Characterisation of the teleostean κB-Ras family: The two members NKIRAS1 and NKIRAS2 from rainbow trout influence the activity of NF-κB in opposite ways. Issue 106 (November 2020)

• Record Type: Journal Article
• Authors: Sarais, Fabio; Rebl, Henrike; Verleih, Marieke; Ostermann, Sven; Krasnov, Aleksei; Köllner, Bernd; Goldammer, Tom; Rebl, Alexander
• Is Part Of: Fish & shellfish immunology. Issue 106(2020)
• Page Start: 1004
• Page End: 1013
• Publication Date: 2020-11
• Subjects: NF-kappaB -- IκBα -- Innate immunity -- Immune regulation -- kappaB-Ras -- p65/RelA; aa amino acid -- CDS coding sequence -- CXCL8 C-X-C motif chemokine ligand 8 -- GDP guanosine diphosphate -- GFP green fluorescent protein -- GTP guanosine-triphosphate -- hpi hours post infection -- IL interleukin -- MAb monoclonal antibody -- mPlum mutant variant of red fluorescent protein -- NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells -- NKIRAS NF-κB-inhibitor-interacting Ras-like proteins -- nt nucleotide(s) -- ORF open-reading frame -- PEST proline (P), glutamic acid (E), serine (S), and threonine (T)-rich domain -- Ras Rat sarcoma -- SNP single-nucleotide polymorphism -- TGFB transforming growth factor beta -- TNF tumour necrosis factor -- qPCR quantitative polymerase-chain reaction -- WGD whole-genome duplication; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2020.08.052
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: Two structurally similar NF-κB-inhibitor-interacting Ras-like proteins (NKIRAS) regulate the activity of the transcription factor NF-κB and thereby control several early immune mechanisms in mammals. We identified the orthologous sequences of NKIRAS1 and NKIRAS2 from the rainbow trout Oncorhynchus mykiss . The level of sequence identity was similarly high (≥68%) between the two and in comparison to their mammalian counterparts. Strikingly, NKIRAS2 was present as four transcript variants. These variants differed only in length and in the nucleotide composition of their 5' termini and were most likely generated by splicing along unconventional splice sites. The shortest NKIRAS2 variant was most strongly expressed in a lymphocyte-enriched population, while NKIRAS1 was most strongly expressed in cells of myeloid origin. Fluorescent-labelled NKIRAS1 and NKIRAS2 proteins from rainbow trout were detected in close association with the p65 subunit of NF-κB in the nucleus and cytoplasm of CHSE-214 cells. Subsequent reporter-gene experiments revealed that NKIRAS1 and a longer NKIRAS2 variant in rainbow trout decreased the level of activated NF-κB, while the two shortest NKIRAS2 variants increased the NF-κB activity. In addition, the overexpression of the shortest NKIRAS2 variant in CHSE-214 cells induced a stronger transcription of the genes encoding the pro-inflammatory cytokines TNF, CXCL8, and IL1B compared to non-transfected control cells. This is the first characterisation of NKIRAS orthologues in bony fish and provides additional information to the as yet underexplored inhibition pathways of NF-κB in lower vertebrates. Highlights: We identified one NKIRAS1 and four NKIRAS2 variants in rainbow trout . NKIRAS1 and NKIRAS2 are localised together with NF-κB p65 in CHSE-214 cells. The investigated NKIRAS variants modulate the NF-κB activity in different ways. The shortest NKIRAS2 variant induced a stronger expression of cytokines in vitro.