A Time-Resolved Cryo-EM Study of Saccharomyces cerevisiae 80S Ribosome Protein Composition in Response to a Change in Carbon Source.

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol. Using cryo-EM combined with the computational classification approach, it is found that a fraction of the yeast cells' 80S ribosomes lack ribosomal proteins at the entrance and exit sites for tRNAs, including uL16(RPL10), eS1(RPS1), uS11(RPS14A/B), and eS26(RPS26A/B). This fraction increased after a change from glucose to glycerol medium. The quantitative structural analysis supports the hypothesis that ribosomes are dynamic complexes that alter their composition in response to changes in growth or environmental conditions.

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

A cost-sensitive online learning method for peptide identification.

BACKGROUND: Post-database search is a key procedure in peptide identification with tandem mass spectrometry (MS/MS) strategies for refining peptide-spectrum matches (PSMs) generated by database search engines. Although many statistical and machine learning-based methods have been developed to improve the accuracy of peptide identification, the challenge remains on large-scale datasets and datasets with a distribution of unbalanced PSMs. A more efficient learning strategy is required for improving the accuracy of peptide identification on challenging datasets. While complex learning models have larger power of classification, they may cause overfitting problems and introduce computational complexity on large-scale datasets. Kernel methods map data from the sample space to high dimensional spaces where data relationships can be simplified for modeling. RESULTS: In order to tackle the computational challenge of using the kernel-based learning model for practical peptide identification problems, we present an online learning algorithm, OLCS-Ranker, which iteratively feeds only one training sample into the learning model at each round, and, as a result, the memory requirement for computation is significantly reduced. Meanwhile, we propose a cost-sensitive learning model for OLCS-Ranker by using a larger loss of decoy PSMs than that of target PSMs in the loss function. CONCLUSIONS: The new model can reduce its false discovery rate on datasets with a distribution of unbalanced PSMs. Experimental studies show that OLCS-Ranker outperforms other methods in terms of accuracy and stability, especially on datasets with a distribution of unbalanced PSMs. Furthermore, OLCS-Ranker is 15-85 times faster than CRanker.

AS03-Adjuvanted H5N1 Avian Influenza Vaccine Modulates Early Innate Immune Signatures in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.

Identification of Changing Ribosome Protein Compositions using Mass Spectrometry.

The regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to environmental stimuli. MS is applied to identify quantitative changes in the protein composition of S. cerevisiae 80S ribosomes in response to different environmental stimuli. Using quantitative MS, it is found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast is switched from growth in glucose to glycerol. By using yeast genetics and polysome profiling, it is shown that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. The quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions.

Critical Role for Saccharomyces cerevisiae Asc1p in Translational Initiation at Elevated Temperatures.

The eukaryotic ribosomal protein RACK1/Asc1p is localized to the mRNA exit channel of the 40S subunit but lacks a defined role in mRNA translation. Saccharomyces cerevisiae deficient in ASC1 exhibit temperature-sensitive growth. Using this null mutant, potential roles for Asc1p in translation and ribosome biogenesis are evaluated. At the restrictive temperature the asc1Δ null mutant has reduced polyribosomes. To test the role of Asc1p in ribosome stability, cryo-EM is used to examine the structure of 80S ribosomes in an asc1Δ yeast deletion mutant at both the permissive and nonpermissive temperatures. CryoEM indicates that loss of Asc1p does not severely disrupt formation of this complex structure. No defect is found in rRNA processing in the asc1Δ null mutant. A proteomic approach is applied to survey the effect of Asc1p loss on the global translation of yeast proteins. At the nonpermissive temperature, the asc1Δ mutant has reduced levels of ribosomal proteins and other factors critical for translation. Collectively, these results are consistent with recent observations suggesting that Asc1p is important for ribosome occupancy of short mRNAs. The results show the Asc1 ribosomal protein is critical in translation during heat stress.

Proteomics show antigen presentation processes in human immune cells after AS03-H5N1 vaccination.

Adjuvants enhance immunity elicited by vaccines through mechanisms that are poorly understood. Using a systems biology approach, we investigated temporal protein expression changes in five primary human immune cell populations: neutrophils, monocytes, natural killer cells, T cells, and B cells after administration of either an Adjuvant System 03 adjuvanted or unadjuvanted split-virus H5N1 influenza vaccine. Monocytes demonstrated the strongest differential signal between vaccine groups. On day 3 post-vaccination, several antigen presentation-related pathways, including MHC class I-mediated antigen processing and presentation, were enriched in monocytes and neutrophils and expression of HLA class I proteins was increased in the Adjuvant System 03 group. We identified several protein families whose proteomic responses predicted seroprotective antibody responses (>1:40 hemagglutination inhibition titer), including inflammation and oxidative stress proteins at day 1 as well as immunoproteasome subunit (PSME1 and PSME2) and HLA class I proteins at day 3 in monocytes. While comparison between temporal proteomic and transcriptomic results showed little overlap overall, enrichment of the MHC class I antigen processing and presentation pathway in monocytes and neutrophils was confirmed by both approaches.

