Search

In aerobic life forms, reactive oxygen species (ROS) are produced by the partial reduction of oxygen during energy-generating metabolic processes. In plants, ROS production increases during periods of both abiotic and biotic stress, severely overloading the antioxidant systems. Hydrogen peroxide (H2O2) plays a central role in cellular redox homeostasis and signaling by oxidising crucial cysteines to sulfenic acid, which is considered a biologically relevant post-translational modification (PTM). Until now, the impact of the nucleus on cellular redox homeostasis has been relatively unexplored. The regulation of histone-modifying enzymes by oxidative PTMs at redox-sensitive cysteine or tyrosine residues is particularly intriguing because it allows the integration of redox signaling mechanisms with chromatin control of transcriptional activity. One of the most extensively studied histone acetyltransferases is the conserved GENERAL CONTROL NONDEPRESSIBLE 5 (GCN5) complex. This study investigated the nuclear sulfenome in Arabidopsis thaliana by expressing a nuclear variant of the Yeast Activation Protein-1 (YAP1) probe and identified 225 potential redox-active proteins undergoing S-sulfenylation. Mass spectrometry analysis further confirmed the S-sulfenylation of GCN5 at cysteines 293, 368, and 400, and their functional significance and impact on the GCN5 protein-protein interaction network were assessed using cysteine-to-serine mutagenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Membrane interaction constitutes to be an essential parameter in the mode of action of entities such as proteins, as well as cell-penetrating and antimicrobial peptides, resulting in noninvasive or lytic activities depending on the membrane compositions and interactions. Recently, a nanobody able to interact with the top priority, multidrug-resistant bacterial pathogen Acinetobacter baumannii was discovered, although binding took place with fixed cells only. To potentially overcome this limitation, linear peptides corresponding to the complementarity-determining regions (CDR) were synthesized and fluorescently labeled. Microscopy data indicated clear membrane interactions of the CDR3 sequence with living A. baumannii cells, indicating both the importance of the CDR3 as part of the parent nanobody paratope and the improved binding ability and thus avoiding the need for permeabilization of the cells. In addition, cyclization of the peptide with an additionally introduced rigidifying 1,2,3-triazole bridge retains its binding ability while proteolytically protecting the peptide. Overall, this study resulted in the discovery of novel peptides binding a multidrug-resistant pathogen.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Anthrax is a highly resilient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis . The bacterium presents a complex and dynamic composition of its cell envelope, which changes in response to developmental and environmental conditions and host-dependent signals. Because of their easy to access extracellular locations, B. anthracis cell envelope components represent interesting targets for the identification and development of novel therapeutic and vaccine strategies. This review will focus on the novel insights regarding the composition, physiological role, and clinical relevance of B. anthracis cell envelope components.

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1-β2-β5), the immunoproteasome (β1i-β2i-β5i) and the two intermediate proteasomes (β1-β2-β5i and β1i-β2-β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three β5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, β5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three β5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

Cells can communicate through special "messages in the bottle", which are recorded in the bloodstream inside vesicles, namely exosomes. The exosomes are nanovesicles of 30-100 nm in diameter that carry functionally active biological material, such as proteins, messanger RNA (mRNAs), and micro RNA (miRNAs). Therefore, they are able to transfer specific signals from a parental cell of origin to the surrounding cells in the microenvironment and to distant organs through the circulatory and lymphatic stream. More and more interest is rising for the pathological role of exosomes produced by cancer cells and for their potential use in tumor monitoring and patient follow up. In particular, the exosomes could be an appropriate index of proliferation and cancer cell communication for monitoring the minimal residual disease, which cannot be easily detectable by common diagnostic and monitoring techniques. The lack of unequivocal markers for tumor-derived exosomes calls for new strategies for exosomes profile characterization aimed at the adoption of exosomes as an official tumor biomarker for tumor progression monitoring.

