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Caspase-mediated processing of the Drosophila NF-κB factor Relish
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 10, 5991-5996 p.Article in journal (Refereed) Published
Abstract [en]

The NF-κB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-κB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IκB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IκB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IκB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IκB kinase β. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.

Place, publisher, year, edition, pages
2003. Vol. 100, no 10, 5991-5996 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-24103DOI: 10.1073/pnas.1035902100OAI: oai:DiVA.org:su-24103DiVA: diva2:196753
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2011-03-28Bibliographically approved
In thesis
1. Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity
Open this publication in new window or tab >>Regulation of antimicrobial peptide gene expression in Drosophila melanogaster: Involvement of POU and NF-kB/Rel factors in innate immunity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fruit fly, Drosophila melanogaster, has a well-developed immune response, and microbial assault induces a rapid production of potent antimicrobial peptides (AMPs). The aim of this thesis work was to gain deeper knowledge of the regulation of AMPs in Drosophila, by the isolation and characterization of transcription factors involved in AMP gene expression. A yeast screen was designed and used to isolate Drosophila cDNAs coding for novel regulators of the CecropinA1 (CecA1) gene. Three transcription factors belonging to the POU domain (Pdm) family were isolated, Pdm1, Pdm2, and Drifter (Dfr), and subsequently verified as regulators of CecA1 in Drosophila cells. POU proteins are known to regulate a range of developmental processes, but this is the first finding of POU factors controlling AMP gene expression. Dfr and Pdm1 were further analyzed with respect to their in vivo function as AMP gene regulators. Over-expression of Dfr activated several AMP genes in non-infected flies, suggesting that Dfr is involved in constitutive expression of AMP genes. Dfr was shown to bind to a CecA1 upstream enhancer, to which the homeodomain protein Caudal (Cad) previously had been shown to bind. Co-expression of Dfr and Cad promoted very high CecA1 expression, indicating that these two transcription factors act synergistically on CecA1 in tissues where both are expressed. In Pdm1 mutant flies, several AMP genes were highly expressed prior to infection, indicating that Pdm1 functions as a repressor of those genes. However, at least one gene, AttacinA, required Pdm1 for its expression suggesting that Pdm1 has dual functions, acting both as a repressor and activator. Finally, the post-translational activation of the NF-κB/Rel protein Relish in response to infection was investigated in detail. Deletion mapping revealed different functional domains of Relish, and site-directed mutagenesis was used to exactly determine the residues required for endoproteolytic cleavage by a caspase.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2007. 136 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-6614 (URN)91-7155-370-3 (ISBN)
Public defence
2007-02-22, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00
Opponent
Supervisors
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2011-03-28Bibliographically approved
2. Relish and the Regulation of Antimicrobial Peptides in Drosophila melanogaster
Open this publication in new window or tab >>Relish and the Regulation of Antimicrobial Peptides in Drosophila melanogaster
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family.

We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal.

Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region.

By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum.

This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2004. 49 p.
Keyword
Drosophila immunity, NF-kappaB, Relish, antimicrobial peptides
National Category
Developmental Biology
Identifiers
urn:nbn:se:su:diva-170 (URN)91-7265-897-5 (ISBN)
Public defence
2004-06-02, hörsalen, Frescati backe, Svante Arrhenius väg 21 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2011-03-28Bibliographically approved

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