Over the past 30 years, transcription factor nuclear factor kappa B (NF-κB) has been extensively characterized in organisms ranging from flies to humans, where it is known to play key roles in developmental and immune-related processes. More recently, DNA sequencing approaches have identified homologs of NF-κB and many upstream signaling components in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. However, little is known about the activity and regulation of NF-κB proteins in these basal organisms. In this dissertation, the structure, activity, and biology of NF-κB in three basal phyla is examined and the extent of conservation with more derived organisms as well as phylum-specific properties are investigated. In the coral Orbicella faveolata (Of) a simplified but nearly complete Toll-like receptor (TLR)-to-NF-κB pathway exists, but basal to cnidarians, there are fewer upstream signaling molecules present. For example, in the poriferan Amphimedon queenslandica (Aq) and the protist Capsaspora owczarzaki (Co), singular NF-κBs and some upstream signaling proteins are encoded in their genomes, but no canonical TLRs exist. In contrast, the expanded family of choanoflagellates, including the choanoflagellate Acanthoeca spectabilis (As), contains TLR-like and up to three NF-κB-like homologs, although their domain structures differ from NF-κB pathway members of higher organisms. Of-NF-κB, Aq-NF-κB, and Co-NF-κB all resemble the mammalian NF-κB protein p100 in that they contain an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and similar DNA binding-site profiles. C-terminal truncation results in translocation of these basal NF-κBs to the nucleus and increases their DNA-binding and transcriptional activation activities. Nevertheless, unlike mammalian NF-κB p100, the C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. The three As-NF-κB-like proteins all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ from one another in DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Expression of IKKs induce proteasome-dependent C-terminal processing of Of-NF-κB and Aq-NF-κB in human cells, and processing requires C-terminal serines. In contrast, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells and no IKK homolog exists in the Co genome, suggesting that IKK-mediated processing of NF-κB is a mechanism that evolved solely in animals. Treatment of Of and sponge tissue with lipopolysaccharide (LPS), a ligand for mammalian innate immunity, results in gene expression changes consistent with NF-κB pathway mobilization in Of and increases both DNA-binding activity and processing of sponge NF-κB. Furthermore, sponge tissue contains constitutive NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Moreover, exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. Together, these data suggest that the mechanism as well as level of activation of NF-κB in basal organisms is different from what is observed in higher organisms. Additionally, NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. Overall, these data represent the first functional characterization of NF-κB signaling proteins in an endangered coral, in any organisms basal to cnidarians (i.e., an evolutionary important sponge), and outside the Kingdom Animalia (protists). These findings suggest that these seemingly simple organisms contain conserved innate immune-like pathways that may be regulated by NF-κB and provide information about the evolution and diversification of this biologically important transcription factor.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43027 |
| Date | 17 September 2021 |
| Creators | Williams, Leah Michele |
| Contributors | Gilmore, Thomas D. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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