Determination of in vivo disulfide-bonded proteins in Arabidopsis

Protein thiol-disulfide oxidoreduction plays an important role in redox regulation of cellular processes. Here we present a proteomic approach to visualize and map in vivo disulfide-bonded proteins in plants. A proteomic map of the disulfide-bonded proteins was achieved using 2D gel electrophoresis of Arabidopsis protein extract. Along with novel proteins identified as potentially redox regulated, we have also shown the feasibility of mapping some of the cysteines involved in the formation of disulfide bonds. This study presents an important tool for characterizing redox-regulated proteins.     

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.     

Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis

Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.     

Depletion of m-type thioredoxin impairs photosynthesis,carbon fixation,and oxidative stress in cyanobacteria

Thioredoxins (Trxs) are disulfide oxidoreductases that regulate many biological processes. The m-type thioredoxin (TrxA) is the only Trx present in all oxygenic photosynthetic organisms. Extensive biochemical and proteomic analyses have identified many TrxA target proteins in different photosynthetic organisms. However, the precise function of this essential protein in vivo is still poorly known. In this study, we generated a conditional Synechocystis sp. PCC 6803 mutant strain (STXA2) using an on-off promoter that is able to survive with only 2% of the TrxA level of the wild-type (WT) strain. STXA2 characterization revealed that TrxA depletion results in growth arrest and pronounced impairment of photosynthesis and the Calvin–Benson–Bassham (CBB) cycle. Analysis of the in vivo redox state of the bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase showed higher levels of oxidation that affected enzyme activity in STXA2. This result implies that TrxA-mediated redox regulation of the CBB cycle is conserved in both cyanobacteria and chloroplasts, although the targets have different evolutionary origins. The STXA2 strain also accumulated more reactive oxygen species and was more sensitive to oxidative stress than the WT. Analysis of the in vivo redox state of 2-Cys peroxiredoxin revealed full oxidation, corresponding with TrxA depletion. Overall, these results indicate that depletion of TrxA in STXA2 greatly alters the cellular redox state, interfering with essential processes such as photosynthetic machinery operativity, carbon assimilation, and oxidative stress response. The TrxA regulatory role appears to be conserved along the evolution of oxygenic photosynthetic organisms.

A cyanobacterial mutant strain with very low TrxA (m-type thioredoxin) content is unable to maintain cellular redox state, affecting the function of essential processes.     

Using quantitative redox proteomics to dissect the yeast redoxome

To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.     

Large‐Scale Analysis of Redox‐Sensitive Conditionally Disordered Protein Regions Reveals Their Widespread Nature and Key Roles in High‐Level Eukaryotic Processes

Recently developed quantitative redox proteomic studies enable the direct identification of redox‐sensing cysteine residues that regulate the functional behavior of target proteins in response to changing levels of reactive oxygen species. At the molecular level, redox regulation can directly modify the active sites of enzymes, although a growing number of examples indicate the importance of an additional underlying mechanism that involves conditionally disordered proteins. These proteins alter their functional behavior by undergoing a disorder‐to‐order transition in response to changing redox conditions. However, the extent to which this mechanism is used in various proteomes is currently unknown. Here, a recently developed sequence‐based prediction tool incorporated into the IUPred2A web server is used to estimate redox‐sensitive conditionally disordered regions at a large scale. It is shown that redox‐sensitive conditional disorder is fairly widespread in various proteomes and that its presence strongly correlates with the expansion of specific domains in multicellular organisms that largely rely on extra stability provided by disulfide bonds or zinc ion binding. The analyses of yeast redox proteomes and human disease data further underlie the significance of this phenomenon in the regulation of a wide range of biological processes, as well as its biomedical importance.     

Interspecies Comparison of the Bacterial Response to Allicin Reveals Species‐Specific Defense Strategies

Allicin, a broad‐spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity‐conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram‐negative species, protein synthesis of the majority of proteins is downregulated while the Gram‐positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy‐based assays further indicate that in B. subtilis cell wall integrity is impaired.     

Thioredoxin affinity chromatography: a useful method for further understanding the thioredoxin network

Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.     

Metabolic Changes in Klebsiella oxytoca in Response to Low Oxidoreduction Potential,as Revealed by Comparative Proteomic Profiling Integrated with Flux Balance Analysis

Oxidoreduction potential (ORP) is an important physiological parameter for biochemical production in anaerobic or microaerobic processes. However, the effect of ORP on cellular physiology remains largely unknown, which hampers the design of engineering strategies targeting proteins associated with ORP response. Here we characterized the effect of altering ORP in a 1,3-propanediol producer, Klebsiella oxytoca, by comparative proteomic profiling combined with flux balance analysis. Decreasing the extracellular ORP from −150 to −240 mV retarded cell growth and enhanced 1,3-propanediol production. Comparative proteomic analysis identified 61 differentially expressed proteins, mainly involved in carbohydrate catabolism, cellular constituent biosynthesis, and reductive stress response. A hypothetical oxidoreductase (HOR) that catalyzes 1,3-propanediol production was markedly upregulated, while proteins involved in biomass precursor synthesis were downregulated. As revealed by subsequent flux balance analysis, low ORP induced a metabolic shift from glycerol oxidation to reduction and rebalancing of redox and energy metabolism. From the integrated protein expression profiles and flux distributions, we can construct a rational analytic framework that elucidates how (facultative) anaerobes respond to extracellular ORP changes.     

