Sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)), play a crucial role as signaling molecules in the establishment and functioning of the nitrogen-fixing legume-Rhizobium symbiosis. The regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Protein cysteine residues are known to be sensitive targets of H(2)O(2), in a posttranslational modification called sulfenylation. We trapped and identified sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis, by combining the use of chemical and genetic probes with mass spectrometry analysis. We identified 44 M. truncatula proteins sulfenylated in inoculated roots (two days post infection, 2dpi) and 65 such proteins in the functioning symbiotic organ, the nodule (four weeks post infection, 4wpi); 18 proteins were identified at both time points. However, the largest functional groups at 2dpi and 4wpi were different: redox state-linked proteins early in the interaction and proteins involved in amino-acid and carbohydrate metabolism in the nodule. Twenty proteins from S. meliloti, including some directly involved in nitrogen fixation, were also identified as sulfenylated. These results suggest that sulfenylation may regulate the activity of proteins playing major roles in the development and functioning of the symbiotic interaction.     

Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

Analysis of pupal head proteome and its alteration in diapausing pupae of Helicoverpa armigera

The proteomic approach has proven to be an useful tool for understanding insect diapause processes. Using 2D gel electrophoresis and matrix assisted laser/desorption ionization (MALDI) time of flight (TOF), we identified 24 proteins in the head of Helicoverpa armigera pupae with diverse functional characteristics, including cytoskeleton proteins, heat-shock proteins, insect development regulation factors, ATPases, proteins regulating signal pathway and enzymes involved in metabolism, etc. A proteomic comparison between nondiapausing and diapausing pupae revealed three proteins that were present only in nondiapausing pupae, and six proteins represented ≥2.0-fold or ≤0.5-fold changes. The differentially expressed proteins, including heat-shock protein 90, chitin deacetylase, alpha-tubulin and transitional endoplasmic reticulum ATPase, etc. were reported for the first time in H. armigera. Identification of these proteins will enable us to further characterize the regulated functions of diapause in this important species.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Differential Proteome Analysis: Two-Dimensional Nano-LC/MS of E. Coli Proteome Grown on Different Carbon Sources

Different sugars provided to bacteria as single sources of carbon and energy require the induction of different metabolic enzymes, transporters, and uptake systems in order to support growth and cell survival. Using a nano–high-performance liquid chromatography/mass spectrometry (nano-HPLC/MS) system we constructed comprehensive peptide maps for Escherichia coli grown with either lactose or glucose in minimal medium. Digested bacterial samples were separated in a two-dimensional manner by combining strong cation exchange (SCX) and reversed-phased (RP) chromatography. Peptides were eluted online to an iontrap MS instrument and further analyzed by tandem MS fragmentation. Bacterial proteins originating from the differing samples were analyzed by searching the Swiss Prot Database. Data are presented that show the ability to detect several hundred different proteins significantly expressed under both conditions. Several enzymes and binding proteins related to the lactose metabolism were only identified in the sample grown with this carbon source.     

Utility of Aspergillus niger citrate synthase promoter for heterologous expression

Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5′ upstream sequence (0.9 kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.     

Proteome insights into the symbiotic relationship between a captive colony of Nasutitermes corniger and its hindgut microbiome

We analyzed the metaproteome of the bacterial community resident in the hindgut paunch of the wood-feeding ‘higher'' termite (Nasutitermes) and identified 886 proteins, 197 of which have known enzymatic function. Using these enzymes, we reconstructed complete metabolic pathways revealing carbohydrate transport and metabolism, nitrogen fixation and assimilation, energy production, amino-acid synthesis and significant pyruvate ferredoxin/flavodoxin oxidoreductase protein redundancy. Our results suggest that the activity associated with these enzymes may have more of a role in the symbiotic relationship between the hindgut microbial community and its termite host than activities related to cellulose degradation.     

