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Quantitative profile of lysine methylation and acetylation of histones by LC-MS/MSGallardo Alcayaga, Karem Daniela 29 March 2017 (has links) (PDF)
Histone post-translational modifications (PTMs), as the histone code assumes, are related with regulation of gene transcription, an important mechanism of cells in the differentiation process. Many PTMs are simultaneously present in histone proteins, and changes in the PTM stoichiometric ratios can have several effects, like changes in the chromatin structure leading to a transcriptionally active or repressive state. Significant progresses were made to map variations of histone PTMs by mass spectrometry (MS), and although many protocols were developed there are still some drawbacks. Incomplete and side reactions were identified, which can directly affect the quantification of histone PTMs, because both (incomplete and side reactions) can be misinterpreted as endogenous histone post translational modifications.
Therefore, a protocol for derivatization of histones with no noticeable undesired reactions (<10%) was required. In this thesis a new chemical modification methodology is presented, which allows the improvement of sequence coverage by acylation with propionic anhydride of lysine residues and N-terminal (free ε- and α- amino groups) and trypsin digestion. more than 95% of complete reaction was achieved with the new derivatization methodology. This strategy (chemical derivatization of histones), in combination with bottom-up MS approach, allows the quantification of lysine methylation (Kme) and acetylation (Kac) in histones from Saccharomyces cerevisiae (S.cerevisiae), mouse embryonic stem cells (mESCs) and human cell lines. The results showed histone H3 PTM pattern as the most variable profile regarding histone Kme and Kac across the three different organisms and experimental conditions. Therefore, it was concluded that quantification of H3 PTM pattern can be used to examine changes in chromatin states when cells are subjected to any kind of perturbation.
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Post-translational Regulation of Plant Fatty Acid Desaturases as Expressed in Saccharomyces cerevisiaeBourassa, Linda 16 May 2008 (has links)
Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.
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Characterization of the PIAS family (protein inhibitors of activated STATs) of the sumoylation E3 ligases.January 2005 (has links)
Ma Kit Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 189-206). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of Contents --- p.iii / Abstract --- p.xi / 摘要 --- p.xiv / Abbreviation List --- p.xv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ubiquitination --- p.1 / Chapter 1.1.1 --- Ubiquitin --- p.1 / Chapter 1.1.2 --- Ubiquitin Pathway --- p.3 / Chapter 1.1.3 --- Functions of Ubiquitination --- p.5 / Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8 / Chapter 1.2 --- SUMO Proteins --- p.10 / Chapter 1.2.1 --- SUMO Isoforms --- p.10 / Chapter 1.2.2 --- SUMO Structure --- p.11 / Chapter 1.3 --- Sumoylation --- p.14 / Chapter 1.3.1 --- Functions of Sumoylation --- p.14 / Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15 / Chapter 1.3.1.2 --- Function of Sumoylation on Transcription Factors / Chapter 1.3.1.3 --- Specific Function of SUMO-2/3 Conjugation / Chapter 1.3.2 --- Sumoylation Pathway --- p.19 / Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24 / Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23 / Chapter 1.4.2 --- Structure of PI AS --- p.23 / Chapter 1.4.3 --- Function of PI AS --- p.27 / Chapter 1.5 --- Aims of Study --- p.29 / Chapter Chapter 2 --- Materials & Methods --- p.30 / Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30 / Chapter 2.1.1 --- Primer Design --- p.30 / Chapter 2.1.2 --- Semi-quantitative PCR --- p.31 / Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31 / Chapter 2.1.2.2 --- PCR --- p.32 / Chapter 2.2 --- DNA Cloning --- p.34 / Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34 / Chapter 2.2.1.1 --- Primer Design --- p.34 / Chapter 2.2.1.2 --- PCR --- p.36 / Chapter 2.2.1.3 --- Purification of PCR Product --- p.37 / Chapter 2.2.2 --- Restriction Digestion --- p.37 / Chapter 2.2.3 --- Ligation --- p.40 / Chapter 2.2.4 --- Transformation --- p.40 / Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40 / Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41 / Chapter 2.2.5 --- Plasmid Preparation --- p.42 / Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43 / Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43 / Chapter 2.3 --- Subcellular Localization Study --- p.45 / Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45 / Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46 / Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46 / Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47 / Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48 / Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48 / Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49 / Chapter 2.3.6 --- Western Blotting --- p.49 / Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49 / Chapter 2.3.6.2 --- Protein Extraction --- p.50 / Chapter 2.3.6.3 --- Protein Quantification --- p.51 / Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51 / Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52 / Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54 / Chapter 2.4.1 --- Sample Preparation --- p.54 / Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54 / Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55 / Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55 / Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57 / Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57 / Chapter 2.4.3.2 --- Silver Staining --- p.58 / Chapter 2.4.4 --- Image Analysis --- p.59 / Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60 / Chapter 2.4.5.1 --- Sample Preparation --- p.60 / Chapter 2.4.5.2 --- Data Acquisition --- p.62 / Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62 / Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63 / Chapter 2.5.1 --- RT-PCR Analysis --- p.63 / Chapter 2.5.1.1 --- RNA Extraction --- p.63 / Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64 / Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64 / Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65 / Chapter 2.5.2 --- Western Blotting --- p.66 / Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67 / Chapter 2.6.1 --- Preparation of Competent Cells --- p.67 / Chapter 2.6.2 --- Small Scale Expression --- p.67 / Chapter 2.6.2.1 --- Transformation --- p.67 / Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68 / Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70 / Chapter 2.6.3.1 --- Transformation --- p.70 / Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70 / Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71 / Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71 / Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73 / Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73 / Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73 / Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74 / Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74 / Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74 / Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75 / Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76 / Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76 / Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77 / Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77 / Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77 / Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78 / Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78 / Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79 / Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79 / Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80 / Chapter 2.6.7.1 --- Transformation --- p.80 / Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80 / Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80 / Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80 / Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81 / Chapter Chapter 3 --- Results --- p.82 / Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82 / Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82 / Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82 / Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83 / Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83 / Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83 / Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84 / Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84 / Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90 / Chapter 3.2.1 --- Overexpression Confirmation --- p.90 / Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91 / Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94 / Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94 / Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95 / Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95 / Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96 / Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97 / Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112 / Chapter 3.3.1 --- Protein Expression Profiles after Transient Transfection / Chapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113 / Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124 / Chapter 3.4.1 --- RT-PCR Analyses --- p.124 / Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124 / Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125 / Chapter 3.4.2 --- Western Blotting --- p.128 / Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128 / Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129 / Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129 / Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133 / Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133 / Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135 / Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137 / Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137 / Chapter 3.5.3.2 --- Cleavage after Elution --- p.137 / Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138 / Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138 / Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141 / Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141 / Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141 / Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143 / Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147 / Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148 / Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148 / Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153 / Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153 / Chapter 3.5.6.2 --- Purification by Heparin --- p.153 / Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154 / Chapter Chapter 4 --- Discussion --- p.157 / Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157 / Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157 / Chapter 4.1.2 --- Importance of Sumoylation --- p.158 / Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159 / Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160 / Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162 / Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162 / Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163 / Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164 / Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165 / Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167 / Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169 / Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169 / Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170 / Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172 / Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172 / Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174 / Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175 / Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176 / Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178 / Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178 / Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180 / Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180 / Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181 / Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182 / Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182 / Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183 / Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184 / Chapter 4.5 --- Conclusion --- p.185 / Chapter 4.6 --- Future Perspectives --- p.187 / Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187 / Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187 / Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188 / References --- p.189 / Appendix --- p.207
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Etude des facteurs structuraux influençant la carbonatation de la lysine 70 chez la beta-lactamase OXA-10 de Pseudomonas aeruginosa/Study of structural factors influencing the lysine 70 carboxylation of OXA-10 beta-lactamaseVercheval, Lionel 21 January 2010 (has links)
Throughout this thesis, we studied the biochemical and structural impact of the essential residues on the activity of class D beta-lactamases. The production of these enzymes plays a major role in the bacterial resistance. Our work is subdivided in two parts : the study of the post-translational modification of lysine 70 and the screening of new potential inhibitors for the class D β-lactamases.
The first part concerns the impact of the residues tryptophan 154 and valine 117 located in the hydrophobic core. Our data indicate that the mutation of tryptophan 154 in alanine or glycine lead to a large decrease of the catalytic efficiencies of the beta-lactamase. The apo-enzyme structures of these mutants show that the lysine 70 is not carboxylated. This absence of carboxylate group induces a modification of the hydrogen network of the active site. The analysis of the complex structure of W154A-benzylpenicillin demonstrates that the deacylation step is clearly the most affected by the mutation. The mutation of tryptophan 154 in histidine leads to a slight decrease of catalytic efficiencies because the imidazol group of histidine mimics the indole group of tryptophan 154. The apo-enzyme structure reveals that lysine 70 is partially carboxylated and stabilized by an hydrogen bond between the carboxylate group and the imidazol group.
In the case of the V117T mutant, a strong increase of the catalytic constant values is observed at 50 mM in NaHCO3. The structure of this mutant at pH 8.0 shows that the lysine 70 is partially carboxylated in the monomer A.
The determination of individual rate constants of acylation and deacylation steps indicates that the deacylation is the limiting step for the class D beta-lactamase. The k2/k3 ratio is similar between the V117T mutant and the wild-type enzyme. The mutation of lysine 70 in alanine or cysteine leads to a large decrease of the deacylation constants inducing a poorly efficient enzyme. The obtaining of the K70C-Ampicillin complex by X-ray cristallography and the trapping of acyl-enzyme by reaction with fluorescent ampicillin are supplemental proofs that the deacylation step is the limiting rate.
By crystallographic and kinetic studies, we demonstrate that the chloride inhibition of the class D beta-lactamases is due to a competition between the carboxylate group of lysine 70 and the chloride ions. At high concentration in bicarbonate, this inhibition is abolished for the wild-type enzyme.
The second part of this work concerns the screening of the citrate and aminophosphonate derivated molecules for the class D beta-lactamases. In the case of OXA-10, a citrate molecule is strongly stabilized by hydrogen bonds in the active site. The benzyl esters derivatives of citrate inhibits OXA-10(KI = 20 µM) but the hydrophobic substituents are necessary to obtain a good inhibition.
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Investigating the inhibitor and substrate diversity of the JmjC histone demethylasesSchiller, Rachel Shamo January 2016 (has links)
Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N<sup>ε</sup>-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.
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Déterminants moléculaires non-apoptotiques de l'activité oncogénique de Bcl-xL : rôle de la monodéamidation de Bcl-xL / Non apoptotic molecular of oncogenic activity of Bcl-xL : role of Bcl-xL monodeamidationEL Dhaybi, Mohamad 24 October 2017 (has links)
Bcl-xL est un oncogène surexprimé dans plusieurs types de cancers et qui joue un rôle important dans la survie cellulaire en régulant deux processus: l'apoptose et l'autophagie. Récemment, nous avons identifié l'existence d'une nouvelle forme de Bcl-xL qui subit une simple déamidation sur le résidu Asn52. Cette forme monodéamidée est exprimée en conditions contrôles et apparaît spontanément in vitro et in vivo. La déamidation de Bcl-xL produit un mélange de protéines contenant en position 52 soit un résidu Asp, soit un résidu isoAsp. L'objectif de cette thèse est de caractériser les fonctions de ces deux espèces protéiques, et de déterminer comment la monodéamidation de Bcl-xL modifie les fonctions de survie de cet oncogène. Nous avons montré que le mutant déamido-mimétique Bcl-xL N52DN66A conserve la même fonction anti-apoptotique que Bcl-xL native, mais présente une activité autophagique plus grande, et des propriétés oncogéniques et tumorigéniques altérées in vitro, ex vivo et in vivo. Nous avons étudié certains des mécanismes impliqués dans la régulation de l'autophagie et les propriétés oncogéniques comme la voie mTor, les voies de signalisation médiées par l'oncogène Ras, ainsi que l'activité métabolique et l'état souche des cellules. D'autre part, nous avons aussi développé des tests in vitro pour analyser les interactions établies par les formes déamidées de Bcl-xL comportant un isoAsp. L'ensemble de nos données permet de suggérer une régulation des fonctions de Bcl-xL par des mécanismes indépendants de l'apoptose, et renforce l'importance d'explorer les fonctions non apoptotiques de cette protéine pour mettre en évidence sa capacité à promouvoir la survie cellulaire et entraîner la progression du cancer. / Bcl-xL is an oncogene overexpressed in many types of cancer and which promotes cell survival by regulating two cellular processes : apoptosis and autophagy. We have recently identified a new form of this oncogene, which results from the deamidation of Asn52. This monodeamidated form is expressed under control conditions and is ubiquitously found in vitro and in vivo. Bcl-xL monodeamidation produces a mixture of proteins containing either an Asp residue or an IsoAsp residue in position 52. Our goal is to caracterise the functions of both species, and to determine how Bcl-xL monodeamidation modifies the survival functions of this oncogene. We have shown that the deamidomimetic mutant Bcl-xL N52DN66A retains the same anti-apoptotic function as the native protein, but exhibits enhanced autophagic activity and impaired clonogenic and tumorigenic properties in vitro, ex-vivo, and in vivo. We have studied certain of the mechanisms which can be involved in the regulation of autophagy and oncogenic properties of Bcl-xL such as mTor, Ras oncogene signaling pathway, metabolic activity measurement and stemness. We also implement in vitro assays to analyse the interactions established by isoAsp containing forms of Bcl-xL. Altogether our results support the view that deamidation regulates Bcl-xL oncogenic properties through apoptosis-independent mechanisms, and reinforce the importance of deciphering the non apoptotic functions of this protein to tackle its ability to sustain cell survival and drivecancer progression.
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Development of a ‘tool box’ for generating designer nucleosomes in high throughput fashionMahler, Henriette 22 December 2016 (has links)
No description available.
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Régulation des aquaporines et réponse des racines d'Arabidopsis thaliana à des stimuli abiotiques et nutritionnels. / Regulation of aquaporins and response of Arabidopsis thaliana roots to abiotic and nutritional stimuli.Di Pietro, Magali 13 December 2011 (has links)
La conductivité hydraulique racinaire (Lpr) traduit la facilité du passage de l'eau au travers des racines. Ce paramètre, majoritairement contrôlé par l'activité de canaux hydriques membranaires (aquaporines), est modulable par diverses contraintes environnementales. Ce travail a permis de caractériser, sur un même organisme (Arabidopsis), les effets d'un ensemble de contraintes abiotiques et biotiques, représentatives de situations environnementales, sur la Lpr. Alors que la flagelline n'affecte pas la Lpr, les contraintes osmotiques (NaCl, mannitol), oxydantes (H2O2, NO) et nutritionnelles (carence en phosphate, en nitrate, culture des plantes en nuit prolongée) inhibent la Lpr. Par contre, la réalimentation en phosphate ainsi que l'addition de saccharose à des plantes cultivées en nuit prolongée stimulent la Lpr. Une approche phosphoprotéomique quantitative, basée sur l'analyse par MS de protéines microsomales racinaires purifiées à partir de plantes cultivées dans trois de ces contextes (NaCl, NO, phosphate) a permis de quantifier les variations d'abondance de l'ensemble des aquaporines racinaires ainsi que de leur état de MPT. D'un point de vue qualitatif, 22 aquaporines ont été identifiées dans la racine ainsi que plusieurs types de MPTs, incluant des nouveaux sites de phosphorylation (7), de méthylation (13 à 15), de formylation (4) et de déamidation (25 à 26). D'un point de vue quantitatif, cette étude a permis de conclure que les observations réalisées au niveau de la Lpr sont la résultante de mécanismes multifactoriels incluant l'état de phosphorylation des trois sites de l'extrémité C terminale de PIP2;1/2;2/2;3, l'état de phosphorylation de l'extrémité N terminale de PIP1;1/1;2, ainsi que les aquaporines TIPs. Ce travail permet donc de proposer de nouveaux mécanismes moléculaires impliqués dans la régulation de la Lpr en réponse à des contraintes de l'environnement / The water uptake capacity of plant roots (root hydraulic conductivity, Lpr) is mainly determined by water channels (aquaporins) and is modulated by environmental constraints. The present work characterised, in a unique organism (Arabidopsis), effects on Lpr of abiotic and biotic constraints representative of environmental situations. Whereas flagelline does not affect Lpr, osmotic (NaCl or mannitol), oxidative (H2O2 or NO) and nutritional (phosphate or nitrate starvation, prolonged night) stimuli inhibit Lpr. However, phosphate and sucrose resupply stimulate Lpr. A phosphoproteomics approach based on MS analysis of microsomal proteins extracted from roots of plants cultivated in different environmental constraints (NaCl, NO,phosphate starvation and resupply) allowed to quantify variations of abundance of roots aquaporins and of their PTMs. As a qualitative point of view, 22 aquaporins were identified in roots as well as several post-translational modifications including new sites of phosphorylation (7), methylation (13 to 15), formylation (4) and deamidation (25 to 26). From a quantitative point of view, the present work drove to the conclusion that the modulations of Lpr result from multifactorial mechanisms including the phosphorylation status of the C terminal part of PIP2;1/2;2/2;3 and of the N-terminal part of PIP1;1/1;2 and TIP aquaporins. This study proposes new molecular mechanisms implicated in Lpr regulation in response to various environmental situations.
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Next-generation Protein Sequencing (NGPS) For Determining Complete Sequences for Unknown Proteins and AntibodiesHoward, Alexis S. 01 January 2021 (has links)
Next-Generation protein sequencing (NGPS) creates newfound ways of fully identifying every protein species in a single biological organism. It is an effort to use technology to determine proteomic data. The purpose of this research project is to use the current technology to sequence proteins and potentially find treatments for some diseases that are common today. Through NGPS, scientists can identify low abundant proteins including those that go through post-translational modifications (PTM) [1]. NGPS will allow us to fully determine protein sequences from protein samples using mass spectrometry with the ultimate goal of being able to determine the primary sequence of the protein in the given sample [1]. Antibodies are a specific class of proteins that aid our bodies in the immune response. Due to their variability in the complementary-determining region (CDR), NGPS will be used to determine the heavy and light chain sequences [2]. The goal of this technology is to fully determine the primary sequence of a protein in a given sample. The randomness of an antibody’s variable (V), diversity (D), and joining (J) genes (VDJ recombination) makes each protein unique. VDJ recombination refers to the process of T cells and B cells randomly assembling different gene segments. This process allows the antibody to make specific receptors that can recognize different molecules presented on the surface of antigens. Proteases are enzymes that break down proteins and peptides. By using different proteases with varying cutting rules, we can digest the antibody and run it through high mass accuracy determining instrument [1]. NGPS allows us to utilize mass spectrometry technology to measure proteins or polypeptides. Because of these measurements, post-translation modifications, including glycosylation, can be detected, unlike in DNA sequencing technology. Protein sequencing has the opportunity to play a major role in the fight against the COVID-19 outbreak and serve as curative measures for the treatment and Type 2 Diabetes [3]. Proteomics can serve as the basis of vaccine development as well as monitoring treatment. Utilizing techniques such as mass spectrometry could reveal the structure of the virus and ultimately allow for engineered tissues to produce the protein in large amounts in a lab setting. Currently, many companies are utilizing highly sensitized technology to carry out the goals of NGPS. The Oxford Nanopore is a company that uses technology to develop and explore more ways to undergo protein analysis. The methods used by this company involve using protein nanopores to mutate residues in pores to determine the overall sequence. The company utilizes modified aptamers that are drawn to the nanopore current. These aptamers can bind with some, but not all pores, allowing for the differentiation between target and non-target proteins. Nicoya Life Sciences is another company that uses Open Surface Plasmon Resonance (SPR) to detect molecular interactions. SPR uses an analyte (a mobile molecule) to bind to a ligand and observe changes in the refractive index. SPR allows researchers the ability to characterize the binding kinetics and affinities of monoclonal antibodies. SPR is an extremely promising technique to sequence proteins due to its flexibility in being able to work with a variety of molecules including lipids, nucleic acids, cells, viruses, nanoparticles, proteins, antibodies, carbohydrates, and more. The original goal behind NGPS was to establish a method to sequence proteins to aid in the early detection of common diseases such as Type 2 Diabetes. After significant research, it is now known that NGPS can be done in a variety of ways to accomplish a common goal—sequencing proteins and understanding how amino acids affect the human body. In the case of diseased states, NGPS can help researchers find ways to diagnose, treat, and cure diseases early on. Focusing on antibodies allows us to manipulate the body’s immune response to rid the host of pathogens. NGPS, however, is advancing at a much slower rate than anticipated by companies due to its many limitations including not being able to sequence large peptides, difficulties in material and composition of the sample, and needing to label small peptides to begin degradation. Ideally, finding a way to combine the high accuracy and specificity of certain techniques, the ability to detect low abundant proteins in others, and the flexibility of Open SPR would allow researchers and companies to create the standard for NGPS. Creating effective antibodies is precisely why NGPS has such great potential today. Ultimately, I found that as a standalone, Open SPR is the most effective method. However, as the research shows, there are limitations with each method, including Open SPR. The conclusion shows that it is necessary to find a combination of these techniques and create an accurate method, potentially using different technologies, to establish the most effective way to sequence proteins.
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MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY OF BACTERIAL RIBOSOMAL PROTEINS AND RIBOSOMESSUH, MOO-JIN 27 May 2005 (has links)
No description available.
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