A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.     

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.     

硫氧还蛋白-过氧化物氧还蛋白与过氧化氢构成环路参与肿瘤的发生与发展

组织细胞可经过多种途径产生氧自由基（ROS）,而肿瘤组织由于多种应激因素会产生大量ROS,其中最重要的是过氧化氢（H2O2).H2O2对细胞发挥着致损伤及亚毒性信使的双重作用,作为信使其不仅参与调节正常细胞信号通路,重要的是促进肿瘤的发生及进展. ROS作为一种应激刺激信号激活细胞内的AP-1（activator protein 1）、Nrf-2（NF-E2-related factor 2）等核转录因子,活化后的AP-1、Nrf-2会结合到硫氧还蛋白（sulfiredoxin, SRX）基因启动子上游的调控序列,促进SRX基因的表达.SRX的表达上调则影响其下游的抗氧化蛋白,即特定亚型的过氧化物氧还蛋白（peroxiredoxin, PRX）的活性状态,最终使细胞内H2O2浓度受到调节. 由SRX-PRX轴与H2O2形成1个环路,通过调节H2O2含量来参与细胞众多信号通路.本文对H2O2、SRX及PRX各自的功能进行综述,还进一步探讨三者构成的信号环路对肿瘤的调控机制,从而了解该环路在肿瘤发生发展中所发挥的作用.     

Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM). However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).     

Type II peroxiredoxin C,a member of the peroxiredoxin family of Arabidopsis thaliana: its expression and activity in comparison with other peroxiredoxins

Plants express a set of peroxiredoxins (PRX) in a cell-, development- and stress-specific manner. In the genomic database of Arabidopsis thaliana, 10 putative prx genes were identified and could be ordered in the four subgroups: the 1-Cys prx, the 2-Cys prx, prx Q and type II prx. Based on sequence analysis, four PRX proteins appear to be targeted to the chloroplast, one PRX each to the nucleus and mitochondria, respectively. The other PRX proteins contain no apparent targeting address. The paper describes the features of the Arabidopsis PRX proteins and their putative function in plant cellular antioxidant metabolism. The expression of seven prx genes was compared with RT-PCR in dependence of leaf age and salinity stress. Emphasis is placed on the type II PRX C, which is shown to be highly active in decomposing H₂O₂ when investigated as heterologously expressed protein.     

A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.     

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic,calorimetric and computational molecular methods

Abstract

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.

Communicated by Ramaswamy H. Sarma     

Distribution of the stress-related anionic peroxidases in different cucumber organs

The distribution of the stress-related anionic peroxidase (srPRX) activity was investigated in various organs of cucumber (Cucumis sativus L. cv. Laura) during their development by activity staining and immunoblotting. In shoots, including cotyledon, leaf, stem and tendril, three stress-related peroxidase isoenzymes were present, particularly in old ones. The PRX1 was the only srPRX isoenzyme found in both young and old roots. As fruits became mature, srPRX activity increased dramatically and was particularly enriched in the external parts of the fruit. The PRX1 isoenzyme was highly accumulated in the course of seed germination, while the absence of other two srPRX isoenzymes (PRX2 and PRX3) was recorded. The possible function of the srPRX is discussed, with respect to this spatio-temporal distribution.     

江南卷柏Phi类谷胱甘肽S-转移酶的分子特性

谷胱甘肽S-转移酶(Glutathione S-transferase,GST)在帮助植物抵抗各种胁迫中发挥重要作用。该研究从江南卷柏Selaginella moellendorffii中克隆到两个Phi类GST基因,分别命名为Sm GSTF1和Sm GSTF2,两个基因均编码215个氨基酸残基的蛋白质。表达模式分析发现,这两个基因在江南卷柏根、茎和叶中均有表达。将这两个基因在大肠杆菌中诱导表达重组蛋白并纯化,酶学性质分析表明Sm GSTF1和Sm GSTF2对CDNB、NBD-Cl和NBC等3种底物都有活性。Sm GSTF1对Fluorodifen和Cum-OOH也有活性,而Sm GSTF2对它们没有活性。酶动力学分析表明Sm GSTF1和Sm GSTF2对GSH有较高的亲和力,而对CDNB的亲和力都相对较低。在不同p H及温度条件下对Sm GSTF1和Sm GSTF2重组蛋白进行活性测定,发现这两个蛋白在p H 7-8.5,45-55℃温度范围内有较高的催化活性。研究推测,Sm GSTF1和Sm GSTF2可能在江南卷柏的抗逆生理过程中有重要的作用。     

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide signaling in vitro, reverses hypotension in canine septic shock