Isolation of two Locust protein targets of a protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

In entomopathogenic fungi, secretory protein phosphatases might function in the utilization of phosphoproteins from the environment. But if secreted into the host, secretory protein phosphatases might play a role in pathogenesis by dephosphorylation of host phosphoproteins. Our group purified a novel phosphatase from entomopathogenic fungi, Metarhizium anisopliae. The substrate specificity and inhibitor sensitivity indicate that the phosphatase is a protein tyrosine phosphatase (PTPase). In order to analyze the targets of the PTPase in Locusta migratoria hemolymph, two-dimensional electrophoresis and mass spectrometry were used. The results indicated that the PTPase could specifically dephosphorylate two phosphoproteins from L. migratoria hemolymph. One phosphoprotein was identified as trans-Golgi p230. Previous studies have shown that trans-Golgi p230 participates in vesicular transport of functional proteins from the distal Golgi compartment. trans-Golgi p230 can be inactivated by dephosphorylation, which implies that M. anisopliae could interfere with the correct transportation of functional proteins by secreting extracellular PTPase into the hemolymph. There are some secretion proteins, such as transferrin, have been thought to participate in the insect innate immune against microbial infection, therefor M. anisopliae could interfere with immune defenses of L. migratoria by secreting extracellular PTPase into the hemolymph.     

The MT-ND1 and MT-ND5 genes are mutational hotspots for Chinese families with clinical features of LHON but lacking the three primary mutations

LHON is one of the most common and primary causes of acute blindness in young male adults. Over 95% of LHON cases are caused by one of the three primary mutations (m.11778G>A, m.14484T>C, and m.3460G>A). In contrast to these genetically diagnosed LHON patients, there are many patients with clinical features of LHON but without the three primary mutations, and these patients have been insufficiently analyzed. We reported 10 suspected Chinese LHON families without the three primary mutations. The overall penetrance (53.4%) in these families is significantly higher than in those families with m.11778G>A (33.3%) or m.3460G>A (25.6%). Complete mtDNA genome sequencing of the 10 families showed that they belonged to different haplogroups and all identified variants (excluding m.12332A>G in mt-tRNA^Leu) were previously reported. Eight of 12 private non-synonymous variants in the probands are located in the MT-ND1 and MT-ND5 genes, which is substantially higher than that of individuals from general Chinese populations. Comparison of the private variants in the 10 families and in 10 randomly selected mtDNAs from general Chinese populations using resampling simulation strategy further confirmed this pattern. Our results suggest that the MT-ND1 and MT-ND5 genes are mutational hotspots for Chinese families with suspected LHON lacking the common primary mutations. Variants m.3736G>A (p.V144I) in family Le1235 and m.10680G>A (p.A71T) in Le1107 can be the pathogenic mutations for LHON.     

Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology

Commercially, lettuce (Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.     

北长山岛森林乔木层碳储量及其影响因子

人工林是庙岛群岛典型的陆地生态系统的组成部分,对维护海岛地区生态环境具有重要作用。采用现有生物量相对生长方程和样地调查数据相结合的方法,以庙岛群岛中北长山岛为研究区,对海岛黑松纯林与黑松×刺槐混交林两种林型的碳储量进行了估算,并分析了土壤质地及其理化性质对海岛乔木层碳储量的影响。结果表明:黑松乔木层平均碳储量为84.00 t/hm²,接近于世界平均水平(86.00 t/hm²);黑松×刺槐混交林乔木层平均碳储量为29.60 t/hm²,高于山东省乔木层的平均碳储量(27.62 t/hm²)。应用因子分析法研究影响乔木层碳储量的主要因子,结果表明:土壤质地、pH值、含水量及含盐量是影响海岛乔木碳储量重要的影响因子。北长山岛土壤全氮、总磷、土壤有机质、碳氮比等其他理化性质对乔木层碳储量影响不是非常明显。     

Biochemical characterization and structure–function relationship of two plant NCS2 proteins,the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent K_M values were determined with 10.12 μM, 4.85 μM and 19.95 μM, respectively for NAT3 and 1.74 μM, 2.44 μM and 29.83 μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton–nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1–7 and TM8–14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure–function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.     

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.