Comparative proteomics of dehydration response in the rice nucleus: New insights into the molecular basis of genotype‐specific adaptation

Dehydration is the most crucial environmental factor that considerably reduces the crop harvest index, and thus has become a concern for global agriculture. To better understand the role of nuclear proteins in water‐deficit condition, a nuclear proteome was developed from a dehydration‐sensitive rice cultivar IR‐64 followed by its comparison with that of a dehydration‐tolerant c.v. Rasi. The 2DE protein profiling of c.v. IR‐64 coupled with MS/MS analysis led to the identification of 93 dehydration‐responsive proteins (DRPs). Among those identified proteins, 78 were predicted to be destined to the nucleus, accounting for more than 80% of the dataset. While the detected number of protein spots in c.v. IR‐64 was higher when compared with that of Rasi, the number of DRPs was found to be less. Fifty‐seven percent of the DRPs were found to be common to both sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. Further, we constructed a functional association network of the DRPs of c.v. IR‐64, which suggests that a significant number of the proteins are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress‐responsive signaling and the molecular basis of dehydration tolerance in plants.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation‐induced DNA damage

The nucleoids of radiation‐resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid‐enriched fractions from unirradiated and irradiated Deinococcus were identified and semiquantified by shotgun proteomics. The ssDNA‐binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament‐like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post‐irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196 ( http://proteomecentral.proteomexchange.org/dataset/PXD000196 ).     

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50 μg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 μg/L Cu. Differential soluble proteome profiling for each strain exposed to sub‐lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter‐strain comparison of stress‐related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn‐stabilizing protein and a Fucoxanthine chlorophyll a–c binding protein. Es524 also expressed specific stress‐related enzymes such as RNA helicases from the DEAD box families and a vanadium‐dependent bromoperoxidase. These observations were supported by RT‐qPCR for some of the identified genes and an enzyme activity assay for vanadium‐dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.     

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

iTRAQ‐based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies

Male and female poplars (Populus cathayana Rehd.) respond differently to nitrogen (N) and phosphorus (P) deficiencies. In this study, an iTRAQ‐based quantitative proteomic analysis was performed. N and P deficiencies caused 189 and 144 proteins to change in abundance in males and 244 and 464 in females, respectively. Compared to N‐ and P‐deficient males, both N‐ and P‐deficient females showed a wider range of changes in proteins that are involved in amino acid, carbohydrate and protein metabolism, and the sexual differences were significant. When comparing the effects of N‐ and P‐deficiencies, N‐deficient females expressed more changes in proteins that are involved in stress responses and gene expression regulation, while P‐deficient females showed more changes in proteins that are involved in energy and lipid metabolism, stress responses and gene expression regulation. The quantitative RT‐PCR analysis of stress‐related proteins showed that males have a better expression correlation between mRNA and protein levels than do females. This study shows that P. cathayana females are more sensitive and have more rapid metabolic mechanisms when responding to N and P deficiencies than do males, and P deficiency has a wider range of effects on females than does N deficiency.     

感染布氏杆菌后的THP-1细胞的蛋白质组学研究

在布氏杆菌(Brucella)的感染免疫过程中,单核巨噬细胞的应答起着非常关键的作用,而毒力不同的布氏杆菌引起的宿主反应截然不同。用双向电泳技术对THP-1单核细胞受毒力不同的布氏杆菌株侵袭后的全细胞蛋白谱进行差异比较和分析,共发现了38个差异表达的蛋白质点。这些点经过胶内酶切后进行MALDI-TOF质谱鉴定,每个蛋白质点的肽质量指纹图谱都在人类的蛋白质组数据库中用Mascot进行检索后,发现这些差异表达的蛋白主要集中在结构蛋白,信号传导途径和物质代谢等领域,还有一些功能未知的蛋白。这一结果为研究布氏杆菌的感染与致病机制提供了方向,对深入探讨病原菌-宿主的相互作用模式具有参考价值。     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).     

Populus cathayana males are less affected than females by excess manganese: Comparative proteomic and physiological analyses

The comprehension of sexually different responses in dioecious plants to excess manganese (Mn) stress requires molecular explanation. Physiological and proteomic changes in leaves of Populus cathayana males and females were analyzed after 4 wk of exposure to Mn stress. Under excess Mn conditions, shoot height and photosynthesis decreased more in females than in males. Females also showed severe browning and subcellular damage, higher Mn²⁺ absorption, and different antioxidant enzyme activities compared with males. There were ten differently regulated protein spots induced by excess Mn stress. They were mainly related to photosynthesis, ROS cleaning, and cell signaling associated to ROS, plant cell death, heat shock, cell defense and rescue, and gene expression and regulation. Variation in protein expression between the sexes clearly showed that males have evolved more efficient photosynthesis capacity, more stable gene expression and regulation, and better cell defense and rescue to prevent further injury under excess Mn stress.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