In 1989, Normark and coworkers reported on fibrous surface structures called curli on strains of Escherichia coli that were suspected of causing bovine mastitis. Subsequent work by many groups has revealed an elegant and highly regulated curli biogenesis pathway also referred to as the type VIII secretion system. Curli biogenesis is governed by two divergently transcribed operons, csgBAC and csgDEFG . The csgBAC operon encodes the structural subunits of curli, CsgA and CsgB, along with a chaperone-like protein, CsgC. The csgDEFG operon encodes the accessory proteins required for efficient transcription, secretion, and assembly of the curli fiber. CsgA and CsgB are secreted as largely unstructured proteins and transition to β-rich structures that aggregate into regular fibers at the cell surface. Since both of these proteins have been shown to be amyloidogenic in nature, the correct spatiotemporal synthesis of the curli fiber is of paramount importance for proper functioning and viability. Gram-negative bacteria have evolved an elegant machinery for the safe handling, secretion, and extracellular assembly of these amyloidogenic proteins.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Regulated intramembrane proteolysis by members of the site-2 protease family (S2P) is an essential signal transduction mechanism conserved from bacteria to humans. There is some evidence that extra-membranous domains, like PDZ and CBS domains, regulate the proteolytic activity of S2Ps and that some members act as dimers. Here we report the crystal structure of the regulatory CBS domain pair of S2P from Archaeoglobus fulgidus, AfS2P, in the apo and nucleotide-bound form in complex with a specific nanobody from llama. Cross-linking and SEC-MALS analyses show for the first time the dimeric architecture of AfS2P both in the membrane and in detergent micelles. The CBS domain pair dimer (CBS module) displays an unusual head-to-tail configuration and nucleotide binding triggers no major conformational changes in the magnesium-free state. In solution, MgATP drives monomerization of the CBS module. We propose a model of the so far unknown architecture of the transmembrane domain dimer and for a regulatory mechanism of AfS2P that involves the interaction of positively charged arginine residues located at the cytoplasmic face of the transmembrane domain with the negatively charged phosphate groups of ATP moieties bound to the CBS domain pairs. Binding of MgATP could promote opening of the CBS module to allow lateral access of the globular cytoplasmic part of the substrate., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H 2 O 2 ) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis . We conclude that dimedone can penetrate the cell wall, does not stress plants, and can "read" the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H 2 O 2 was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H 2 O 2 -stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants.

Next Generation Sequencing is dramatically increasing the number of known protein sequences, with related experimentally determined protein structures lagging behind. Structural bioinformatics is attempting to close this gap by developing approaches that predict structure-level characteristics for uncharacterized protein sequences, with most of the developed methods relying heavily on evolutionary information collected from homologous sequences. Here we show that there is a substantial observational selection bias in this approach: the predictions are validated on proteins with known structures from the PDB, but exactly for those proteins significantly more homologs are available compared to less studied sequences randomly extracted from Uniprot. Structural bioinformatics methods that were developed this way are thus likely to have over-estimated performances; we demonstrate this for two contact prediction methods, where performances drop up to 60% when taking into account a more realistic amount of evolutionary information. We provide a bias-free dataset for the validation for contact prediction methods called NOUMENON.

Respiratory complex I transfers electrons from NADH to quinone, utilizing the reaction energy to translocate protons across the membrane. It is a key enzyme of the respiratory chain of many prokaryotic and most eukaryotic organisms. The reversible NADH oxidation reaction is facilitated in complex I by non-covalently bound flavin mononucleotide (FMN). Here we report that the catalytic activity of E. coli complex I with artificial electron acceptors potassium ferricyanide (FeCy) and hexaamineruthenium (HAR) is significantly inhibited in the enzyme pre-reduced by NADH. Further, we demonstrate that the inhibition is caused by reversible dissociation of FMN. The binding constant (K d ) for FMN increases from the femto- or picomolar range in oxidized complex I to the nanomolar range in the NADH reduced enzyme, with an FMN dissociation time constant of ~5s. The oxidation state of complex I, rather than that of FMN, proved critical to the dissociation. Such dissociation is not observed with the T. thermophilus enzyme and our analysis suggests that the difference may be due to the unusually high redox potential of Fe-S cluster N1a in E. coli. It is possible that the enzyme attenuates ROS production in vivo by releasing FMN under highly reducing conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses., (© 2016 American Society of Plant Biologists. All rights reserved.)

An interesting asset of diagonal chromatography, which we have introduced for contemporary proteome research, is its high versatility concerning proteomic applications. Indeed, the peptide modification or sorting step that is required between consecutive peptide separations can easily be altered and thereby allows for the enrichment of specific, though different types of peptides. Here, we focus on the application of diagonal chromatography for the study of modifications of plant proteins. In particular, we show how diagonal chromatography allows for studying proteins processed by proteases, protein ubiquitination, and the oxidation of protein-bound methionines. We discuss the actual sorting steps needed for each of these applications and the obtained results. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In many established methods, identification of hydrogen bonds (H-bonds) is primarily based on pairwise comparison of distances between atoms. These methods often give rise to systematic errors when sulfur is involved. A more accurate method is the non-covalent interaction index, which determines the strength of the H-bonds based on the associated electron density and its gradient. We applied the NCI index on the active site of a single-cysteine peroxiredoxin. We found a different sulfur hydrogen-bonding network to that typically found by established methods, and we propose a more accurate equation for determining sulfur H-bonds based on geometrical criteria. This new algorithm will be implemented in the next release of the widely-used CHARMM program (version 41b), and will be particularly useful for analyzing water molecule-mediated H-bonds involving different atom types. Furthermore, based on the identification of the weakest sulfur-water H-bond, the location of hydrogen peroxide for the nucleophilic attack by the cysteine sulfur can be predicted. In general, current methods to determine H-bonds will need to be reevaluated, thereby leading to better understanding of the catalytic mechanisms in which sulfur chemistry is involved.

