47 research outputs found
The role of interfacial lipids in stabilizing membrane protein oligomers
Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways1 but is often difficult to define2 or predict3. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors
Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation
Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes
Sorting Signals, N-Terminal Modifications and Abundance of the Chloroplast Proteome
Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that ‘spectral counting’ can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models
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Final Technical Report
Two primary technologies have been employed for analysis and measurement of the Synechocystis proteome. (1) 2D-gel electrophoresis. Currently one of the most reliable options in quantitative proteomics, typical 2D-gel experiments use isoelectric focusing (IEF) in the first dimension. In the case of membrane proteins, detergents must be added to maintain their solubility though only neutral/zwitterionic surfactants are compatible with the IEF process. We have optimized 2D gel separations for Synechocystis proteins extracted and separated into soluble and membrane subfractions. The resolution and coverage of integral membrane proteins is only marginally satisfactory and alternatives to the first dimension are being considered. Size-exclusion chromatography under non-denaturing conditions was one option that was explored but resolution was insufficient for subfractionation of the membrane-bound proteome. A more highly resolving technique, the ''Blue-native gel'' has proven excellent for Synechocystis and we plan to set up this technology in the near future. Proteins with altered expression are being identified through standard LCMSMS technologies. The analysis of PSI, PSII and SDH deficient mutants is completed, establishing the comparative aspect of the project for integration with the ultrastructural and metabolomic experiments at ASU. We are also looking forward to receiving ftsZ and VIPP1 interruption mutants to explore the effects on the proteome of cell enlargement and disruption of thylakoid biogenesis, respectively. (2) 2D liquid chromatography with mass spectrometry of intact proteins. Early experiments with total membrane protein extracts of Synechocystis showed that the spatial resolution of the reverse-phase separation used in front of the mass spectrometer limited detection to the one hundred or so most abundant proteins. The intact mass tags (IMTs) measured in this experiment represent the first of these measurements that will ultimately define the entire proteome. While some of the IMTs were matched to masses calculated from translations of genomic open-reading frames allowing reasonably confident identification of about half of them (hypothetical IMTs), we are currently validating identifications using a combination of peptide mass fingerprinting after cyanogen bromide cleavage and LC-MSMS after trypsin, of protein in fractions collected during LC-MS+. In order to gain more complete proteome coverage we are applying a liquid separation in front of the LC-MS+ experiment. Size-exclusion chromatography is the first separation technology to be employed, yielding immediate benefits, while still not satisfactory for overall resolution of complexes. Total membranes were solubilized with dodecyl maltoside (1.5%) and separated on deactivated silica (G 4000 SW). LC-MS+ analysis of less-retained chlorophyll-containing fractions, using reverse-phase and size-exclusion technologies, yielded intact protein mass spectra of the two large photosystem I subunits PsaA and PsaB as well as many other IMTs (Figures 1 & 2). These integral membrane proteins have eleven transmembrane helices and, at 81 and 83 kDa, represented one of the most significant challenges to the intact protein molecular weight approach. The identities of the proteins were confirmed by peptide mass fingerprinting and while there is good general agreement between measured and calculated masses it is noted that modest post-translational modifications are necessary to account for the measured molecular weights of the intact proteins. Whether these discrepancies are due to genuine post-translational modifications or DNA sequence errors remains to be determined. The data have been published allowing us to claim to be the first to have completed high-resolution electrospray-ionization mass spectrometry of the core subunits of Photosystem II, Photosystem I and the cytochrome b{sub 6}f complex providing effective proof-of-principle for application of the intact mass approach to the integral membrane proteome. Significantly, we reported greater integral membrane proteome coverage than a colleague studying thylakoids of Arabidopsis illustrating the benefits of the technique over sequential organic extraction of membrane proteins and 1D-gel analysis. The homogeneity of the PsaA and PsaB protein mass spectra attest to the quality of material grown at ASU and the viability of extraction and work up of the material after transport to UCLA
Structure of the Cytochrome b 6 f Complex: n-Side Donor Pathway to the Plastoquinone Pool
Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus
Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes (Clinical and Vaccine Immunology (2010) 17, 8, (1274-1281))
Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes.
Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 x 10(6) inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein