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Functional conservation of an ancestral Pellino protein in helminth species - PubMed

  • ️Thu Jan 01 2015

Functional conservation of an ancestral Pellino protein in helminth species

Christopher D Cluxton et al. Sci Rep. 2015.

Abstract

The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans.

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Figures

Figure 1
Figure 1. Schematic of Pellino proteins.

Pellino proteins contain an N-terminal FHA domain and a C-terminal RING-like domain. The FHA domain contains residues for activation and IRAK binding, while the C-terminal RING-like domain confers E3-ligase activity of the protein. TRAF proteins potentially bind the RING-like domain, as RING domains are known to bind each other in vivo.

Figure 2
Figure 2. Phylogenetic analysis of the full-length sequences of known mammalian, nematode and arthropod Pellinos with helminth Pellino orthologues.

A maximum likelihood tree was formulated using the LG model and gamma correction. 100 bootstraps were calculated. The scale bar represents mutations per site.

Figure 3
Figure 3. Homology modeling of helminth Pellino proteins.

(A) Topology diagrams of Pellino2 and S. mansoni Pellino with beta-strands of core FHA domain in blue and of non-canonical wing in green. (B) FHA domain of the Pellino 2 (PDB:3EGB) crystal structure (left image), comparative model of SmPellino modeled as an FHA domain (middle image). (C) Pellino 2 (PDB:3EGB) crystal structure in red overlaid with the comparative model of SmPellino modeled as an FHA domain (in blue). Visualisation was performed with PyMOL (De Lano Scientific, USA).

Figure 4
Figure 4. Analysis of the helmith Pellino RING-like domain by multiple sequence alignment with human, nematode and arthropod Pellino proteins.

(A) Schematic representation of the RING-like domain of the helminth protein. The RING-like domain has an atypical CHC2CHC2 conformation. (B) Multiple sequence alignment of the RING-like domain containing region of human Pellino 1, 2, 3, D. melanogaster Pellino, Brugia malayi Pellino, C. elegans Pellino and various helminth Pellino proteins. The highlighted residues form the RING-like domain. The RING-like domain is conserved in all species analyzed.

Figure 5
Figure 5. Functional characterization of SmPellino in human cells.

(A) Transient over-expression of SmPellino-Myc with HsIRAK-1 or HsTRAF-6-Flag. SmPellino was immunoprecipitated (IP) with immunoblotting (IB) for HsIRAK-1 and HsTRAF-6 to detect protein-protein interactions. (B) In vivo ubiquitination assay was performed by co-transfecting HsIRAK-1 with or without SmPellino, HsPellino3 (HsPel3) or HsPellino3-RING-mutant (HsPel3-RM), and HA-tagged ubiquitin. IRAK-1 was IP and ubiquitin-HA detected by IB with anti-HA antibody. Expression of IRAK-1 was controlled by IB for IRAK-1 post-IP. (C) HEK 293-TLR4 cells were co-transfected with empty expression vector (EV) or constructs encoding HsPellino1, 2, 3S or SmPellino and NF-κB-luciferase (80 ng) and pGL3-Renilla luciferase (20 ng). Cells were stimulated with either IL-1β or LPS. (D) HEK-293 cells were transfected with increasing concentrations of EV or SmPellino in the presence/absence of LPS. (E) HEK-293 cells were co-transfected with either EV or SmPellino and vector for the overexpression of MyD88, IRAK-1, TRAF-6, TAK-1/TAB-1 or IKKβ to induce downstream signaling. (C–E) In total, 24 h post-transfection, lysates were assayed for firefly and pGL3-Renilla luciferase activity. Data are presented relative to cells transfected with empty vector alone and were subjected to a paired t-test. The asterisk (*) indicates that SmPellino reduces the corresponding TLR-signalling component-induced NF-kB activation with p < 0.05. Results represent mean+S.E.M. of three independent experiments, each performed in triplicate.

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References

    1. Henry J. Q., Martindale M. Q. & Boyer B. C. The unique developmental program of the acoel flatworm, Neochildia fusca. Dev Biol 220, 285–295 (2000). - PubMed
    1. Adoutte A., Balavoine G., Lartillot N. & de Rosa R. Animal evolution. The end of the intermediate taxa? Trends Genet 15, 104–108 (1999). - PubMed
    1. Lemaitre B., Nicolas E., Michaut L., Reichhart J. M. & Hoffmann J. A. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983 (1996). - PubMed
    1. O’Neill L. A. Plant science. Innate immunity in plants goes to the PUB. Science 332, 1386–1387 (2011). - PubMed
    1. Fallon P. G., Allen R. L. & Rich T. Primitive Toll signalling: bugs, flies, worms and man. Trends Immunol 22, 63–66 (2001). - PubMed

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