The genes coding for the core metabolic enzymes of the photorespiratory pathway that allows plants with C3-type photosynthesis to survive in an oxygen-rich atmosphere, have been largely discovered in genetic screens aimed to isolate mutants that are unviable under ambient air. As an exception, glycolate oxidase (GOX) mutants with a photorespiratory phenotype have not been described yet in C3 species. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the peroxisomal CATALASE2 (cat2-2) that display stunted growth and cell death lesions under ambient air, we isolated a second-site loss-of-function mutation in GLYCOLATE OXIDASE1 (GOX1) that attenuated the photorespiratory phenotype of cat2-2 Interestingly, knocking out the nearly identical GOX2 in the cat2-2 background did not affect the photorespiratory phenotype, indicating that GOX1 and GOX2 play distinct metabolic roles. We further investigated their individual functions in single gox1-1 and gox2-1 mutants and revealed that their phenotypes can be modulated by environmental conditions that increase the metabolic flux through the photorespiratory pathway. High light negatively affected the photosynthetic performance and growth of both gox1-1 and gox2-1 mutants, but the negative consequences of severe photorespiration were more pronounced in the absence of GOX1, which was accompanied with lesser ability to process glycolate. Taken together, our results point toward divergent functions of the two photorespiratory GOX isoforms in Arabidopsis and contribute to a better understanding of the photorespiratory pathway., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (-SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H2 O2 reduction, a sulfenic acid (-SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S-S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx-MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general., (© 2015 John Wiley & Sons Ltd.)

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In plants, fluctuation of the redox balance by altered levels of reactive oxygen species (ROS) can affect many aspects of cellular physiology. ROS homeostasis is governed by a diversified set of antioxidant systems. Perturbation of this homeostasis leads to transient or permanent changes in the redox status and is exploited by plants in different stress signalling mechanisms. Understanding how plants sense ROS and transduce these stimuli into downstream biological responses is still a major challenge. ROS can provoke reversible and irreversible modifications to proteins that act in diverse signalling pathways. These oxidative post-translational modifications (Ox-PTMs) lead to oxidative damage and/or trigger structural alterations in these target proteins. Characterization of the effect of individual Ox-PTMs on individual proteins is the key to a better understanding of how cells interpret the oxidative signals that arise from developmental cues and stress conditions. This review focuses on ROS-mediated Ox-PTMs on cysteine (Cys) residues. The Cys side chain, with its high nucleophilic capacity, appears to be the principle target of ROS. Ox-PTMs on Cys residues participate in various signalling cascades initiated by plant stress hormones. We review the mechanistic aspects and functional consequences of Cys Ox-PTMs on specific target proteins in view of stress signalling events., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Motivation: Cysteine residues have particular structural and functional relevance in proteins because of their ability to form covalent disulfide bonds. Bioinformatics tools that can accurately predict cysteine bonding states are already available, whereas it remains challenging to infer the disulfide connectivity pattern of unknown protein sequences. Improving accuracy in this area is highly relevant for the structural and functional annotation of proteins., Results: We predict the intra-chain disulfide bond connectivity patterns starting from known cysteine bonding states with an evolutionary-based unsupervised approach called Sephiroth that relies on high-quality alignments obtained with HHblits and is based on a coarse-grained cluster-based modelization of tandem cysteine mutations within a protein family. We compared our method with state-of-the-art unsupervised predictors and achieve a performance improvement of 25-27% while requiring an order of magnitude less of aligned homologous sequences (∼10(3) instead of ∼10(4))., Availability and Implementation: The software described in this article and the datasets used are available at http://ibsquare.be/sephiroth., Contact: wvranken@vub.ac.be, Supplementary Information: Supplementary material is available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

In this issue of Structure, Glatt and colleagues report the structure of the Kti11/Kti13 heterodimer. This study reveals how dimerization and Fe(2+) binding are required for modification of both tRNA and EF2, thus suggesting a mechanism for regulation of translation elongation via two different pathways., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1-like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼ 100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of dehydroascorbate reductase2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage.

The 2'-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2'-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level., (© 2014 Somme et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)