A proteomics study of the mung bean epicotyl regulated by brassinosteroids under conditions of chilling stress

Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77% identity with the Arabidopsis CPD gene. It was strongly suppressed by chilling stress. This implies that exogenous treatment with BR could allow the plant to recover from the inhibited growth caused by chilling. In this study, we used proteomics to investigate whether the mung bean epicotyl can be regulated by brassinosteroids under conditions of chilling stress. Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24-epibrassinolde; 17 proteins down-regulated by this chilling were re-up-regulated. These up-regulated proteins are involved in methionine assimilation, ATP synthesis, cell wall construction and the stress response. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment with DL-methionine. This is the first proteome established for the mung bean species. The regulatory relationship between brassinosteroids and chilling conditions was investigated, and possible mechanisms are discussed herein.     

Neurogenesis and neuronal regeneration in the adult fish brain

Fish are distinctive in their enormous potential to continuously produce new neurons in the adult brain, whereas in mammals adult neurogenesis is restricted to the olfactory bulb and the hippocampus. In fish new neurons are not only generated in structures homologous to those two regions, but also in dozens of other brain areas. In some regions of the fish brain, such as the optic tectum, the new cells remain near the proliferation zones in the course of their further development. In others, as in most subdivisions of the cerebellum, they migrate, often guided by radial glial fibers, to specific target areas. Approximately 50% of the young cells undergo apoptotic cell death, whereas the others survive for the rest of the fish’s life. A large number of the surviving cells differentiate into neurons. Two key factors enabling highly efficient brain repair in fish after injuries involve the elimination of damaged cells by apoptosis (instead of necrosis, the dominant type of cell death in mammals) and the replacement of cells lost to injury by newly generated ones. Proteome analysis has suggested well over 100 proteins, including two dozen identified ones, to be involved in the individual steps of this phenomenon of neuronal regeneration.     

Whole Genome Phage Display Selects for Proline-rich Boi Polypeptides against Bem1p

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second ``src homology region 3' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1_{[Asn142-Ile551]}, which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained ~7.7 × 10⁷ independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.     

Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis

With the recent publication of the Cryptosporidium genome, investigation of the proteins expressed by Cryptosporidium parvum will provide complementary information on the biology of this complex organism. Proteomic studies on this apicomplexan parasite have been hampered due to the inability to culture or isolate high numbers required for 2D gel analysis. Neonatal calves are a common source of Cryptosporidium oocysts and we report on the development of a sucrose-Percoll purification procedure which produced the high yield and purity (free from faecal and bacterial contaminants) that is required for successful proteomic studies from neonatal calves. We report on the development of quantitative and qualitative flow cytometric methods which were confirmed by epifluorescence microscopy. A comparison of five common purification procedures was carried out to determine the efficiency of the sucrose-Percoll gradient. 2D-PAGE results strongly support the sucrose-Percoll procedure as the most suitable method for applications like proteomics which require the recovery of high numbers of isolated oocysts with minimal faecal and bacterial contaminants.     

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.     

Facile synthesis toward the construction of an activity probe library for glycosidases

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-d-Glc, alpha- and beta-d-Gal, alpha-d-Man, and alpha-l-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-d-glucosidase, alpha-d-galactosidase, and alpha-l-fucosidase, respectively.     

Prediction of protease types in a hybridization space

Regulating most physiological processes by controlling the activation, synthesis, and turnover of proteins, proteases play pivotal regulatory roles in conception, birth, digestion, growth, maturation, ageing, and death of all organisms. Different types of proteases have different functions and biological processes. Therefore, it is important for both basic research and drug discovery to consider the following two problems. (1) Given the sequence of a protein, can we identify whether it is a protease or non-protease? (2) If it is, what protease type does it belong to? Although the two problems can be solved by various experimental means, it is both time-consuming and costly to do so. The avalanche of protein sequences generated in the post-genetic era has challenged us to develop an automated method for making a fast and reliable identification. By hybridizing the functional domain composition and pseudo-amino acid composition, we have introduced a new method called "FunD-PseAA predictor" that is operated in a hybridization space. To avoid redundancy and bias, demonstrations were performed on a dataset where none of the proteins has >or=25% sequence identity to any other. The overall success rate thus obtained by the jackknife cross-validation test in identifying protease and non-protease was 92.95%, and that in identifying the protease type was 94.75% among the following six types: (1) aspartic, (2) cysteine, (3) glutamic, (4) metallo, (5) serine, and (6) threonine. Demonstration was also made on an independent dataset, and the corresponding overall success rates were 98.36% and 97.11%, respectively, suggesting the FunD-PseAA predictor is very powerful and may become a useful tool in bioinformatics and proteomics.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.