Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

Abstract

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

The first embryo, the origin of cancer and animal phylogeny. IV. The neoplastic basis for the formation of the innate immune system

“…NF-kB is a molecule extensively studied for its role in embryonic development and immunity, from the first animal to modern humans (Gilmore, 2006;Ghosh and Hayden, 2012;Williams and Gilmore, 2020). It should be noted, however, that functional NF-kB-like proteins (DNA binding and transcription activators) vary considerably in general structure, activity, and regulation mechanisms, both between protists and in comparison with animal NF-kB (Williams et al, 2021). The emergence of metazoans seems to have been essential for the definitive configuration of the NF-kB protein, further supporting the view that the emergence of the first embryo was a fundamental step in the development of the immune system.…”

An innate ability: How do basal invertebrates manage their chronic exposure to microbes?

“…AP-1-like proteins have been identified in poriferans and cnidarians, but there is little data regarding their biological functions and regulation in these organisms. In the single-celled protist Capsaspora owczarzaki , no obvious TLR proteins have been found; however, Capsaspora does contain homologs to some TLR pathway intracellular signaling components and to NF-κB [ 18 , 21 ]. On the other hand, many single-celled choanoflagellate species have multiple TLR-like proteins with the following types of structures: (1) prototypical, mammalian-like joined LRR-TIR domain proteins; (2) a transmembrane protein with an extracellular LRR and an internal kinase domain; and (3) some TIR-only domain proteins [ 22 ].…”

“…Upon activation of mammalian noncanonical NF-κB signaling, the C-terminal domain of p100 is phosphorylated by an IκB kinase and this phosphorylation promotes proteasome-dependent processing of p100 to the active N-terminal p52 protein, which then enters the nucleus to affect gene expression. Bipartite NF-κB proteins of corals, anemones, sponges, and the protist Capsaspora can undergo proteasome-dependent processing and nuclear translocation when expressed in mammalian cells in culture [ 6 , 7 , 16 , 18 ]. However, it is still not clear whether and under what circumstances such induced processing of NF-κB occurs in the native animals.…”

“…In addition to their roles in immunity, TLR->NF-κB pathways appear to play species-specific roles in early development in many basal organisms [ 2 , 3 ]. Roles for TLRs and NF-κB in development have been suggested by experiments in the anemone Nematostella , the sponge Amphimedon queenslandica , and the protist Capsaspora [ 5 , 18 , 29 ]. In Nematostella , knockdown of its single TLR results in embryos that fail to gastrulate [ 5 ], and knockdown of NF-κB results in lack of development of the immune stinging cells (cnidocytes) present in members of this phylum [ 30 ].…”

“…In the sponge Amphimedon , NF-κB transcripts are expressed throughout the early embryo shortly after cleavage and then primarily in large granular cells in later stage embryos [ 29 ]. In Capsaspora , NF-κB expression and DNA-binding activity levels vary in different life stages, suggesting specific roles for NF-κB in each life stage [ 18 ]. We wish to point out, however, that the early developmental roles of TLRs and NF-κB in many organisms appear to be independently derived activities, where the pathways and molecules have been retooled for specific purposes in many organisms and these developmental activities have not been retained through evolutionary history.…”