Development and optimization of a workflow to enable mass spectrometry-based quantitative membrane proteomics of mature and tolerogenic dendritic cells

PhD ThesisTolerogenic dendritic cells are monocyte-derived dendritic cells (DC) cultured such that they adopt an immunoregulatory phenotype. In vitro, these cells are able to induce and maintain T cell tolerance through deviation of naive T cells to an anti-inflammatory phenotype and induction of anergy in memory T cells. Equivalent cells suppress established arthritis in murine models and tolerogenic DC are presently the subject of a phase I safety and efficacy trial at Newcastle University as part of the AutoDeCRA study. However, in spite of these promising data, we are yet to rigorously explore the basis of the phenotype of tolerogenic DC and lack markers to unequivocally distinguish them from other types of DC. This body of work is concerned with the development of a workflow to enable these questions to be addressed using mass spectrometry-based quantitative proteomics. Specifically, methods have been optimized and validated to enable a) enrichment and proteolytic digestion of membrane proteins, favouring their detection over more abundant cytoplasmic and nuclear proteins in LC/MS; b) differential stable isotope labelling of peptide N- and C-termini, enabling ‘isobaric peptide termini labelling’-based relative quantitation at the MS2 level; c) pipette-tip based anion exchange fractionation of IPTL-labelled peptides prior to LC/MS analysis, broadening depth of proteome coverage. Efforts to apply aspects of the workflow to perform quantitative comparisons of the whole cell proteomes and qualitative profiling of the membrane proteomes of mature and tolerogenic DC are also documented. It is envisaged that future application of this optimized workflow as a whole will enable the identification and relative quantitation of significant numbers of mature and tolerogenic DC plasma membrane proteins. Differentially expressed proteins of interest identified through this approach may then be further investigated for putative roles in tolerance induction

The Structure and Function of Glycoforms

Glycoproteins generally consist of populations of glycosylated variants of a single protein (glycoforms). While the potential oligosaccharide processing pathways available to a protein are dictated by the cell in which it is expressed, the final glycosylation pattern reflects constraints imposed by the three dimensional structure of the individual protein. This thesis explores the extent to which a protein controls its own glycosylation by comparing the glycosylation patterns of soluble forms of leucocyte surface antigens (CD2, CD5d1, CD59, CD5/CD4d3,4 and CD48) expressed in Chinese Hamster Ovary cells (ch.2). In chapter 3 the Nand 0-linked and anchor glycans of human erythrocyte CD59 were characterised; the data suggested that the individual protein may influence the processing of glycosylphosphatidyl inositol anchor glycans. In chapter 4-7 consequences of variable glycosylation were explored by preparing defined sets of glycoforms for testing in functional assays. Individual glycoforms of RNase B reduced the susceptibility of the protein to proteases and modulated the activity of the enzyme (ch.4). IgG rheumatoid factor, from patients with rheumatoid arthritis, contained a specific subset of IgG glycoforms with Fc oligosaccharides terminating in GlcNAc (IgGO) and an extra-glycosylated heavy chain. Exposed GlcNAc residues on multiply presented IgGO bound serum mannose binding protein, suggesting a role for Fc sugars in recognition (ch.6). In chapter 7 a subset of highly active tissue plasminogen activator (tPA) glycoforms, type II dimers, was identified. In the fibrin stimulated activation of plasminogen, glycosylation of tPA site 184 (type I glycoforms) decreased kcat, while the Km was decreased by glycosylation at the variably occupied plasminogen site, Asn289. Chapter 8, which deals with technology, explores applications of HPLC and mass spectrometry to the analysis of oligosaccharides and in chapter 5 capillary electrophoresis was used to resolve transferrin glycoforms, suggesting a rapid diagnostic for carbohydrate deficient glycoprotein syndrome

NUTRITIONAL PEPTIDOMICS: DISCOVERY, QUANTIFICATION, AND FUNCTIONAL ANALYSIS OF PLANT PROTEIN DERIVED PEPTIDES

NUTRITIONAL PEPTIDOMICS: DISCOVERY, QUANTIFICATION, AND FUNCTIONAL ANALYSIS OF PLANT PROTEIN DERIVED PEPTIDES Sector CHIM/10 - Food chemistry Introduction and aims of thesis The study of bioactive peptides is a central issue in the development of innovative therapies. The increased attention for fresher and \u2018greener\u2019 foods and nutraceuticals possessing health-preventing or health-promoting properties makes bioactive peptides suitable candidates for a new era of pharmaceutical products. Analysing and understanding nature and bioactivity of nutritional peptides, typically delivered from parent food proteins, means comprehending an important level of environmental regulation of the human genome: diet is the environmental factor having the most profound life-long influence on health. Although a remarkable progress has been done in protein analysis, as a consequence of proteomic research, and in small molecule analysis, as a consequence of drug discovery/development initiatives, the field of nutritional peptidomic is still quite unexplored and some drawbacks should be addressed. The pharmacological applications of bioactive peptides depend primarily on their ability to be absorbed in order to exert their bioactivity. In addition, it is very likely that peptide sequences are subjected to structural alterations before performing their final activity in vivo due to different events, such as the attack of gastrointestinal enzymes, brush border peptidases, absorption through the intestinal barrier, and attack of intracellular peptidases in the intracellular absorption. Therefore, all of these different aspects about the bioavailability have attracted a growing interest in the last years. In addition, the possibility of the peptides breakdown during the gastrointestinal digestion is one of the most important factor to be considered when evaluating food-derived peptides for the promotion of human health. Chemical stability is also crucial for proper assay development, since these peptides could lose stability when placed in solution or in biological fluids or even before absorption. Once they are delivered, the biodistribution of bioactive peptides may be sometimes hampered as a result of proteolytic attack, primarily due to the action of brush border peptidases overexpressed at the microvilli surface of intestinal cells. However, before evaluating aspects such as the bioavailability, the optimization of hydrolytic conditions and the chemical identification of protein hydrolysates are other important aspects to be highlighted. The broad variety of physiological activities attributed to protein hydrolysates are determined by the type, number, position, and properties of amino acids present in the sequence of bioactive peptides. The optimization of the hydrolytic conditions for the obtainment of bioactive hydrolysates was also addressed in this work. Since peptides derive from proteins, the integration of peptidomics and proteomics methodologies permitted the enlargement of proteomic databank, which may facilitate the improvement of peptidomics platform libraries. In this context, advanced analytical techniques such as those based on mass spectrometry (MS) have emerged as indispensable and irreplaceable tools in the discovery, identification, quantification and functional analysis of bioactive peptides arising from proteolysis. Among the toolkit of techniques developed to investigate proteins at the proteome-wide scale, MS has gained popularity especially because of its ability to handle the hierarchical complexity associated with the biological systems. In addition, MS-based approaches coupled to cell culturing and bioinformatics tools set a new standard in peptide research. Based on these premises, the aim of my PhD project was to set-up and apply MS strategies in order to evaluate: I. The absorption at intestinal level of peptic and tryptic hydrolysates from lupin protein using an in vitro model based on Caco-2 cells, providing for the first time this kind of data on peptides from this seed. II. The modulation of protein-protein interaction (PPI) of proprotein convertase subtilisin/kexin type 9 (PCSK9) with the low density lipoprotein receptor (LDL-R) in HepG2 cells induced by lupin peptides as well as the quantification by MS of the absorbed peptides that had been predicted to be the best inhibitors of the PCSK9-LDLR PPI by molecular modeling. III. The absorption and metabolism of authentic samples of peptides from soy proteins using Caco-2 cells. IV. The extensive investigation of the hempseed proteome including the identification of minor protein components by CPLL methodology. V. The production of some hydrolysates from hempseed protein endowed of hypocholesterolemic properties using different proteases. VI. The enlargement of apricot seed protein databank through an extensive proteome characterization followed by an in silico driven approach for the prediction of the peptides released by simulated gastrointestinal digestion. General conclusion. The results obtained in this work allowed the enlargement of the bioactive-peptide library platform adding a new dimension to the potential health benefits derived from protein from the seeds of different plants of agronomic importance. In details, a growing body of reports on novel peptide sequences and function-structure relationships contributed to the improvement of plant protein and peptide knowledge. Information obtained from characterizing structural components of plant hydrolysates offers useful technological and functional implications for food ingredient formulation or pharmacological use

Proteomická analýza leukemických buněčných linií po ozáření

(CZE) Název dizertační práce: Proteomická analýza leukemických buněčných linií po ozáření V této dizertační práci jsme se zaměřili na objasnění molekulárních mechanismů radiosensibilizace leukemické buněčné linie MOLT-4 specifickou inhibicí kináz z rodiny fosfatidylinositol-3-kináza příbuzných kináz (PIKKs). Byly testovány dva vysoce účinné a selektivní inhibitory VE-821 (inhibitor ATR) a KU55933 (inhibitor ATM), pro jejich účinky na proliferaci, viabilitu a buněčný cyklus neozářených a ozářených buněk MOLT4. Aplikace obou inhibitorů způsobila radiosensibilizaci MOLT-4 buněk a 10 µM VE-821 navíc působil jako silné antiproliferativní agens i v neozářených MOLT-4 buňkách. K dalšímu popisu mechanismů, které jsou zodpovědné za radiosensibilizaci MOLT-4 buněk VE-821 inhibitorem byly použity hmotnostně spektrometrické metody. Pomocí metod kvantitativní proteomiky jsme identifikovali a kvantifikovali změny v proteomu a fosfoproteomu (tj. změny na úrovni fosforylace proteinů) buněk, které byly způsobeny účinkem inhibitoru v ozářených buňkách. Protože detekce a kvantifikace fosforylovaných peptidů v komplexních vzorcích je komplikována mimo jiné jejich relativně nízkým zastoupením, zaměřili jsme se nejprve na výběr optimální metody pro jejich selektivní izolaci ze směsi s nemodifikovanými peptidy....(ENG) Title of the dissertation: Proteomic analysis of gamma-irradiated human leukemic cells In the presented doctoral thesis, we aimed to elucidate molecular mechanisms underlying radiosensitization of MOLT-4 cell line (T-ALL) by specific inhibition of kinases from the phosphatidylinositol-3 kinase-related kinases (PIKKs) family. We tested two highly potent inhibitors of ATR and ATM, VE-821 and KU55933, respectively, for their effects on proliferation, viability, and cell cycle of sham-irradiated and irradiated MOLT-4 cells. Both inhibitors proved to radiosensitize MOLT-4 cells and furthermore, 10 µM VE-821 was shown to act as a strong antiproliferative agent in sham-irradiated MOLT-4 cells. To further describe cellular mechanisms underlying the VE-821-mediated radiosensitization of MOLT-4 cells, we employed high-resolution mass spectrometry to identify and quantify changes in proteome and phosphoproteome of irradiated VE-821-treated cells. As the detection and quantification of phosphorylated peptides in complex biological samples is challenging due to their low stoichiometry, we first compiled and optimized protocol for their enrichment. The protocol was then applied to study changes in radiosensitized MOLT-4 cells. In concordance with our expectations, VE-821 did not cause any significant...Department of Medical BiochemistryÚstav lékařské biochemieFaculty of Medicine in Hradec KrálovéLékařská fakulta v Hradci Králov

Iron uptake and homeostasis in the veterinary pathogen Rhodococcus equi: an integrated omics approach

