Characterization of new D-beta-aspartate-containing proteins in a lens-derived cell line

Although proteins are generally composed of l-alpha-amino acids, biologically uncommon D-beta-aspartic acid (Asp)-containing proteins have been reported in various tissues from elderly individuals. Our previous study indicated that the N/N1003A cell line, derived from rabbit lens, includes D-beta-Asp-containing proteins of approximately 50 kDa by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-beta-Asp-containing proteins. In this study, we identified the D-beta-Asp-containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. The results indicate that one of these 50 kDa proteins is an enolase showing homology with tau-crystallin. Other D-beta-Asp-containing proteins, which we have recently discovered include lamin A/C, cytoplasmic NADP+-dependent isocitrate dehydrogenase, fructose-bisphosphate aldolase A, aldose reductase, L-lactate dehydrogenase A or calponin H2, phosphoglycerate mutase 1, phosphatidylethanolamine-binding protein, alpha-B-crystallin, and peptidyl-prolyl cis-trans isomerase A (PPlase).     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.     

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior.     

Enzyme activity in the crowded milieu

The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.     

Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two-dimensional electrophoresis

Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.     

Proteomic Analysis of the Response of Liangyoupeijiu （Super High-Yield Hybrid Rice） Seedlings to Cold Stress

Liangyoupeijiu is a super high-yield hybrid rice. Despite its advantages with respect to yield and grain quality, it is sensitive to cold, which keeps it from being widely cultivated. We subjected Liangyoupeijiu seedlings to 4 ℃ cold treatment, then extracted the leaf proteins. After 2-D gel electrophoresis separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, a series of differentially displayed proteins were identified. Some metabolism-associated proteins were found among the downregulated proteins, such as carbamoyl phosphate synthetase, transketolase 1, dihydrolipoamide dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase. The upregulated proteins included both stress-resistance proteins such as nucleoside diphosphate kinase I and proteins that are negative for rice growth, such as FtsH-like protein, plastid fusion and/or translocation factor （Pftf） and actin. Our results indicate that cold may inhibit Liangyoupeijiu growth through decreasing metabolic activity and damaging cell structure.     

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

Proteome map of Aspergillus nidulans during osmoadaptation

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.     

Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat

Chronic restraint stress induces cardiac dysfunction as well as cardiomyocyte injury including severe ultrastructural alteration and cell death, but its mechanism and molecular basis remain unclear. Mitochondria play a key role in regulating cell life. For exploring mitochondrial proteins which correlate with stress-induced injury, two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) were applied. After comparing the protein profiles of myocardial mitochondria between a chronic restraint stress group and a control group, 11 protein spots were found altered, seven of which were identified by MALDI-TOF MS. Among the seven proteins, five proteins involved in the Krebs cycle and lipid metabolism in mitochondria decreased after chronic restraint stress. They were identified as carnitine palmitoyltransferase 2, mitochondrial acyl-CoA thioesterase 1, isocitrate dehydrogenase 3 (NAD+) alpha, fumarate hydratase 1 and pyruvate dehydrogenase beta. The last two proteins, creatine kinase and prohibitin, increased after chronic restraint stress. Biochemical tests for energy metabolism in mitochondria also supported the proteomic results. These findings provide clues for understanding the mechanism of dysfunction or injury in cardiomyocytes induced by chronic stress.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Aldose reductase and macrophage migration inhibitory factor are associated with epididymosomes and spermatozoa in the bovine epididymis

During the epididymal transit, mammalian spermatozoa acquire new surface proteins necessary for male gamete function. We have previously shown that membranous vesicles, called epididymosomes, interact with spermatozoa allowing the transfer of some proteins to sperm surface within the epididymal lumen. The protein composition of those vesicles has been investigated to document the mechanisms of protein transfer from epididymosomes to spermatozoa. Electrophoretic analysis revealed that protein composition is different from the epididymal soluble compartment as well as from similar vesicles present in the semen. Protein association with epididymosome is very strong as revealed by resistance to extraction with detergent. Matrix-assisted laser desorption ionization time-of-flight as well as immunodetection techniques have been used to identify some proteins associated to epididymosomes and spermatozoa. An aldose reductase known for its 20alpha-hydroxysteroid dehydrogenase activity and the cytokine (macrophage migration inhibitory factor) have been identified. These two proteins have been immunolocalized in principal cells of the epididymal epithelium, a more intense signal being detected in the distal epididymal segment as well as in the vas deferens. Database search revealed that these two proteins are characterized by the lack of a signal peptide. These results are discussed with regard to a possible apocrine mode of secretion of these proteins acquired by spermatozoa during the epididymal transit.     

