Proteomics analysis revealed changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure

Exposure to oil mist has been associated with a variety of acute and chronic respiratory effects. Using proteomics approaches to investigate exposure-associated proteins may provide useful information to understand the mechanisms of associated respiratory effects. The aim of this study was to investigate changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure using nano-HPLC-ESI-MS/MS. The results revealed that 29 proteins exhibited significant changes after exposure. These proteins included surfactant-associated proteins (SP-A and SP-D), inflammatory proteins (complement component 3, immunoglobulins, lysozyme, etc.), growth factors (e.g., transforming growth factor alpha (TGF-alpha)), calcium-binding proteins (calcyclin, calgranulin A, calreticulin, and calvasculin), and other proteins (e.g., cathepsin D, saposin, and intestinal trefoil factor). To further evaluate changes in protein levels, a simple quantitative strategy was developed in this study. A large decrease in protein levels of SP-A and SP-D (0.24- and 0.38-fold, respectively) following exposure was observed. In contrast, protein levels of TGF-alpha and calcium-binding proteins were significantly increased (4.46- and 1.4-1.8-fold, respectively). Due to the diverse functions of these proteins, the results might contribute to understand the mechanisms involved in lung disorders induced by oil mist exposure.     

Proteomics reveals new salt responsive proteins associated with rice plasma membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

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.     

Proteomics of CaCO3 biomineral‐associated proteins: How to properly address their analysis

In a recent editorial (Proc. Natl. Acad. Sci., 2013 110, E2144–E2146) and elsewhere, questions have been raised regarding the experimental practices in relation to the proteomic analysis of organic matrices associated to the biomineralized CaCO₃ skeletons of metazoans such as molluscan shells and coral skeletons. Indeed, although the use of new high sensitivity MS technology potentially allows to identify a greater number of proteins, it is also equally (or even more) sensitive to contamination of residual proteins from soft tissues, which are in close contact with the biomineral. Based on our own past and present experimental know‐how—observations that are reproducible and coherent with the current understanding of extracellular biomineralization processes—we are convinced that a careful and appropriate cleaning of biominerals prior to any analysis is crucial for accurate proteomic investigations and subsequent pertinent interpretation of the results. Our goal is to alert the scientific community about the associated bias that definitely should be avoided, and to provide critical recommendations on sample preparation and experimental design, in order to better take advantage of the aptness of proteomic approaches aiming at improving our understanding of the molecular mechanisms in biomineralization.     

Proteomics analysis of two heat shock proteins in insects

Heat shock proteins (HSPs) are found in all living organisms, from bacteria to humans, are expressed under stress. In this study, characterization of two families of HSP including HSP60 and HSP70 protein was compared in different insect species from different orders. According to the conserved motifs analysis, none of the motifs were shared by all insects of two protein families but each family had their own common motifs. Functional and structural analyses were carried out on seven different insect species from each protein family as the representative samples. These analyses were performed via ExPASy database tools. The tertiary structure of Drosophila melanogater as the sample of each protein family were predicted by the Phyre2 and TM-score servers then their qualities were verified by SuperPose and PROCHECK. The tertiary structures were predicted through the “c4pj1E” model (PDB Accession Code: 4pj1) in HSP60 family and “c3d2fC” model (PDB Accession Code: 3d2f) in HSP70 family. The protein phylogenetic tree was constructed using the Neighbor-joining (NJ) method by Molecular Evolutionary Genetic Analysis (MEGA) 6.06. According to the results, there was a high identity of HSP60 and HSP70 families so that they should be derived from a common ancestor however they belonged to separate groups. In protein–protein interaction analysis by STRING 10.0, 10 common enriched pathways of biological process, molecular function and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified in D. melanogaster in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of insects and other organisms.

Communicated by Ramaswamy H. Sarma     

Proteomics enhances evolutionary and functional analysis of reproductive proteins

Reproductive proteins maintain species‐specific barriers to fertilization, affect the outcome of sperm competition, mediate reproductive conflicts between the sexes, and potentially contribute to the formation of new species. However, the specific proteins and molecular mechanisms that underlie these processes are understood in only a handful of cases. Advances in genomic and proteomic technologies enable the identification of large suites of reproductive proteins, making it possible to dissect reproductive phenotypes at the molecular level. We first review these technological advances and describe how reproductive proteins are identified in diverse animal taxa. We then discuss the dynamic evolution of reproductive proteins and the potential selective forces that act on them. Finally, we describe molecular and genomic tools for functional analysis and detail how evolutionary data may be used to make predictions about interactions among reproductive proteins.     

Proteomics analysis of the temporal changes in axonal proteins during maturation

After the initial primary projection, axons undergo various structural and functional changes to establish mature neural circuits. The changes in protein expression associated with this maturation were investigated in lateral olfactory tract axons using two‐dimensional gel electrophoresis. The most prominent group upregulated during the period consisted of calcium‐dependent membrane‐binding proteins including VILIP1, neurocalcin δ, copine 6, and annexin A6 from three structurally different families. During maturation of primary cultured neurons, annexin A6 gradually became concentrated on the axon initial segment, and its overexpression significantly enhanced axon branching. On the other hand, overexpression of VILIP1 and neurocalcin δ reduced axon outgrowth and branching. The second group upregulated during axon maturation comprised tubulin‐ and microtubule‐binding proteins including CRMP2, guanine deaminase, MAP1B, and fibronectin type3 SPRY domain‐containing protein. Because the maturation of lateral olfactory axons involves massive extension of secondary collateral branches, the augmentation of these proteins during these stages may underlie the drastic restructuring of the axon cytoskeleton. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:523–537, 2010     

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.     

Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies.

We have shown that hypergravity (35g) stimulates production of inositol 1,4,5-trisphosphate (IP3) and decreases adenosine 3',5'-cyclic monophosphate (cAMP) levels in HeLa cells. IP3 production rapidly increased 1.5- and 2.1-fold greater (P less than 0.05) than the control after 2- and 5-min exposures to 35g, respectively. The intracellular cAMP levels, determined in the presence of isobutylmethylxanthine, were decreased by 11% (P less than 0.05) and 16% (P less than 0.01) relative to the control after 10- and 20-min exposures to 35g, respectively. The phosphorylation of proteins which were immunoprecipitated by antibodies recognizing microtubule-associated proteins (ipMAPs) was also apparent after exposure of these cells to hypergravity. In the detergent-insoluble fraction, phosphorylation of a 115-kDa protein was significantly enhanced compared to the control after a 5-min exposure to 35g. In the detergent-soluble fraction, phosphorylation of a 200-kDa protein was observed served after a 20-min exposure to 35g. Our study suggests that IP3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of ipMAPs in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.     

An oncogenic role of sphingosine kinase

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.     

Protein kinase and specific phosphate acceptor proteins associated with tupaia herpesvirus.

A protein kinase activity was found to be associated with tree shrew (tupaia) herpesvirus. The protein kinase was characterized with respect to its requirements for enzymatic activity. A divalent cation such as Mg2+ or Mn2+ was necessary as well as ATP as the phosphate donor. Distinct tupaia herpesvirus polypeptides (molecular weights of 100,000 [100K], 82K, and 53K) were found to be phosphate acceptor proteins when 5 mM Mg2+ was used. At a higher Mg2+ concentration (20 mM), additional viral proteins (220K, 71K, 31K, and 20K) were phosphorylated. The viral phosphoproteins were analyzed by chemical and enzymatic hydrolyses. The predominant sites of phosphorylation were the beta-OH groups of the serine and threonine residues of these tupaia herpesvirus proteins. Kinase activity was not stimulated by cyclic nucleoside monophosphates. Endogenously added proteins did not enhance protein kinase activity. Protein kinase activity was inhibited by 5'-p-fluorosulfonylbenzoyladenosine.     

Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics.

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.     

Interaction of oncogenic papillomavirus E6 proteins with fibulin-1

Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calcium-binding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6.     

Proteomics of Chlamydomonas reinhardtii light-harvesting proteins

With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.     

Characterization of two high molecular weight proteins immunoprecipitated with an antibody against rat liver cytochrome c oxidase

Isolated rat hepatocytes were labelled with [35S]methionine, dissolved in Triton X-100-containing buffer, and incubated with antibodies against rat liver cytochrome c oxidase. After separation by dodecyl sulfate-gel electrophoresis the fluorogram of immunoprecipitated proteins showed two labelled bands with apparent molecular weights of 52000 and 182000. The immunological relationship of the two proteins to cytochrome c oxidase was demonstrated by immunocompetition with the isolated enzyme and with purified subunits IV-VIII. Although the precursor nature of the two described proteins for cytoplasmically synthesized subunits of cytochrome c oxidase cannot be excluded, the following observations do not support this assumption: 1) The amount of incorporated radioactivity is too high; 2) they are exclusively located with the microsomal fraction; 3) the turnover is rather slow, compared to that of known precursor proteins.     

Proteomics analysis reveals marker proteins for minor vein initiation in rice leaf

Leaf veins play a critical role in resource supplication and photosynthate translocation; thus, it is considered as an important agricultural trait for crop breeding. The rice minor veins are parallelly grown along all the parts of the leaf from base to tip. To understand the process of minor vein development, anatomy analysis was performed to reveal the initiation and development of minor veins in rice leaf. The frequency of minor vein initiation follows a decreased tendency from leaf base to tip. An iTRAQ-based proteomics analysis was performed in rice leaf sections. Photosynthesis- and carbon fixation-related proteins accumulated a high level in the middle part of leaves. Furthermore, marker proteins involved in sucrose degradation and starch synthesis were accumulated into initiation and mature parts of minor veins, respectively. It suggests a different source-sink activity in the initiation and mature parts of minor veins in terms of photosynthate translocation. The identified proteins are candidate markers for small vein initiation in rice leaves.     

Proteomics analysis of human serum of patients with non-small-cell lung cancer reveals proteins as diagnostic biomarker candidates

Non-small-cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has a poor prognosis, because overall survival after 5 years is 20–25% for all stages. Thus, it is extremely important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study, we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, α2 macroglobulin, and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, α2 macroglobulin, and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, αmicroglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.