Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...

Nitric oxide (NO) overproduction is correlated with numerous human diseases, such as arthritis, asthma, diabetes, inflammation and septic shock. The enzyme activities of both NO synthase (NOS) and dimethylarginine dimethylaminohydrolase-1 (DDAH-1) promote NO production. DDAH-1 mainly colocalizes in the same tissues as the neuronal isoform of NOS and catabolizes the endogenously-produced competitive inhibitors of NOS, N[omega]-monomethyl-L-arginine (NMMA) and asymmetric N[omega], N[omega]-dimethyl-L-arginine (ADMA). Inhibition of DDAH-1 leads to elevated concentrations of NMMA and ADMA, which subsequently inhibit NOS. To better understand DDAH-1, I first characterized the catalytic mechanism of human DDAH-1, where Cys274, His173, Asp79 and Asp127 form a catalytic center. Particularly, Cys274 is an active site nucleophile and His173 plays a dual role in acid/base catalysis. I also studied an unusual mechanism for covalent inhibition of DDAH-1 by S-nitroso-L-homocysteine (HcyNO), where an N-thiosulfoximide adduct is formed at Cys274. Using a combination of site directed mutagenesis and mass spectrometry, we found that many residues that participate in catalysis also participate in HcyNO mediated inactivation. Following these studies, I then screened a small set of known NOS inhibitors as potential inhibitors of DDAH-1. The most potent of these, an alkylamidine, was selected as a scaffold for homologation. Stepwise lengthening of the alkyl substituent changes an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-L-ornithine for neuronal NOS (1.7, 3, 20, >1,900 [mu]M, respectively) and DDAH-1 (990, 52, 7.5, 110 [mu]M, respectively). X-ray crystal structures suggest that this selectivity is likely due to active site size differences. To rank the inhibitors' in vivo potency, we constructed a click-chemistry based activity probe to detect inhibition of DDAH-1 in live mammalian cell culture. In vivo IC50 values for representative alkylamidine based inhibitors were measured in living HEK293T cells. Future application of this probe will address the regulation of DDAH-1 activity in pathophysiological states. In summary, this work identifies a versatile scaffold for developing DDAH targeted inhibitors to control NO overproduction and provides useful biochemical tools to better understand the etiology of endothelial dysfunction. / text

Inositolpyrophosphate (PP InsPs) sind Signalmoleküle in eukaryotischen Zellen, die u.a. als Sensoren für ATP- und Phosphat fungieren, und insbesondere durch allosterische Regulation und posttranslationale Modifikationen (PTMs) wirken. Diese Arbeit ist in zwei Teile unterteilt, die sich auf zwei verschiedene ungewöhnliche Eigenschaften dieser Moleküle konzentrieren. Der erste Teil untersucht PP-InsPs in Lösung, mit Schwerpunkt auf ihrer Fähigkeit, abhängig von pH und Metallionen ihre Konformation zu ändern. Diese Eigenschaft ist einzigartig unter biologisch vorkommenden kleinen Molekülen. Drei eng verwandte Moleküle, InsP6, 5PP InsP5 und InsP8, wurden mittels NMR Spektroskopie charakterisiert, um herauszufinden, ob sie unter annähernd zytosolischen Bedingungen ihre Konformation ändern können. Dies war der Fall für InsP8, welches deshalb bezüglich Protonierung und Komplexbildung genauer untersucht wurde. Zu guter Letzt konnten ITC Experimente demonstrieren, dass eine Lösungsumgebung, die die Konformationsänderung von InsP8 begünstigt, auch seine Bindung an eine damit interagierende Proteindomäne verstärkt. Der zweite Teil beschäftigt sich mit der Pyrophosphorylierung von Proteinen, einer PTM, die nach derzeitigem Wissen non-enzymatisch von PP InsPs auf phosphorylierte Aminosäurereste übertragen wird – im Gegensatz zur enzymatischen Phosphorylierung durch Kinasen. Ein Probenvorbereitungsprotokoll zum Nachweis von endogener Pyrophosphorylierung in Zellen wurde entwickelt und mit synthetischen Standardpeptiden validiert. Anschließend wurde es an drei menschlichen Zelllinien erprobt, und konnte über einhundert Modifikationsstellen identifizieren, zumeist auf Proteinen im Zellkern. Dies beweist zum ersten Mal die Existenz von endogener Pyrophosphorylierung. Proteomics an Knockout-Zelllinien bestätigten die Hypothese, dass Pyrophosphorylierung von 5PP-InsP5 (InsP7) abhängig ist. Mikroskopie und qPCR-Experimente lieferten Hinweise auf eine Funktion in der Regulation der Ribosomenbiogenese. / Inositol pyrophosphates (PP-InsPs) are messenger molecules in eukaryotic cells, that serve as sensors of phosphate and ATP, among other functions, signaling e.g. through allosteric regulation and posttranslational modifications. This work is structured into two parts, focusing on two different unusual features of these molecules. The first part investigates PP-InsPs in solution, with emphasis on the messengers’ ability to undergo a pH and metal ion dependent conformational change, a feature unique among biological small molecules. Three closely related molecules, InsP6, 5PP InsP5 and InsP8 were characterized by NMR, to determine if they could change conformation under conditions approximating a cytosolic environment. This was the case for InsP8, which was therefore studied in more detail regarding protonation and complexation. Finally, ITC experiments showed that solution conditions favoring the conformational change of InsP8 also improved its binding to a known interacting protein domain. The second part of the thesis is concerned with protein pyrophosphorylation, a post-translational modification thought to be transferred non-enzymatically from PP InsPs to phosphorylated amino acid residues – opposed to the usual enzymatic phosphorylation through kinases. A sample preparation workflow for detection of endogenous pyrophosphorylation in cells has been developed and validated using synthetic standard peptides. It was then applied to three human cell lines, discovering more than one hundred modified sites, mostly on nuclear proteins, and proving for the first time the existence of endogenous pyrophosphorylation. Proteomics on knockout cell lines confirmed the hypothesis that pyrophosphorylation depends on 5PP-InsP5 (InsP7). Finally, microscopy and qPCR experiments suggested a regulatory role in ribosome biogenesis.