Caspase Mediated Cleavage, IAP Binding, Ubiquitination and Kinase Activation : Defining the Molecular Mechanisms Required for \u3cem\u3eDrosophila\u3c/em\u3e NF-кB Signaling: A Dissertation

Innate immunity is the first line of defense against invading pathogens. Vertebrate innate immunity provides both initial protection, and activates adaptive immune responses, including memory. As a result, the study of innate immune signaling is crucial for understanding the interactions between host and pathogen. Unlike mammals, the insect Drosophila melanogasterlack classical adaptive immunity, relying on innate immune signaling via the Toll and IMD pathways to detect and respond to invading pathogens. Once activated these pathways lead to the rapid and robust production of a variety of antimicrobial peptides. These peptides are secreted directly into the hemolymph and assist in clearance of the infection. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophilashow strong homology to those of vertebrates making them ideal for the study of activation, regulation and mechanism. Currently a number of questions remain regarding the activation and regulation of both vertebrate and insect innate immune signaling. Over the past years many proteins have been implicated in mammalian and insect innate immune signaling pathways, however the mechanisms by which these proteins function remain largely undetermined. My work has focused on understanding the molecular mechanisms of innate immune activation in Drosophila. In these studies I have identified a number of novel protein/protein interactions which are vital for the activation and regulation of innate immune induction. This work shows that upon stimulation the Drosophila protein IMD is cleaved by the caspase-8 homologue DREDD. Cleaved IMD then binds the E3 ligase DIAP2 and promotes the K63-polyubiquitination of IMD and activation of downstream signaling. Furthermore the Yersinia pestis effector protein YopJ is able to inhibit the critical IMD pathway MAP3 kinase TAK1 by serine/threonine-acetylation of its activation loop. Lastly TAK1 signaling to the downstream Relish/NF-κB and JNK signaling pathways can be regulated by two isoforms of the TAB2 protein. This work elucidates the molecular mechanism of the IMD signaling pathway and suggests possible mechanisms of homologous mammalian systems, of which the molecular details remain unclear

Specificity and Signaling in the Drosophila Immune Response

The Drosophila immune response is characterized by the rapid and robust production of a battery of antimicrobial peptides immediately following infection. The genes encoding these antimicrobial peptides are controlled by two NF-κB signaling pathways that respond to microbial infection. The IMD pathway is triggered by DAP-type peptidoglycan, from the cell wall of most Gram-negative and certain Gram-positive bacteria, and activates the NF-κB precursor protein Relish. The Toll pathway, on the other hand, is stimulated by lysine-type peptidoglycan from many Gram-positive bacteria, β 1,3 glucans from many fungi, as well as by microbial proteases. Toll signaling leads to the activation and nuclear translocation of DIF or Dorsal, two other NF-κB homologs. This review presents our current understanding of the molecular mechanisms involved in microbial recognition and signal transduction in these two innate immune pathways

Serine/threonine acetylation of TGFbeta-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-kappaB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-kappaB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition

Effects of ajulemic acid on synoviocytes in vitro

In this project the effects of a cannabinoid, Ajulemic Acid (AjA), on different types of rheumatoid tissues were studied. Cultured synovial cells, synovial fluid cells and peripheral blood mononucleocytes, were treated with AjA prior to stimulation with TNF-. The results showed a marked decrease in the cytokines Interleukin-6 and Interleukin-8. As both IL-6 and IL-8 play a part in the pathogenesis of rheumatoid arthritis, reduction of their secreted levels could serve as an effective treatment for the disease

Transgenic animals

The ability to create transgenic organisms has been present since the 1970's. Since then this power has increased greatly, and current uses of this technology include pharmaceutical production, organ transplants and disease research. Because this science involves creating new animals, various issues regarding the moral acceptability of this technology have arisen. Our recommendations include increasing public awareness about the benefits of the technology so that educated decisions can be made about its future

NF-kappaB/Rel Proteins and the Humoral Immune Responses of Drosophila melanogaster

Nuclear Factor-kappaB (NF-kappaB)/Rel transcription factors form an integral part of innate immune defenses and are conserved throughout the animal kingdom. Studying the function, mechanism of activation and regulation of these factors is crucial for understanding host responses to microbial infections. The fruit fly Drosophila melanogaster has proved to be a valuable model system to study these evolutionarily conserved NF-kappaB mediated immune responses. Drosophila combats pathogens through humoral and cellular immune responses. These humoral responses are well characterized and are marked by the robust production of a battery of anti-microbial peptides. Two NF-kappaB signaling pathways, the Toll and the IMD pathways, are responsible for the induction of these antimicrobial peptides. Signal transduction in these pathways is strikingly similar to that in mammalian TLR pathways. In this chapter, we discuss in detail the molecular mechanisms of microbial recognition, signal transduction and NF-kappaB regulation, in both the Toll and the IMD pathways. Similarities and differences relative to their mammalian counterparts are discussed, and recent advances in our understanding of the intricate regulatory networks in these NF-kappaB signaling pathways are also highlighted

Bacterial effectors: learning on the fly

A common defining characteristic of pathogenic bacteria is the expression of a repertoire of effector molecules that have been named virulence factors. These bacterial factors include a -variety of proteins, such as toxins that are internalized by receptors and translocate across endosomal membranes to reach the cytosol, as well as others that are introduced directly into the cell by means of bacterial secretory apparatuses. Given the importance of these effectors for understanding bacterial pathogenicity, significant effort has been made to dissect their molecular mechanisms of action and their respective roles during infection. Herein we will discuss how Drosophila have been used as a model system to study these important microbial effectors, and to understand their contribution to pathogenicity

Bug Versus Bug: Humoral Immune Responses in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Insects mount a robust innate immune response against a wide array of microbial pathogens. For example, the fruit fly Drosphila melanogaster uses both cellular and humoral innate immune responses to combat pathogens. The hallmark of the Drosophila humoral immune response is the rapid induction of antimicrobial peptide genes in the fat body, the homolog of the mammalian liver. Expression of these antimicrobial peptide genes is rapidly induced by two immune signaling pathways, which respond to distinct microorganisms. The Toll pathway is activated by fungal and Gram-positive bacterial infections, whereas the IMD pathway responds to Gram-negative bacteria. In this chapter, we discuss recent advances in understanding the mechanisms involved in microbial recogni-tion, signal transduction, and immune protection mediated by these pathways, highlighting similarities and differences between Drosophila immune responses and mammalian innate immunity