A Mammalian Mediator Subunit that Shares Properties with Saccharomyces cerevisiae Mediator Subunit Cse2

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193open reading frame. In this study, we present direct biochemical evidence that the FLJ10193protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex

Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen

Abstract Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO2 concentrations and CO2 concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO2 assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO2. The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.</jats:p

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato(Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration)and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762516/Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.This work was supported by NIAID Clinical Proteomics Center Grant HHSN272200800048C (to A.R.B.), University of Texas Medical Branch Clinical and Translational Science Award (CTSA) UL1TR000071 (to A.R.B.), and NIEHS Grant P30 ES006676 (to A.R.B.).info:eu-repo/semantics/publishedVersio

The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers

Rim1 is the mitochondrial single-stranded DNA binding protein in Saccharomyces cerevisiae and functions to coordinate replication and maintenance of mtDNA. Rim1 can form homo-tetramers in solution and this species has been assumed to be solely responsible for ssDNA binding. We solved structures of tetrameric Rim1 in two crystals forms which differ in the relative orientation of the dimers within the tetramer. In testing whether the different arrangement of the dimers was due to formation of unstable tetramers, we discovered that while Rim1 forms tetramers at high protein concentration, it dissociates into a smaller oligomeric species at low protein concentrations. A single point mutation at the dimer–dimer interface generates stable dimers and provides support for a dimer–tetramer oligomerization model. The presence of Rim1 dimers in solution becomes evident in DNA binding studies using short ssDNA substrates. However, binding of the first Rim1 dimer is followed by binding of a second dimer, whose affinity depends on the length of the ssDNA. We propose a model where binding of DNA to a dimer of Rim1 induces tetramerization, modulated by the ability of the second dimer to interact with ssDNA

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

PPDB, the Plant Proteomics Database at Cornell

The Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.edu), launched in 2004, provides an integrated resource for experimentally identified proteins in Arabidopsis and maize (Zea mays). Internal BLAST alignments link maize and Arabidopsis information. Experimental identification is based on in-house mass spectrometry (MS) of cell type-specific proteomes (maize), or specific subcellular proteomes (e.g. chloroplasts, thylakoids, nucleoids) and total leaf proteome samples (maize and Arabidopsis). So far more than 5000 accessions both in maize and Arabidopsis have been identified. In addition, more than 80 published Arabidopsis proteome datasets from subcellular compartments or organs are stored in PPDB and linked to each locus. Using MS-derived information and literature, more than 1500 Arabidopsis proteins have a manually assigned subcellular location, with a strong emphasis on plastid proteins. Additional new features of PPDB include searchable posttranslational modifications and searchable experimental proteotypic peptides and spectral count information for each identified accession based on in-house experiments. Various search methods are provided to extract more than 40 data types for each accession and to extract accessions for different functional categories or curated subcellular localizations. Protein report pages for each accession provide comprehensive overviews, including predicted protein properties, with hyperlinks to the most relevant databases

Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92060/1/pmic7070.pd

Update on chloroplast research

Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional characterization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowledge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire complement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants

Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes