Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules

Nodulin-35, a protein specific to soybean root nodules, was purified under non-denaturing conditions (DEAE-cellulose followed by Sephacryl S-200 chromatography) to homogeneity. The holoprotein showed uricase (EC 1.7.3.3) activity. Analytical ultracentrifugation under non-denaturing conditions revealed a molecule of 124 kd, S°_20W = 8.1; however, under denaturing conditions a value of 33 kd, S°_20W = 1.9, was obtained. This indicated that nodulin-35 is the 33-kd subunit of a specific soybean root nodule uricase (uricase II) and that the enzyme contains four similar subunits. The native molecule contains ˜1.0 mol Cu²⁺ per mol, has an isoelectric point of ˜9.0 and a pH optimum for uricase activity at 9.5. Uricase activity found in young uninfected soybean roots is due to another form of enzyme (uricase I) which is of ˜190 kd, has maximum activity at pH 8.0 and does not contain any subunit corresponding in size to nodulin-35. Uricase I, also present in young infected roots, declines at a time when nodulin-35 appears. Monospecific antibodies prepared against uricase II (nodulin-35) showed no cross-reactivity. Uricase II was localized in the uninfected cells of the nodule tissue. These results are consistent with the concept that a nodule-specific ureide metabolism takes place in peroxisomes of uninfected cells, and suggest the participation of uricase II in this pathway.     

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.     

Soybean Nodule-Specific Uricase (Nodulin-35) Is Expressed and Assembled into a Functional Tetrameric Holoenzyme in Escherichia coli

A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent K_m values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase.     

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

Peroxisome biogenesis in the yeast Yarrowia lipolytica

Extensive perexisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis. A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast. The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glyco-sylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes. The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles. A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes. The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome. These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.     

Peroxisome biogenesis

Peroxisome biogenesis conceptually consists of the (a) formation of the peroxisomal membrane, (b) import of proteins into the peroxisomal matrix and (c) proliferation of the organelles. Combined genetic and biochemical approaches led to the identification of 25 PEX genes-encoding proteins required for the biogenesis of peroxisomes, so-called peroxins. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported into the organelle in an unknown fashion. The protein import into the peroxisomal matrix and the targeting and insertion of peroxisomal membrane proteins is performed by distinct machineries. At least three peroxins have been shown to be involved in the topogenesis of peroxisomal membrane proteins. Elaborate peroxin complexes form the machinery which in a concerted action of the components transports folded, even oligomeric matrix proteins across the peroxisomal membrane. The past decade has significantly improved our knowledge of the involvement of certain peroxins in the distinct steps of the import process, like cargo recognition, docking of cargo-receptor complexes to the peroxisomal membrane, translocation, and receptor recycling. This review summarizes our knowledge of the functional role the known peroxins play in the biogenesis and maintenance of peroxisomes. Ideas on the involvement of preperoxisomal structures in the biogenesis of the peroxisomal membrane are highlighted and special attention is paid to the concept of cargo protein aggregation as a presupposition for peroxisomal matrix protein import. Electronic Publication     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Immunogold localization of nodule-specific uricase in developing soybean root nodules

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurr's epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurr's. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.Abbreviations BSA bovine serum albumin - Da dalton - ER endoplasmic reticulum - IgG immunoglobulin G     

Unexpected diversity of ferredoxin-dependent thioredoxin reductases in cyanobacteria

Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.     

Peroxisome degradation in mammals

This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Biogenesis of peroxisomes in regenerating rat liver. I. Sequential changes of catalase and urate oxidase detected by ultrastructural cytochemistry

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".     

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.     

The Arabidopsis Chloroplast Division Protein DYNAMIN-RELATED PROTEIN5B Also Mediates Peroxisome Division

Peroxisomes are highly dynamic organelles involved in various metabolic pathways. The division of peroxisomes is regulated by factors such as the PEROXIN11 (PEX11) proteins that promote peroxisome elongation and the dynamin-related proteins (DRPs) and FISSION1 (FIS1) proteins that function together to mediate organelle fission. In Arabidopsis thaliana, DRP3A/DRP3B and FIS1A (BIGYIN)/FIS1B are two pairs of homologous proteins known to function in both peroxisomal and mitochondrial division. Here, we report that DRP5B, a DRP distantly related to the DRP3s and originally identified as a chloroplast division protein, also contributes to peroxisome division. DRP5B localizes to both peroxisomes and chloroplasts. Mutations in the DRP5B gene lead to peroxisome division defects and compromised peroxisome functions. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we further demonstrate that DRP5B can interact or form a complex with itself and with DRP3A, DRP3B, FIS1A, and most of the Arabidopsis PEX11 isoforms. Our data suggest that, in contrast with DRP3A and DRP3B, whose orthologs exist across plant, fungal, and animal kingdoms, DRP5B is a plant/algal invention to facilitate the division of their organelles (i.e., chloroplasts and peroxisomes). In addition, our results support the notion that proteins involved in the early (elongation) and late (fission) stages of peroxisome division may act cooperatively.     

Bacillus anthracis Thioredoxin Systems,Characterization and Role as Electron Donors for Ribonucleotide Reductase

Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.