水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light‐ and dark‐grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress‐related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light‐grown cells were more sensitive to heavy metal toxicity than dark‐grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism‐related proteins increased in relative abundance both in light‐ and dark‐grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti‐oxidative processes were decreased both in light‐ and dark‐grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light‐dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.     

Quantitative changes in protein expression of cadmium-exposed poplar plants

Cadmium (Cd) pollution is a worldwide major concern having, among others, deleterious effects on plants. In the present work, the effects of a 20 microM Cd exposure in hydroponics culture during 14 days were evaluated in young poplar leaves. Proteins were analysed by 2-D DIGE, followed by MALDI-TOF-TOF identification. Additionally, growth and other physiological parameters were monitored during the experiment. Treated plants exhibited an inhibition of growth and visual symptoms appeared after 7 days. A significant accumulation of Cd in all organs was recorded by ICP-MS analysis. A number of changes in the expression of proteins with various functions were identified; in particular a decreased abundance of oxidative stress regulating proteins, whereas pathogenesis-related proteins showed a drastic increase in abundance. Furthermore, a large number of proteins involved in carbon metabolism showed a decrease in abundance, while proteins involved in remobilizing carbon from other energy sources were upregulated. In conclusion, the negative effect of Cd could be explained by a deleterious effect on protein expression from the primary carbon metabolism and from the oxidative stress response mechanism. Accumulation of Cd in stems of poplar, coupled with a low impact of Cd on physiological parameters, promotes the use of poplar trees for phytoremediation purposes.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Cd36, a class B scavenger receptor, functions as a monomer to bind acetylated and oxidized low-density lipoproteins

Cd36 is a small-molecular-weight integral membrane protein expressed in a diverse, but select, range of cell types. It has an equally diverse range of ligands and physiological functions, which has implicated Cd36 in a number of diseases including insulin resistance, diabetes, and, most notably, atherosclerosis. The protein is reported to reside in detergent-resistant microdomains within the plasma membrane and to form homo- and hetero-intermolecular interactions. These data suggest that this class B scavenger receptor may gain functionality for ligand binding, and/or ligand internalization, by formation of protein complexes at the cell surface. Here, we have overexpressed Cd36 in insect cells, purified the recombinant protein to homogeneity, and analyzed its stability and solubility in a variety of nonionic and zwitterionic detergents. Octylglucoside conferred the greatest degree of stability, and by analytical ultracentrifugation we show that the protein is monomeric. A solid-phase ligand-binding assay demonstrated that the purified monomeric protein retains high affinity for acetylated and oxidized low-density lipoproteins. Therefore, no accessory proteins are required for interaction with ligand, and binding is a property of the monomeric fold of the protein. Thus, the highly purified and functional Cd36 should be suitable for crystallization in octylglucoside, and the in vitro ligand-binding assay represents a promising screen for identification of bioactive molecules targeting atherogenesis at the level of ligand binding.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.     

In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells

Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.     

A comparison of 60, 70, and 90 kDa stress protein expression in normal rat NRK-52 and human HK-2 kidney cell lines following in vitro exposure to arsenite and cadmium alone or in combination

Arsenite and cadmium are two potent nephrotoxicants and common Superfund site elements. These elements are included among the stress protein inducers, but information regarding relationships between toxicity produced by combinations of these agents to the stress protein response is lacking. In this study, the immortalized cell lines normal rat kidney NRK-52E and human kidney HK-2 were exposed in vitro to arsenite (As(3+)), cadmium (Cd(2+)), or to equimolar As(3+) plus Cd(2+) mixture combinations for 3 and 5 h over a concentration range of 0.1-100 microM. After a 12-h recovery period, cultured cells were then evaluated for expression of the 60, 70, and 90 kDa major stress protein families. Results indicated that expression of stress proteins varied depending on the species of kidney cells exposed, the exposure concentrations, and the length of exposure to each element on an individual basis and for combined mixtures. For the HK-2 kidney cell line, increased levels of the 70 kDa stress protein was observed for single and combined element exposures whereas there was no change or a decrease of stress proteins 60 and 90 kDa. Increased 70 kDa expression was observed for 10-microM doses of single elements and for a lower dose of 1 microM of the As plus Cd mixture at 3- and 5-h exposures. NRK-52 kidney cells exposed to equivalent doses of As(3+) and Cd(2+) alone or in combination showed increased levels of all stress proteins 60, 70, and 90 kDa. This increase was seen for 10 microM of the As plus Cd mixture at 3 h whereas for single element exposures, increased stress protein levels were generally observed for the 100-microM doses. At 5 h- exposure, 60 and 90 kDa levels increased for 10 microM of Cd(2+) and 60 kDa levels increased for 1 microM of As(3+). However, exposures to 10 microM of the As plus Cd mixture decreased 60 kDa protein expression to control levels at 5 h. For both kidney cell lines, there was a decrease in the stress protein expression levels for all three stress protein families for 100-microM doses of the mixture combination for 3- and 5-h exposures. These data indicate a dose- and combination-related correlation between depression of the stress protein response and the onset of overt cellular toxicity and/or cell death. The threshold for these changes was cell line specific.     

