Genomic analysis of biochemical function

Establishing the linkage between an individual biochemical activity and the gene(s) specifying that activity has been facilitated by advances in mass spectrometry and affinity purification methods. In addition, a genomic protein array has been produced in yeast by fusing each yeast open reading frame to glutathione-S-transferase, thus linking each protein with its cognate gene. Purification and biochemical assay of pools of glutathione-S-transferase-open-reading-frame proteins allows analysis of the entire proteome for biochemical activities, followed by simple deconvolution to identify the responsible open reading frame. An alternative method to analyze large sets of proteins is the use of protein microarrays in which over 10,000 individual proteins can be immobilized and assayed on a single slide.     

Genome-Wide Identification and Analysis of Expression Profiles of Maize Mitogen-Activated Protein Kinase Kinase Kinase

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.     

酵母双杂交技术及其在蛋白质组研究中的应用

蛋白质组学是后基因组时代出现的一个新兴的研究领域,它的主要任务是识别鉴定细胞,组织或机体的全部蛋白质,并分析蛋白质的功能及其模式。因此,揭示蛋白质组中蛋白质间的相互作用关系也是蛋白质组学的重要内容之一。酵母双杂交技术是用来检测蛋白质间是否相互作用的一个非常有效的手段,该技术在酵母蛋白质组研究中的初步成功应用,表明它有望在人类蛋白质且研究中发挥重要作用。     

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.     

Tandem affinity purification and identification of protein complex components

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.     

Biochemical clustering of monomeric GTPases of the Ras superfamily

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.     

Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database

The proteome is the entire protein complement of the genome expressed in a particular cell, tissue, or organism at a given time under a specific set of environmental conditions. Proteomics is a combinatorial methodology to comprehensively analyze the proteome. The general protocol of the expression proteomics consists of advanced methods of high-resolution protein separation, high-quality image analysis and high-throughput protein identification. Although Epstein-Barr virus-transformed B-lymphoblastoid cell lines (LCLs) have long been believed to be immortalized, recent studies have provided ample evidence that a large proportion of LCLs have limited life spans due to shortening of telomeres, and that part of them are truly immortalized by developing strong telomerase activity to maintain telomeres. Differential proteome analysis of pre- and post-immortal LCLs would provide a powerful tool to analyze proteins participating in the process of immortalization. We focus in this review on cumulative data of proteomic information on pre- and post-immortal LCLs.     

Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase

A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.     

Identification of deletion mutant respiratory syncytial virus strains lacking most of the G protein in immunocompromised children with pneumonia in South Africa

Respiratory syncytial virus (RSV) G protein deletion mutants replicate effectively in vitro but have not been detected in nature. Subtyping of RSV strains in hospitalized children in South Africa identified G protein PCR amplicons significantly reduced in size in 2 out of 209 clinical specimens screened over 4 years. Sequence analysis revealed subtype B strains lacking nearly the entire G protein ectodomain in one HIV-positive and one HIV-exposed child hospitalized with pneumonia. The association of clinical strains lacking most of the G protein with lower respiratory tract infection in immunocompromised children may have implications for RSV vaccine development.     

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.     

Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

The proteome analysis by 2-DE is one of the most potent methods of analyzing the complete proteome of cells, cell lines, organs and tissues in proteomics studies. It allows a fast overview of changes in cell processes by analysis of the entire protein extracts in any biological and medical research projects. New instrumentation and advanced technologies provide proteomics studies in a wide variety of biological and biomedical questions. Proteomics work is being applied to study antibiotics-resistant strains and human tissues of various brain, lung, and heart diseases. It cumulated in the identification of antigens for the design of new vaccines. These advances in proteomics have been possible through the development of advanced high-resolution 2-DE systems allowing resolution of up to 10 000 protein spots of entire cell lysates in combination with protein identification by new highly sensitive mass spectrometric techniques. The present technological achievements are suited for a high throughput screening of different cell situations. Proteomics may be used to investigate the health effects of radiation and electromagnetic field to clarify possible dangerous alterations in human beings.     

Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation

Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E. coli during the process of export. It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded. Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltosebinding protein has reached molecular weight 33,000 (80% of its entire length) and after ribosebinding protein has been fully elongated (molecular weight 29,000). The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains. In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis. This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed.     

Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv

Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.     

Direct evidence for co-localization of adenylate cyclase,dopamine-β-hydroxylase and cytochrome b562 to bovine chromaffin granule membranes

Adenylate cyclase, dopamine-β-hydroxylase and cytochrome b₅₆₂ have been found to co-equilibrate on equilibrium sucrose gradients of lysed chromaffin granule membranes from bovine adrenal medulla. Peak activities for these enzymes, as well as maximum membrane protein concentration, were found to coincide at d = 1.11 gm cm^?3, and the ratios of adenylate cyclase to both dopamine-β-hydroxylase and cytochrome b₅₆₂ were constant across the entire peak. Adenylate cyclase activity has been reported previously to co-purify with chromaffin granule membranes, and we conclude, on the basis of these new data, that adenylate cyclase is also an intrinsic granule membrane enzyme.     

Non-heme iron through the three domains of life

Metalloproteins are proteins capable of binding one or more metal ions, which are often required for their biological function or for regulation of their activities or for structural purposes. In high-throughput genome-level protein investigation efforts, such as Structural Genomics, the systematic experimental characterization of metal-binding properties (i.e. the investigation of the metalloproteome) is not always pursued, and remains far from trivial. In the present work we have applied a bioinformatic approach to investigate the occurrence of (putative) non-heme iron-binding proteins in 57 different organisms spanning the entire tree of life. It is found that the non-heme iron-proteome constitutes between 1% and 10% of the entire proteome of an organism. However, the iron-proteome constitutes a higher fraction of the proteome in archaea (on average 7.1% +/- 2.1%) than in bacteria (3.9% +/- 1.6%) and in eukaryota (1.1% +/- 0.4%). The analysis of the function of each putative iron-protein identified suggests that extant organisms have inherited the large majority of their iron-proteome from the last common ancestor.     

Localization of immunogenic domains in the human immunodeficiency virus type 2 envelope.

Highly immunogenic domains have not yet been defined in the extracellular protein of the human immunodefiency virus type 2 (HIV-2) envelope. In this study, six contiguous segments covering the entire HIV-2ST envelope were amplified and cloned into a bacterial expression vector to localize the relative immunogenic reactivity of different regions of the molecule by Western blot (immunoblot) analysis. Our results demonstrate that the extracellular protein of the HIV-2 envelope contains highly immunogenic epitopes with potential value as type-specific markers for HIV-2 infection.