Investigations on DNA methylation by Dnmt2 and impact of tRNA modifications on TLR7 stimulation

The incorporation of modified nucleotides into ribonucleic acids (RNAs) is important for their structure and proper function. These modifications are inserted by distinct catalytic macromolecules one of them being Dnmt2. It methylates the Cytidine (C) at position 38 in tRNA to 5-methylcytidine (m5C). Dnmt2 has been a paradigm in this respect, because all of its nearest neighbors in evolution are DNA-cytosine C5-methyltransferases and methylate DNA, while its (own) DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. This work determines whether the biochemical potential for DNA methylation is present in the enzyme. It was discovered that DNA fragments, when presented as covalent RNA:DNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Additional minor deviations from a native tRNA structure that were seen to be tolerated by Dnmt2 were used for a stepwise development of a composite system of guide RNAs that enable the enzyme to perform cytidine methylation on single stranded DNA in vitro. Furthermore, a proof-of-principle is presented for utilizing the S-adenosyl methionine-analog cofactor SeAdoYn with Dnmt2 to search for new possible substrates in a SELEX-like approach.rnIn innate immunity, nucleic acids can function as pathogen associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs). The modification pattern of RNA is the discriminating factor for toll-like receptor 7 (TLR7) to distinguish between self and non-self RNA of invading pathogens. It was found that a 2'-O-methylated guanosine (Gm) at position18, naturally occurring at this position in some tRNAs, antagonizes recognition by TLR7. In the second part of this work it is pointed out, that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The immune silencing effect of the ribose methylation is most pronounced if the dinucleotide motif is composed of purin nucleobases whereas pyrimidines diminish the effect. Similar results were obtained when the Gm modification was transposed into other tRNA domains. Point mutations abolishing base pairings important for a proper tertiary structure had no effect on the immune stimulatory potential of a Gm modified tRNA. Taken together these results suggest a processive type of RNA inspection by TLR7.rn

Kostimulatorische Rezeptoren in der Aktivierung humaner neutrophiler Granulozyten

Neutrophile Granulozyten spielen eine wichtige Rolle in der ersten Phase der Inflammation. Sie infiltrieren den Infektionsort um den eingedrungenen Erreger zu bekämpfen und Ihre Effektor Funktion auszuführen. Neben den Mustererkennenden Rezeptoren des angeborenen Immunsystems (pattern recognition receptors) werden weitere Rezeptoren auf der Oberfläche von neutrophilen Granulozyten exprimiert, welche zur Aktivierung der Zelle beitragen können. In dieser arbeit wurden der Herpes Virus Entry Mediator (HVEM) und Triggering Receptor expressed on Myeloid Cells-1 (TREM-1) auf neutrophilen untersucht. Für HVEM konnte eine synergistische Aktivierung von neutrophilen Granulozyten im Zusammenspiel mit Toll like Rezeptor (TLR) Liganden nachgewiesen werden. Für TREM-1 konnte ein Vorhandensein eines Liganden auch Thrombozyten beschrieben. Es wurden weiterhin Mechanismen untersucht und beschrieben, welche für die synergistische Aktivierung von neutrophilen Granulozyten verantwortlich sind, welche nach TREM-1 und TLR Stimulation erkennbar ist.

Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH-terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).