Differential proteomic analysis of cadmium-responsive proteins in wheat leaves

To gain a comprehensive understanding of plant response to Cd, physiological and proteomic changes in wheat (Triticum aestivum L.) leaves exposed to a range of Cd concentrations (10, 100 and 200 μM) were investigated. Leaf elongation was decreased, whereas the H₂O₂ and malondialdehyde content increased significantly at higher Cd concentrations. Changes in protein profiles were analyzed by two-dimensional electrophoresis. Twenty-one proteins which showed 1.5-fold change in protein abundance in response to Cd were identified. These proteins can be functionally grouped into three groups: 1) oxidative stress response, 2) photosynthesis and sugar metabolism and 3) protein metabolism and others. These results provide a new insight into our understanding of the molecular basis of heavy metal response in plants.     

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.     

Annotated proteome of a human T-cell lymphoma.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.     

In vitro assessment of chelating agents with regard to their abstraction efficiency of Cd(2+) bound to plasma proteins

The exposure of various human populations to Cd(2+) is of increasing health concern. After its gastrointestinal absorption into the bloodstream, Cd(2+) binds to α(2)-macroglobulin and serum albumin. Although animal studies have demonstrated that meso-2,3-dimercaptosuccinic acid (DMSA) and diethylenetriamine pentaacetic acid (DTPA) can effectively mobilize Cd(2+) to urine and decrease the Cd concentrations of the kidneys, the liver and the brain, not much is known about the abstraction of Cd(2+) from blood plasma proteins. We prepared a stock of Cd(2+) spiked rabbit plasma (2.0 μg of Cd(2+)/mL) and analyzed aliquots by size exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES) while simultaneously monitoring the emission lines of Ca, Cd, Cu, Fe, and Zn. After the addition of 0.33 mM, 0.66 mM or 0.99 mM of DMSA, DTPA, 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetyl-l-cysteine (NAC) to plasma aliquots, the obtained mixtures were analyzed by SEC-ICP-AES after 5 min and 30 min. None of the investigated compounds adversely affected the plasma distribution of Fe at all investigated doses. At 0.33 mM, DTPA was most effective at mobilizing plasma protein bound Cd(2+) to a ～5 kDa Cd-species (100% removal), followed by DMPS (94%), DMSA (83%) and NAC (3%). All investigated compounds also mobilized Zn(2+) from plasma proteins to ～5 kDa Zn-species (DTPA: 80% removal; DMPS: 63%; DMSA: 29% and NAC: 3%). The addition of DTPA resulted in the dose-dependent elution of a [Ca-DTPA](3-) complex. Based on these results, 0.33 mM DMSA represents the best compromise that can be achieved between maximizing the abstraction of Cd(2+) from plasma proteins (83%), while minimizing the mobilization of Zn(2+) from plasma proteins (29%), and avoiding the complexation of Ca(2+).     

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.     

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT)

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.     

Cadmium-induced synthesis of metallothioneins in human lymphocytes and monocytes

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.     

利用S-层蛋白在苏云金芽胞杆菌细胞表面展示多聚组氨酸肽（简报）

S层(Slayer)是由单一的蛋白或糖蛋白组成的薄层晶状结构,它广泛存在于古细菌和真细菌细胞的最外表面,可包裹整个细胞。S层在结构化学、形态学、遗传学以及物理化学等方面具有独特的性质,使之在生物技术、分子纳米技术和仿生学等领域蕴藏着广泛的应用潜力。近年来,随着微生物表