Sculpting MHC class II-restricted self and non-self peptidome by the class I Ag-processing machinery and its impact on Th-cell responses.

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery - which supplies peptides for presentation by class I molecules - plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4(+) T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4(+) T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4(+) T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4(+) T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.

The sodium channel accessory subunit Navß1 regulates neuronal excitability through modulation of repolarizing voltage-gated K⁺ channels.

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navß1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navß1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navß1. Biochemical and voltage-clamp experiments further demonstrated that Navß1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navß1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.

A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

Peptide identification based on fuzzy classification and clustering.

BACKGROUND: The sequence database searching has been the dominant method for peptide identification, in which a large number of peptide spectra generated from LC/MS/MS experiments are searched using a search engine against theoretical fragmentation spectra derived from a protein sequences database or a spectral library. Selecting trustworthy peptide spectrum matches (PSMs) remains a challenge. RESULTS: A novel scoring method named FC-Ranker is developed to assign a nonnegative weight to each target PSM based on the possibility of its being correct. Particularly, the scores of PSMs are updated by using a fuzzy SVM classification model and a fuzzy silhouette index iteratively. Trustworthy PSMs will be assigned high scores when the algorithm stops. CONCLUSIONS: Our experimental studies show that FC-Ranker outperforms other post-database search algorithms over a variety of datasets, and it can be extended to solve a general classification problem with uncertain labels.

Dap1/PGRMC1 binds and regulates cytochrome P450 enzymes.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

Studies of the mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes.

Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pH-dependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. EPR analyses of spin-labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH.

Assessing the components of the eIF3 complex and their phosphorylation status.

The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC-MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. In Vivo metabolic labeling of proteins with (32)P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry.

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.

Saccharomyces cerevisiae Gis2 interacts with the translation machinery and is orthogonal to myotonic dystrophy type 2 protein ZNF9.

The myotonic dystrophy type 2 protein ZNF9/CNBP is a small nucleic acid binding protein proposed to act as a regulator of transcription and translation. The precise functions and activity of this protein are poorly understood. Previous studies suggested that ZNF9 regulates translation and facilitates the process of cap-independent translation through interactions with mRNA and the translating ribosome. To help determine the role played by ZNF9 in the activation of translation initiation, we combined genetic and biochemical analysis of the putative ZNF9 ortholog GIS2, in the budding yeast Saccharomyces cerevisiae. Purification of the Gis2p protein followed by mass spectrometry based-proteomic analysis identified a large number of co-purifying ribosomal subunits and translation factors, strongly suggesting that Gis2p interacts with the protein translation machinery. Polysome profiling and ribosome isolation experiments confirm that Gis2p physically interacts with the translating ribosome. Interestingly, expression of yeast Gis2p in HEK293T cells activates cap-independent translation driven by the 5'UTR of the ODC gene. These data suggest that Gis2 is functionally orthologous to ZNF9 and acts as a cap-independent translation factor.

Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic.

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men.

Quantitative analysis of the secretome of TGF-beta signaling-deficient mammary fibroblasts.

Transforming growth factor beta (TGF-beta) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-beta type II receptor (TbetaRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TbetaRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TbetaRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TbetaRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-beta signaling pathway in fibroblasts because systemic inhibition of TGF-beta signaling pathways is being considered as a potential cancer therapy.

A proximal activator of transcription in epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

A proteomics analysis of yeast Mot1p protein-protein associations: insights into mechanism.

Yeast Mot1p, a member of the Snf2 ATPase family of proteins, is a transcriptional regulator that has the unusual ability to both repress and activate mRNA gene transcription. To identify interactions with other proteins that may assist Mot1p in its regulatory processes, Mot1p was purified from replicate yeast cell extracts, and Mot1p-associated proteins were identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. Using this approach we generated a catalog of Mot1p-interacting proteins. Mot1p interacts with a range of transcriptional co-regulators as well as proteins involved in chromatin remodeling. We propose that interaction with such a wide range of proteins may be one mechanism through which Mot1p subserves its roles as a transcriptional activator and repressor.