Proteomic characterization of copper stress response in Cannabis sativa roots

Cannabis sativa is an annual herb with very high biomass and capability to absorb and accumulate heavy metals in roots and shoots; it is therefore a good candidate for phytoremediation of soils contaminated with metals. Copper is an essential micronutrient for all living organisms, it participates as an important redox component in cellular electron transport chains; but is extremely toxic to plants at high concentrations. The aim of this work was to investigate copper effects on the root proteome of C. sativa, whose genome is still unsequenced. Copper stress induced the suppression of two proteins, the down-regulation of seven proteins, while five proteins were up-regulated. The resulting differences in protein expression pattern were indicative of a plant adaptation to chronic stress and were directed to the reestablishment of the cellular and redox homeostasis.     

In the beginning, there was the cell: cellular homeostasis

In the past 5 years, the biomedical, scientific community has sequenced the genomes of several organisms (including Homo sapiens), has cloned entire organisms and has determine the molecular structures for several membrane proteins. These advances combined with the advances in technology enabling high-throughput drug screening, gene expression readout using DNA chips and evolving proteomic techniques, make it imperative that physiologist and biomedical professionals understand the basis of cellular function and homeostasis. The Cellular Homeostasis Refresher Course at Experimental Biology 2004 in Washington, DC, was designed to fulfill this need. The specific topics covered were 1) generation of membrane potential, 2) an update on cellular mechanisms of ion homeostasis, channels and transporters, and 3) cellular volume homeostasis, and regulation of intracellular pH.     

Biogenesis of lipid droplets--how cells get fatter

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.     

Redox Biology on the rise

Abstract Redox reactions are at the heart of bioenergetics, yet their biological role is not restricted to metabolism. One specific focus of contemporary Redox Biology is the study of how the folding, stability, activity, and interactivity of proteins are subject to redox control. Key questions pertain to the chemical nature of physiological redox changes and their exact location inside the cell, the nature and distribution of protein redox modifications, and their meaning for cellular physiology. In recent years, Redox Biology has developed novel methodological directions, for example, the proteomic profiling of protein redox modifications and the noninvasive monitoring of redox processes in vivo. These and other approaches allow asking new questions for which the answers are almost completely unknown. To stimulate exchange of technical knowledge and the appreciation of Redox Biology in general, the German Society for Biochemistry and Molecular Biology (GBM) recently founded a Study Group for Redox Biology.     

Heat shock proteins and hormesis in the diagnosis and treatment of neurodegenerative diseases

Modulation of endogenous cellular defense mechanisms via the vitagene system represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. The possibility of high-throughoutput screening using proteomic techniques, particularly redox proteomics, provide more comprehensive overview of the interaction of proteins, as well as the interplay among processes involved in neuroprotection. Here by introducing the hormetic dose response concept, the mechanistic foundations and applications to the field of neuroprotection, we discuss the emerging role of heat shock protein as prominent member of vitagene network in neuroprotection and redox proteomics as a tool for investigating redox modulation of stress responsive vitagenes. Hormetic mechanisms are reviewed as possibility of targeted therapeutic manipulation in a cell-, tissue- and/or pathway-specific manner at appropriate points in the neurodegenerative disease process.     

Plant Lipid Rafts: Fluctuat Nec Mergitur*

Lipid rafts in plasma membranes are hypothesized to play key roles in many cellular processes including signal transduction, membrane trafficking and entry of pathogens. We recently documented the biochemical characterization of lipid rafts, isolated as detergent-insoluble membranes, from Medicago truncatula root plasma membranes. We evidenced that the plant-specific lipid steryl-conjugates are among the main lipids of rafts together with free sterols and sphingolipids. An extensive proteomic analysis showed the presence of a specific set of proteins common to other lipid rafts, plus the presence of a redox system around a cytochrome b₅₆₁ not previously identified in lipid rafts of either plants or animals. Here, we discuss the similarities and differences between the lipids and proteins of plant and animal lipid rafts. Moreover we describe the potential biochemical functioning of the M. truncatula root lipid raft redox proteins and question whether they may play a physiological role in legume-symbiont interactions.Key Words: plasma membrane, Medicago, root, legume-Rhizobium symbiosis, redox, sterol, sphingolipid     

Biogenesis of lipid droplets – how cells get fatter

Abstract

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.