White Spot Syndrome Virus infection in Penaeus monodon is facilitated by housekeeping molecules

White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp aquaculture, and its rampant spread has resulted in great economic loss. Identification of host cellular proteins interacting with WSSV will help in unravelling the repertoire of host proteins involved in WSSV infection. In this study, we have employed one-dimensional and two-dimension virus overlay protein binding assay (VOPBA) followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the host proteins of Penaeus monodon that could interact with WSSV. The VOPBA results suggest that WSSV interacted with housekeeping proteins such as heat shock protein 70, ATP synthase subunit β, phosphopyruvate hydratase, allergen Pen m 2, glyceraldehyde-3-phosphate dehydrogenase, sarcoplasmic calcium-binding protein, actin and 14-3-3-like protein. Our findings suggest that WSSV exploits an array of housekeeping proteins for its transmission and propagation in P. monodon.     

应激心肌损伤的蛋白质组学研究

目的:寻找应激心肌损伤相关蛋白.方法:建立束缚应激心肌损伤模型,制备心室肌2DE蛋白样品和心肌2DE图谱,图像分析软件分析应激后蛋白表达差异点,MALDI-TOF-MS-数据库搜索鉴定蛋白质.结果:应激前后10个蛋白表达量发生改变,其中8个应激后表达显著升高,经质谱鉴定为心肌肌球蛋白、白蛋白、脂蛋白A-I前体等;2个显著降低,经质谱鉴定为线粒体能量代谢酶类和UCP3.结论:这些差异蛋白可能参与应激机体心肌损伤的发生.     

Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging

Aging is accompanied by gradual cellular dysfunction associated with an accumulation of damaged proteins, particularly via oxidative processes. This cellular dysfunction has been attributed, at least in part, to impairment of mitochondrial function as this organelle is both a major source of oxidants and a target for their damaging effects, which can result in a reduction of energy production, thereby compromising cell function. In the present study, we observed a significant decrease in the respiratory activity of rat liver mitochondria with aging, and an increase in the advanced glycation endproduct-modified protein level in the mitochondrial matrix. Western blot analysis of the glycated protein pattern after 2D electrophoresis revealed that only a restricted set of proteins was modified. Within this set, we identified, by mass spectrometry, proteins connected with the urea cycle, and especially glutamate dehydrogenase, which is markedly modified in older animals. Moreover, mitochondrial matrix extracts exhibited a significant decrease in glutamate dehydrogenase activity and altered allosteric regulation with age. Therefore, the effect of the glycating agent methylglyoxal on glutamate dehydrogenase activity and its allosteric regulation was analyzed. The treated enzyme showed inactivation with time by altering both catalytic properties and allosteric regulation. Altogether, these results showed that advanced glycation endproduct modifications selectively affect mitochondrial matrix proteins, particularly glutamate dehydrogenase, a crucial enzyme at the interface between tricarboxylic acid and urea cycles. Thus, it is proposed that glycated glutamate dehydrogenase could be used as a biomarker of cellular aging. Furthermore, these results suggest a role for such intracellular glycation in age-related dysfunction of mitochondria.     

The synovial proteome: analysis of fibroblast-like synoviocytes

The present studies were initiated to determine the protein expression patterns of fibroblast-like synovial (FLS) cells derived from the synovia of rheumatoid arthritis patients. The cellular proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the in-gel digested proteins were analyzed by matrix-assisted laser desorption ionization mass spectrometry. A total of 368 spots were examined and 254 identifications were made. The studies identified a number of proteins that have been implicated in the normal or pathological FLS function (e.g. uridine diphosphoglucose dehydrogenase, galectin 1 and galectin 3) or that have been characterized as potential autoantigens in rheumatoid arthritis (e.g. BiP, colligin, HC gp-39). A novel uncharacterized protein product of chromosome 19 open reading frame 10 was also detected as an apparently major component of FLS cells. These results demonstrate the utility of high-content proteomic approaches in the analysis of FLS composition.