Targeted proteomics reveals strain‐specific changes in the mouse insulin and central metabolic pathways after a sustained high‐fat diet

The metabolic syndrome is a collection of risk factors including obesity, insulin resistance and hepatic steatosis, which occur together and increase the risk of diseases such as diabetes, cardiovascular disease and cancer. In spite of intense research, the complex etiology of insulin resistance and its association with the accumulation of triacylglycerides in the liver and with hepatic steatosis remains not completely understood. Here, we performed quantitative measurements of 144 proteins involved in the insulin‐signaling pathway and central metabolism in liver homogenates of two genetically well‐defined mouse strains C57BL/6J and 129Sv that were subjected to a sustained high‐fat diet. We used targeted mass spectrometry by selected reaction monitoring (SRM) to generate accurate and reproducible quantitation of the targeted proteins across 36 different samples (12 conditions and 3 biological replicates), generating one of the largest quantitative targeted proteomics data sets in mammalian tissues. Our results revealed rapid response to high‐fat diet that diverged early in the feeding regimen, and evidenced a response to high‐fat diet dominated by the activation of peroxisomal β‐oxidation in C57BL/6J and by lipogenesis in 129Sv mice.     

应用差异蛋白质组学的方法分析左氧氟沙星对肺炎链球菌D39的作用

应用差异蛋白质组学的方法,对肺炎链球菌D39在左氧氟沙星作用下蛋白质表达水平的变化进行了研究,利用质谱技术共鉴定到23个差异蛋白质。这些蛋白质主要参与DNA的复制、转录以及蛋白质的翻译过程,为深入了解抗生素的作用机理以及细菌的耐药机制提供重要理论基础。     

Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies

Introduction: Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates.

Areas covered: The review includes the human studies found by a PubMed search using the following terms: ‘depression cerebrospinal fluid biomarker’, ‘major depression biomarker CSF’, ‘depression CSF biomarker’, ‘proteomics depression’, ‘proteomics biomarkers in depression’, ‘proteomics CSF biomarker in depression’, and ‘major depressive disorder CSF’. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies.

Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.     

Comparative analyses of the proteomes of leaves and flowers at various stages of development reveal organ‐specific functional differentiation of proteins in soybean

The functional differentiation of protein networks in individual organs and tissues of soybean at various developmental stages was investigated by proteomic approach. Protein extraction by Mg/NP‐40 buffer followed by alkaline phenol‐based method was optimized for proteomic analysis. Proteome analyses of leaves at various developmental stages showed 26 differentially expressed proteins, wherein proteins in translocon at the outer/inner envelope membrane of chloroplast protein‐transport machineries increased significantly at the first trifoliate. Immunoblot analysis showed chaperonin‐60 expressed abundantly in young leaves, whereas HSP 70 and ATP‐synthase β were constitutively expressed in all tissues. The net photosynthesis rate and chlorophyll content showed an age‐dependent correlation in leaves. These results suggest that proteins involved in carbon assimilation, folding and assembly, and energy may work synchronously and show a linear correlation to photosynthesis at developmental stages of leaves. Comparison of flower bud and flower proteome reveals 29 differentially expressed proteins, wherein proteins involved in mitochondrial protein transport and assembly, secondary metabolism, and pollen‐tube growth were up‐regulated during flower development. Together, these results suggest that during developmental stages, each type of tissue is associated with a specific group of proteins; wherein proteins involved in energy, sugar metabolism, and folding, assembly, and destination may play pivotal roles in the maturation process of each organ or tissue.     

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma‐glutamyl cycle in plant's adaptation to environment

The existence of a gamma‐glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma‐glutamyl transferase (GGT)‐assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild‐type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low‐molecular weight range compared to WT. The combined iTRAQ and LC‐MS/MS‐based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH‐associated genes showed that disruption of gamma‐glutamyl cycle in ggt1 knockout‐leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma‐glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.     

The proteome complement of Nicotiana tabacum Bright‐Yellow‐2 culture cells

The Nicotiana tabacum Bright‐Yellow‐2 (BY2) cell line is one of most commonly used plant suspension cell lines and offers interesting properties, such as fast growth, amenability to genetic transformation, and synchronization of cell division. To build a proteome reference map of BY2 cell proteins, we isolated the soluble proteins from N. tabacum BY2 cells at the end of the exponential growth phase and analyzed them by 2‐DE and MALDI TOF‐TOF. Of the 1422 spots isolated, 795 were identified with a significant score, corresponding to 532 distinct proteins.