Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae
ABSTRACT: Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2 % and 0.1 % (w/v) glucose. A total of eight protein spots exhibiting a minimum 2-fold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are – besides the expected Hxk2 – two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.
Project description:Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2 % and 0.1 % (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are – besides the expected Hxk2 – two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.
Project description:BACKGROUND:Two-dimensional differential gel electrophoresis (2D-DIGE) provides a powerful technique to separate proteins on their isoelectric point and apparent molecular mass and quantify changes in protein expression. Abundantly available proteins in spots can be identified using mass spectrometry-based approaches. However, identification is often not possible for low-abundant proteins. RESULTS:We present a novel computational approach to prioritize candidate proteins for unidentified spots. Our approach exploits noisy information on the isoelectric point and apparent molecular mass of a protein spot in combination with functional similarities of candidate proteins to already identified proteins to select and rank candidates. We evaluated our method on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. Using leave-one-out cross-validation, we show that the true-positive rate for the top-5 ranked proteins is 43.8%. CONCLUSIONS:Our approach shows good performance on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. We expect our method to be highly useful in (re-)mining other 2D-DIGE experiments in which especially the low-abundant protein spots remain to be identified.
Project description:Background:To understand the roles of serum exosomes in rheumatoid arthritis (RA), we comprehensively investigated the protein profiles of serum exosomes in patients with RA. Methods:Exosomes were isolated from serum samples obtained from 33 patients (12 with active RA [aRA], 11 with inactive RA [iRA], 10 with osteoarthritis [OA]) and 10 healthy donors (HLs). Proteins extracted from the exosomes were separated by two-dimensional differential gel electrophoresis (2D-DIGE) and identified by mass spectrometry. Results:In total, 204 protein spots were detected by 2D-DIGE. In the aRA, iRA, and OA groups, 24, 5, and 7 spots showed approximately ≥ ±1.3-fold intensity differences compared with the HL group, respectively. We were able to identify proteins in six protein spots. Among them, the protein spot identified as Toll-like receptor 3 (TLR3) showed approximately 6-fold higher intensity in the aRA group than in the other groups. Conclusions:Patients with active RA possessed considerably different protein profiles of serum exosomes from patients with iRA, patients with OA, and healthy donors. The unique protein profile of serum exosomes, such as the possession of abundant TLR3 fragments, may reflect the pathophysiology of active RA.
Project description:To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.
Project description:Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
Project description:BACKGROUND: Differential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. CONCLUSIONS/SIGNIFICANCE: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.
Project description:Aging is characterized by increase in reactive oxygen (ROS) and nitrogen (RNS) species, key factors of cardiac failure and disuse-induced muscle atrophy. This study focused on serum nitroproteome as a trait of longevity by adopting two complementary gel-based techniques: two-dimensional differential in gel electrophoresis (2-D DIGE) and Nitro-DIGE coupled with mass spectrometry of albumin-depleted serum of aged (A, <i>n</i> = 15) and centenarian (C, <i>n</i> = 15) versus young females (Y, <i>n</i> = 15). Results indicate spots differently expressed in A and C compared to Y and spots changed in A vs. C. Nitro-DIGE revealed nitrosated protein spots in A and C compared to Y and spots changed in A vs. C only (<i>p</i>-value < 0.01). Nitro-proteoforms of alpha-1-antitripsin (SERPINA1), alpha-1-antichimotripsin (SERPINA3), ceruloplasmin (CP), 13 proteoforms of haptoglobin (HP), and inactive glycosyltransferase 25 family member 3 (CERCAM) increased in A vs. Y and C. Conversely, nitrosation levels decreased in C vs. Y and A, for immunoglobulin light chain 1 (IGLC1), serotransferrin (TF), transthyretin (TTR), and vitamin D-binding protein (VDBP). Immunoblottings of alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR) and thioredoxin reductase 1 (TRXR1) indicated lower levels of ADH5 in A vs. Y and C, whereas TRXR1 decreased in A and C in comparison to Y. In conclusion, the study identified putative markers in C of healthy aging and high levels of ADH5/GSNOR that can sustain the denitrosylase activity, promoting longevity.
Project description:Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen ? chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at ~37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at ~50 kDa and spots 526 and 548 at ~37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 × 10?/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.
Project description:The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.
Project description:This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere .