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111	Chemical Approaches to Elucidate Lysine Phosphorylation Hauser, Anett 12 February 2021 (has links) Reversible Phosphorylierung ist die bekannteste posttranslationale Modifikation (PTM) und die O-Phosphomonoester von Serin, Threonin und Tyrosin galten lange als die einzigen relevanten Vertreter. Vor kurzem wurden erste Erkenntnisse über die biologische Relevanz von labilen Phosphorylierungen veröffentlicht, z.B. die Phosphorylierung von Histidin, Arginin und Cystein sowie die Pyrophosphorylierung von Serin und Threonin. Auch die Aufklärung von Phospho-Lysin (pLys) wurde mit der Etablierung einer chemoselektiven Synthese zur Darstellung ortsselektiv phosphorylierter Lysinpeptide und der Entwicklung massenspektrometrischer Protokolle zur eindeutigen pLys-Identifizierung in Angriff genommen. Dennoch wurde bisher kein endogenes pLys beschrieben oder eingehende Untersuchungen mit interagierenden Enzymen durchgeführt. In dieser Arbeit werden mehrere Ansätze zur Erweiterung des Wissens über pLys vorgestellt. Dazu gehören das Design einer alternativen Syntheseroute zu pLys-Peptiden und die Entwicklung sowie Evaluierung von zwei stabilen Analoga als Bausteine für die Peptidsynthese. Weiterhin wurde die Protonierung des Phosphoramidatstickstoffs untersucht. In systematischen Enzymaktivitätsassays wurden die Wechselwirkungen zwischen Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) und verschiedenen Phospho-Substraten untersucht. Dabei zeigte sich eine ausgeprägte Selektivität für pLys, eine hohe Sequenzabhängigkeit der LHPP-Aktivität und ein klares Bindungsmotiv. Darüber hinaus wurden proteomische Methoden hinsichtlich ihrer Eignung für pLys-Peptide evaluiert. Im Laufe dieser Untersuchung wurden mehrere pLys-Immunogene für die Generierung von monoklonalen anti-pLys-Antikörpern und ein Workflow für die Histontrennung und -analyse entwickelt. Des Weiteren wurde die chelationsverstärkte Fluoreszenz von markierten Phospho-Peptiden als Werkzeug zur Bestimmung des Phosphorylierungsgrades in Enzymaktivitäts- oder Stabilitätsassays untersucht. / Reversible phosphorylation is the most prominent post-translational modification (PTM) and the O-phosphomonoesters of serine, threonine and tyrosine have been considered as the only notable forms for long time. Recent developments have paved the way to insights into the biological relevance of labile phosphorylations, e.g. phosphorylation of histidine, arginine and cysteine as well as pyrophosphorylation of serine and threonine. Also, the elucidation of phospho-lysine (pLys) was tackled with the establishment of a chemoselective synthesis to obtain site-selectively phosphorylated lysine peptides and the development of mass spectrometric protocols to unambiguously identify modification sites. Nonetheless, no endogenous pLys site has been described or in-depth investigations of interacting enzymes have been conducted. In this thesis, several approaches to enhance the knowledge about pLys are introduced. These include the design of an alternative synthesis route to pLys peptides and the development as well as evaluation of two stable analogues as building blocks for peptide synthesis. Furthermore, the protonation of the phosphoramidate-nitrogen was investigated. In systematic phosphoramidate hydrolase and phosphatase activity assays, the interactions between phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) and various phospho-substrates were examined. Thereby, distinct selectivity for pLys, high sequence dependence of LHPP activity and a distinct binding motif were revealed. In addition to that, proteomic methods were evaluated regarding their suitability for pLys peptides. Over the course of this investigation, several pLys immunogens for the generation of monoclonal anti-pLys antibodies and a workflow for histone separation and analysis were developed. Furthermore, chelation-enhanced fluorescence of labeled phospho-peptides was studied as a tool for determining the degree of phosphorylation in enzyme activity or stability assays. posttranslationale Modifikation labile Phosphorylierung Phospho-Lysin chemoselektive Reaktionen biochemische Assays post-translational modification phospho-lysine chemoselective reactions biochemical assays labile phosphorylation 547 Organische Chemie VK 8577 VK 8567 ddc:540 ddc:547
112	Decoding Complex Architectures of K48- and K63-linked Ubiquitin Chains Waltho, Anita 07 February 2025 (has links) Ubiquitinierung ist eine posttranslationalen Modifikation (PTM), die nahezu alle zellulären Prozesse reguliert. Diese Fähigkeit wird durch den Ubiquitin (Ub)-Code erreicht, der durch Merkmale wie Verknüpfungstyp, Kettenlänge und Verknüpfungskombinationen definiert wird. Wichtige Komponenten des Ub-Systems sind die Ub-bindenden Proteine (UbBPs), die den Ub-Code lesen und in zelluläre Funktionen übersetzen. Obwohl viele verknüpfungsspezifische UbBPs beschrieben wurden, ist nicht vollständig verstanden, wie sie komplexere Ub-Architekturen entschlüsseln. Ub-Verzweigungen, bei denen ein Ub mit mehr als einem anderen Ub verbunden ist, und die Ub-Kettenlänge sind solche Merkmale, die noch unzureichend erforscht sind. In dieser Arbeit wurden die beiden häufigsten Verknüpfungstypen K48- und K63-gebundenes Ub auf Verzweigungs- und Längenspezifische UbBPs untersucht. Dies erfolgte durch enzymatische Synthese und Reinigung von Ub-Ketten sowie die Etablierung einer Ub-Interaktor-Anreicherung gekoppelt mit Massenspektrometrie. Mit dieser Methode konnten Ub-Ketten-Interaktoren identifiziert und deren Kettentyp-Präferenz ermittelt werden. Einige der ersten K48/K63-gebundenen verzweigten Ub-spezifischen Interaktoren wurden identifiziert, darunter PARP10/ARTD10, E3-Ligasen UBR4 und HIP1. Es wurden auch Interaktoren mit einer Präferenz für Ub3 gegenüber Ub2-Ketten bestimmt, darunter DDI2, CCDC50 und FAF1. Zusätzlich zur Identifizierung von Verzweigungs- und Längenspezifischen UbBPs wurde die Eignung von zwei Deubiquitinase-Inhibitoren getestet. Schließlich wurde die zelluläre Funktion von K48/K63-gebundenem, verzweigtem Ub in Folgeexperimenten untersucht. / Ubiquitination is a post-translational modification (PTM) which regulates almost all cellular processes. The capacity to regulate such a wide range of processes is achieved through the ubiquitin (Ub) code. This denotes the wide array of complex Ub architectures which can be characterised by features such as linkage type, chain length and linkage combinations. Key to the Ub system are the Ub-binding proteins (UbBPs) which read the Ub code and translate it into cellular functions. Although many linkage-specific UbBPs have been described, how UbBPs are able to decode more complex Ub architectures is not fully understood. Ub branches, when a single Ub has more than one other Ub attached to it, and Ub chain-length are two of such architectural features which remain understudied. In this work, the two most abundant linkage types K48- and K63-linked Ub were interrogated for branch- and length-specific UbBPs. This was achieved by Ub chain enzymatic synthesis and purification, followed by the establishment of an Ub interactor enrichment coupled with mass spectrometry method. This method was able to identify Ub chain interactors and using statistical comparisons elucidate their chain type preference. Some of the first K48/K63-linked branched Ub-specific interactors were identified, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4 and huntingtin-interacting protein HIP1. Ub interactors with a preference for Ub3 over Ub2 chains were also determined, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50 and p97-adaptor FAF1. Alongside the identification of branch- and length-specific UbBPs, the suitability of two different deubiquitinase inhibitors for use in this type of binding screen was tested. Finally, the cellular function of K48/K63-linked branched Ub was investigated in follow-up experiments based on the branch-specific interactors identified. Proteine Zell Ubiquitin Massenspektrometrie Huntington Erkrankung post-translational modification Molecular biology Protein Cell Ubiquitin Mass spectrometry Huntington's disease Posttranslational modification Molecular biology 570 Biologie 572 Biochemie ddc:570 ddc:572
113	Récepteurs auto-assemblés sur mesure pour les protéines thérapeutiques / On-demand self-assembled receptors for therapeutic proteins Dumartin, Mélissa 13 January 2017 (has links) Discipline récente, la chimie supramoléculaire est l'un des domaines les plus fertiles de la recherche chimique. Motivé par le défi que représente la reconnaissance moléculaire sur mesure d'édifices complexes, un grand intérêt est apparu pour la conception et la synthèse des récepteurs macrocycliques polyfonctionnalisés. Nous avons récemment décrit une nouvelle classe de récepteurs moléculaires accessibles et polyvalents: les Dyn[n]arenes. Cette nouvelle classe est obtenue à partir de briques moléculaires 1,4-dithiophénols fonctionnalisés en position ortho assemblées par des liaisons disulfures. Cette stratégie de macrocyclisation contrôlée thermodynamiquement permet de produire de grandes quantités de produit final avec un moindre coût synthétique. Des récepteurs sur-mesure pour la reconnaissance d'anions, de cations et de zwitterions ont été obtenus par cette approche polyvalente. En particulier, le Dyn[4]arene octacarboxylate a montré la capacité de reconnaître sélectivement des dérivés de la lysine par via un ajustement induit asymétrique du récepteur lors de l'association. L'utilisation de ce récepteur pour reconnaître des peptides et protéines portant en position N-terminale une lysine a été étudiée. Enfin, la post-fonctionnalisation de ces Dyn[4]arenes a été explorée afin d'améliorer leur solubilité et leur propriétés de reconnaissance vis-à-vis de cibles biologiquement actives, ainsi que pour étudier la possibilité de leur greffage sur phase solide et leur utilisation en chromatographie d'affinité / As a recent discipline, supramolecular chemistry is one of the most active and fast-growing fields of chemical research. Driven by the challenge that tailored molecular recognition of complex molecules represents, a large interest has grown for the design and synthesis of multi-functionalized macrocyclic receptors. We recently described a new class of accessible and versatile molecular receptors: Dyn[n]arenes. This new class is obtained from 1,4-dithiophenols units functionalized in ortho position and assembled by disulfide linkages. This strategy of thermodynamically controlled macrocyclization allows producing large amounts of final product with a low synthetic cost. On demand receptors for anions, cations and zwitterions were obtained by this versatile approach. Particularly, the octacarboxylate-bearing dyn[4]arene showed the ability to selectively recognize Lysine derivatives via an asymmetric induced adjustment of the receptor upon the complexation. The use of this receptor to recognize N-terminal Lysine tagged peptides and proteins have been investigated. Finally, post-functionalization of Dyn[4]arenes have been explored to improve their solubility and recognition properties toward biologically active target and to investigate their solid phase grafting to be implemented in affinity chromatography Chimie Combinatoire Dynamique Modifications post-traductionnelles Dyn[n]arene Transfert d’information Reconnaissance moléculaire Greffage sur phase solide Dynamic combinatorial chemistry Post-translational modification Dyn[n]arene Information transfer Molecular recognition Solid phase grafting 547
114	The role of the CTD phosphatase Rrt1 and post-translational modifications in regulation of RNA polymerase II Cox, Mary L. 07 July 2014 (has links) Indiana University-Purdue University Indianapolis (IUPUI) / RNA polymerase II (RNAPII) is regulated by multiple modifications to the C-terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification. Transcription RNA polymerase II CTD phosphatase Rtr1 MuDPIT AP-MS Cellular signal transduction RNA -- Research RNA polymerases Genetic transcription -- Regulation Transcription factors -- Research Phosphatases -- Research Proteins -- Analysis Proteomics Ubiquitin Phosphorylation Yeast -- Physiology
115	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
116	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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