Although hydrogen peroxide (H2O2) is a well-described reactive oxygen species that is known for its cytotoxic effects and associated tissue injury, H2O2 has recently been established as an important signaling molecule. We previously demonstrated that lysozyme (Lzm-S), a mediator of sepsis that is released from leukocytes, could produce vasodilation in a phenylephrine-constricted carotid artery preparation by H2O2 signaling. We found that Lzm-S could intrinsically generate H2O2 and that this generation activated H2O2-dependent pathways. In the present study, we used this carotid artery preparation as a bioassay to define those antioxidants that could inhibit Lzm-S's vasodilatory effect. We then determined whether this antioxidant could reverse the hypotension that developed in an Escherichia coli bacteremic model. Of the many antioxidants tested, we found that ethyl gallate (EG), a nonflavonoid phenolic compound, was favorable in inhibiting Lzm-S-induced vasodilation. In our E. coli model, we found that EG reversed the hypotension that developed in this model and attenuated end-organ dysfunction. By fluorometric H2O2 assay and electrochemical probe techniques, we showed that EG could scavenge H2O2 and that it could reduce H2O2 production in model systems. These results show that EG, an antioxidant that was found to scavenge H2O2 in vitro, was able to attenuate cardiovascular dysfunction in a canine in vivo preparation. Antioxidants such as EG may be useful in the treatment of hemodynamic deterioration in septic shock.     

On the role of the axial ligand in heme proteins: a theoretical study

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys相似文献   

Quantitative analysis of redox-sensitive proteome with DIGE and ICAT

Oxidative modifications of protein thiols are important mechanisms for regulating protein functions. The present study aimed to compare the relative effectiveness of two thiol-specific quantitative proteomic techniques, difference gel electrophoresis (DIGE) and isotope coded affinity tag (ICAT), for the discovery of redox-sensitive proteins in heart tissues. We found that these two methods were largely complementary; each could be used to reveal a set of unique redox-sensitive proteins. Some of these proteins are low-abundant signaling proteins and membrane proteins. From DIGE analysis, we found that both NF-kappaB-repressing protein and epoxide hydrolase were sensitive to H 2O 2 oxidation. In ICAT analysis, we found that specific cysteines within sacroplasmic endoplamic reticulum calcium ATPase 2 and voltage-dependent anion-selective channel protein 1 were sensitive to H 2O 2 oxidation. From these analyses, we conclude that both methods should be employed for proteome-wide studies, to maximize the possibility of identifying proteins containing redox-sensitive cysteinyl thiols in complex biological systems.     

Increase of yeast survival under oxidative stress by the expression of the laccase gene from Coprinellus congregatus

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress (H2O2) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.     

NADPH oxidase-dependent hydrogen peroxide production, induced by salinity stress, may be involved in the regulation of total calcium in roots of wheat

Hydrogen peroxide (H(2)O(2)) is often generated by cells and tissues under environmental stress. In this work, we provide evidence that plasma membrane (PM) NADPH oxidase-dependent H(2)O(2) production might act as an intermediate step in the NaCl-induced elevation of calcium (Ca) in roots of wheat. Remarkable increases in the content of total Ca were observed not only in roots exposed to NaCl but also in roots of seedlings exposed to exogenous H(2)O(2). In roots, H(2)O(2) production increased upon exposure to salt stress. PM vesicles were isolated from roots, and NADPH oxidase activity was determined by measuring superoxide anion (O(2)(-)) production. NADPH oxidase-dependent O(2)(-) production was 11.6nmolmg(-1)proteinmin(-1) in control vesicles, but 19.6nmol after NaCl treatment (24h), indicating that salt stress resulted in the activation of the PM NADPH oxidase. Furthermore, the NaCl-induced increase in total Ca was partially abolished by the addition of 150U/mL catalase (CAT), a H(2)O(2) scavenger, and also by 10microM diphenylane iodonium (DPI), a NADPH oxidase inhibitor. This data suggest that NADPH oxidase-dependent H(2)O(2) production might be involved in the modulation of the Ca content in wheat roots. In conclusion, our results show that salinity stress increases the total Ca content of wheat roots, which is partly due to PM NADPH oxidase-dependent H(2)O(2) generation.     

Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs.

Chloroplasts contain their own genetic system. Eighteen different split genes have been found among approximately 130 chloroplast genes from higher plants. However, little is known about the chloroplast splicing system. Mammalian heterogeneous nuclear ribonucleoproteins (hnRNP proteins) have been shown to be involved in splicing. We applied a purification procedure developed for HeLa cell hnRNP proteins, which uses a single-stranded DNA (ssDNA) affinity column, directly to the tobacco chloroplast lysate to isolate their chloroplast counterparts. Four proteins (mol. wt approximately 30 kd) bound strongly to the column. The amino-terminal sequences of three of them were determined and their cDNA clones were isolated from a tobacco leaf cDNA library. Sequence analysis of these clones revealed that all three proteins contain two ribonucleoprotein consensus sequences (RNP-CS), confirming their ribonucleoprotein (RNP) nature. The presence of putative transit peptides in their predicted protein sequences, and an in vitro import experiment confirmed they are located in the chloroplast. This is the first report of organellar proteins containing RNP-CS. In addition, these three chloroplast proteins have a very acidic amino-terminal domain, a novel feature among RNP proteins identified so far. They are expressed both in leaves and roots; their mRNA levels showed different light modulation in mature leaves. The three proteins might be involved in splicing and/or processing of chloroplast RNAs.