especially in relation to the glutathionylation status of mitochondrial proteins, highlighting different consequences of Grx2 depletion in the various cell types.
In order to determine the importance of Grx2 in vivo, we also characterized a whole-body mitochondrial Grx2 depleted (mGD) mouse model. These animals become overweight and show increased plasmatic and hepatic lipid levels when fed a standard chow diet. In addition, mGD liver presents lower capacity of glycogen storage, changed expression of enzymes involved in lipid metabolism and dysfunctional mitochondria. Moreover, an altered glutathionylation pattern of mitochondrial proteins was associated to the observed phenotype leading us to the conclusion that the altered glutathionylation of mitochondrial proteins in Grx2 deficient mice affects the mitochondrial physiology especially in the liver, leading to the development of a phenotype resembling the metabolic associated fatty liver disease (MAFLD). 
 Scalcon V. et al. Metallomics. 2019; 11:1241-1251.
 Scalcon V. et al. Redox Biol. 2022; 51:102277.
SFRR-E OP 4.4:
Identification and quantification of nitration and oxidation sites in extracellular matrix from human coronary artery smooth muscle cells treated with peroxynitrous acid (ONOOH)
Shuqi Xu | University of Copenhagen | Denmark
Peroxynitrous acid (ONOOH) is a powerful oxidizing and nitrating agent generated at sites of inflammation by the diffusion-controlled reaction of nitric oxide (NO.) with superoxide radicals (O2.-). Extracellular matrix (ECM) proteins are major targets as these are highly abundant, react rapidly and poorly-protected by antioxidant defenses. The resulting modifications have been associated with multiple pathologies. Previous studies have examined specific ECM components, but data on native ECM targets is lacking. We hypothesized that LC-MS/MS methods could detect and map nitration/oxidation sites on intact ECM generated by human coronary artery smooth muscle cells. Cells, and associated ECM, were exposed to ONOOH (0, 50-, 500- and 5000 µM) before protein extraction, digestion to peptides, and analysis by LC-MS/MS. Nitration occurred on both cell and ECM species in a site-specific manner with modifications detected on multiple proteins. Some ECM proteins were modified to a greater extent (e.g. fibronectin and collagens) than others (laminins), and the extent of alteration increased with higher oxidant concentrations. Nitration occurred mainly at Tyr and Trp residues, with oxidation detected at Met and His. Met oxidation (to the sulfoxide) was facile, and detected even at the lowest ONOOH doses. Tyr nitration predominated over Trp nitration, with 23 Tyr, and only 1 Trp nitrated on fibronectin. The extent of modification at individual residues on individual ECM proteins was quantified, with some proteins showing both large numbers of sites, and high extents of modification (e.g. 20 sites in fibronectin with >10% conversion). The nitration of particular residues did not increase in a linear manner. These data indicate that LC-MS/MS is a powerful tool to unveil, in an unbiased manner, the sites and extents of protein modification in complex systems. The modified species may be useful biomarkers of ongoing inflammation and damage, as these may be released into plasma and urine.
SFRR-E OP 4.5:
SELENBP1 and SEMO-1: copper-dependent H2S -generating enzymes in humans and in the model organism C. elegans
Dr. Holger Steinbrenner | Friedrich-Schiller-Universität Jena, Institut für Ernährungswissenschaften | Germany
Depending on its concentration, hydrogen sulfide (H2S) may act as a toxin, a redox regulator triggering protein persulfidation, or a substrate for the respiratory chain. Recently, selenium-binding protein 1 (SELENBP1) has been identified as a novel H2S-producing enzyme in mammals, by means of its methanethiol oxidase (MTO) activity. To assess MTO-catalyzed H2S production, we developed a coupled assay that is based on in situ-generation of methanethiol through recombinant L-methionine gamma-lyase, and subsequent detection of H2S (1). Applying this assay, we detected a strong increase in MTO activity in cultured Caco-2 cells during differentiation from a proliferating to an enterocyte/colonocyte-like phenotype, occurring in parallel with elevated SELENBP1 expression. MTO activity of recombinant human SELENBP1 required the binding of copper ions but not of selenium. In the nematode Caenorhabditis elegans, we identified an ortholog of human SELENBP1, also showing MTO activity; we therefore named it SEMO-1 (SELENBP1 ortholog with MTO activity) (2). SEMO-1 is involved in the regulation of stress resistance and life span in C. elegans (2, 3). SELENBP1 and SEMO-1 apparently exhibit a similar enzymatic mechanism: MTO activity of both recombinant proteins was copper-dependent, and the introduction of point mutations previously reported to naturally occur in human SELENBP1, causing the loss of MTO activity, also abolished MTO activity of SEMO-1 (2). Supplementation of wild-type worms with copper chloride increased their methanethiol-derived H2S production, whereas a copper chelator attenuated C. elegans MTO activity; moreover, SEMO-1-deficient worms showed suppressed MTO activity. Taken together, SELENBP1 and SEMO-1 are copper-dependent H2S-producing enzymes that degrade methanethiol, a toxic product of sulfur metabolism of bacteria living in the mammalian gut as well as in the soil habitat of nematodes.
(1) Philipp et al., Redox Biol 43:101972, 2021
(2) Philipp et al., BioFactors 2022 (doi: 10.1002/biof.1836)
(3) Köhnlein et al., Redox Biol 28:101323, 2020