Rhodococcus equi, a veterinary pathogen that causes pyogranulomatous pneumonia, can secrete low molecular weight chelators called siderophores to scavenge iron when its bioavailability is limited. When iron is plentiful, synthesis of siderophores and ferri–siderophore transport systems are repressed. Current literature on bacterial iron regulation and homeostasis indicates two distinct protein families of global iron-dependent transcriptional repressor: Fur and DtxR. Gram-negative bacteria produce Fur to regulate iron uptake genes and the biosynthesis of siderophores in response to the iron level in the cell. However, the Gram-positive Corynebacteriaceae produce DtxR-like proteins to regulate analogous genes. Much remains undefined with respect to rhodococcal siderophore biosynthesis and uptake. Detailed analysis of the R. equi 103S genome for genes related to iron homeostasis identified two potential metal regulatory genes each from the Fur and DtxR families: iron dependent regulatory protein (IdeR), Diphtheria toxin repressor (DtxR), Ferric uptake regulator A (FurA) and Ferric uptake regulator B (FurB). Bioinformatic analysis confirmed that this complement of genes was conserved throughout Rhodococcus and the Corynebacteriaceae in general. To investigate their individual roles in metal homeostasis, molecular cloning and gene expression was performed, to facilitate analysis of regulator-metal specificities. Each gene was cloned but over-expression for functional analysis could only be achieved for ideR; thus, a thorough systematic analysis could not be achieved. In order to address their individual roles, homology-based protein modelling was used, and comparisons made with characterised homologues from M. tuberculosis. The geometrical conservation of key ligand amino acid residues strongly suggests R. equi utilises ideR as an iron regulator; furB as a zinc regulator, dtxR as a manganese regulator and furA as an oxidative stress response protein. Most bacteria generate an exaggerated response to iron limitation in vitro, however R. equi produces very small siderophore yields s, which has complicated their characterisation. In-frame deletion of the putative metal regulator genes ideR, dtxR, furA and furB was attempted in order to address the hypothesis that de-repression might generate greater yields. All genes were deleted individually; a marked phenotypic difference was noted only for R. equi-ΔfurA, which significantly upregulated the catalase encoded by the neighbouring gene and was coincidentally hyper-resistant to hydrogen peroxide. Surprisingly, analysis of siderophore production in the mutants indicated no increase in yield. The thesis discusses the relevance of this observation to microbial ecology. The availability of these mutants in combination with their predicted metal specificities facilitates the design of experiments to define their individual roles in metal homeostasis beyond the scope of this thesis. The combination of ‘omic’ analyses was attempted here to initiate the ultimate definition of the complex molecular network associated with iron uptake. The genomic investigation informed hypothesis building for the other omic analyses. It suggested R. equi is capable of synthesising two siderophores, rhequibactin and rhequichelin; up to three had previously been postulated in the literature. Culture optimisation was required to deliver a robust experimental design to impose iron limitation in isolation from other stresses. Once medium composition and biomarker-indicated harvesting criteria were established, biomass and associated secretomes were produced en masse for integrated omics analysis. A comparative untargeted metabolomics study demonstrated an adapted iron-starved metabolome; strong siderophore candidates were then investigated using a targeted strategy. A strong candidate metabolite was identified by mass that appeared to be responsible for a heterobactin-like chromophore, however further biochemical characterisation has been elusive. Interestingly, the metabolite readily precipitates on complexation with iron, an observation also made for heterobactins. Secondly, a transcriptomic study was attempted to study the global gene expression under iron starvation, and the impact of the loss of the IdeR in the deletion mutant generated in this work. However, the RNA extraction proved particularly challenging likely due to difficulties arising from lysis of the mycolic acid-containing cell wall. In the absence of a high-quality transcriptome sample, the study did not advance further and other aspects of the study were prioritised. Finally, a comparative proteomic analysis into iron regulatory mechanisms associated with the rhodococcal cell wall was performed. Current literature deliberates how R. equi uses a range of strategies to overcome iron limitation through proposed uptake mechanisms associated with translocation across the cytoplasmic membrane via ABC transport systems, while no consideration has yet been made with regards to transport across the mycolic acid-containing cell wall structure. In this study no obvious candidate proteins for ferri-sideophore transport across the mycolate region were identified, therefore it is possible that R. equi utilises facilitated diffusion via a porin for entry of ferri-siderophore complexes into the pseudoperiplasm, where a substrate-binding lipoprotein may act as the primary receptor to facilitate cytosolic transfer through an ABC transport system