Doctor of Philosophy

dissertationMicroRNAs (miRNAs) are small, noncoding RNA regulators of gene expression that have many important functions within the immune system. While various critical immunologic functions for specific miRNAs have been uncovered, less is known about the roles of these molecules within the intestinal and adipose microenvironments. Recently, many studies have described the complex intestinal interface, which contains host immune cells and epithelial cells interacting with the microbiota in a manner that promotes symbiosis. Further, there is emerging evidence that miRNAs have evolved to fine tune host gene expression networks and signaling pathways that modulate cellular physiology in the intestinal tract. Here, I first review the present knowledge of the influence miRNAs have on both immune and epithelial cell biology in the mammalian intestines and the impact this has on the microbiota. Next, my work demonstrates the role of one specific miRNA, microRNA-146a (miR-146a), in intestinal homeostasis and disease. miR-146a has previously been shown to have anti-inflammatory function within the immune system and is required to downregulate inflammation in mammals. I find that this miRNA constrains multiple parameters of intestinal immunity and increases murine colitis severity. Further, because miR-146a regulates intestinal homeostasis and populations of the gut microbiota, I hypothesized that this molecule may also be important in regulating immunometabolism in a model of diet-induced obesity. I demonstrate that miR-146a is required to prevent obesity, diabetes, and metabolic disease during high-fat diet. miR-146a was found to regulate multiple networks of gene expression in adipose tissue macrophages both during dietary homeostasis and metabolic disease, and these miR-146a-dependent pathways converge upon inflammation and cell metabolism. Altogether, miR-146a constrains immune responses both within the intestine and adipose tissue, and can both prevent or promote disease, depending on disease and context. This institutes the importance of studying miRNA functions within multiple tissues types and disease contexts, as novel roles for these molecules may be established in various situations

Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression

Massenspektrometrische Untersuchung epigenetischer Proteine

Im Rahmen dieser Dissertation wurden epigenetisch relevante DNA-Protein- sowie Protein-Protein-Interaktionen untersucht. Außerdem wurde ein neuartiges Reagenz für die massenspektrometrische Analyse von Proteinstrukturen und Protein-Protein-Interaktionen etabliert.In the scope of this PhD thesis, epigenetically relevant DNA-protein- as well as protein-protein-interactions were investigated. In addition, a novel reagent for the mass spectrometric analysis of protein structures and protein-protein-interactions was established

A Click‐Chemistry‐Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein–protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter‐ and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low‐abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high‐end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases

Azobenzene-based inhibitors of human carbonic anhydrase II

Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4 '-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electrondonating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with K-i = 25-65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2’-deoxycytidine by methylating 2’-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity toward AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double-strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis efficiently in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response toward AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells

Epistasis between MicroRNAs 155 and 146a during T Cell-Mediated Antitumor Immunity

An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155^(−/−) mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4^+ and CD8^+ T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1β and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1−/− mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.We would like to acknowledge the following grants: the National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to P.G.B., M.G.C., and L.A.J.O.; Principal Research Fellowship 1117602 to J.B.B.; and NHMRC Project Grant APP1156455 to J.B.B., P.G.B., and M.G.C. The O’Neill laboratory acknowledges the following grant support: European Research Council (ECFP7-ERC-MICROINNATE) and Science Foundation Ireland Investigator Award (SFI 12/IA/1531)