The rule of three: structural diversity, differential expression and distinct regulatory output of three MALT...

The paracaspase MALT1 plays a central role in regulating NF-κB and MAPK signalling pathways that, in mammals, control lymphocyte differentiation and survival. Rainbow trout possess three MALT paralogues that show distinct tissue- and cell-specific expression patterns. Malt1 transcripts predominated in the head kidney, malt2 was most highly expressed in the spleen and trunk kidney and malt3 was dominant in the liver and intestine. Integration with publicly available bulk and single-cell RNA-seq datasets supported these patterns and indicated broad expression of malt3 across tissues. At the structural level, Malt3 differs from Malt1 and Malt2 by the absence of a death domain. We explored potential functional differences using CHSE-214 cells as a heterologous system for overexpression studies. Luciferase reporter assays revealed promoter-specific activation of NF-κB signalling, where Malt1 and Malt3 activated the human ELAM promoter and Malt2 and Malt3 induced the teleostean saa promoter, whereas none of the three Malt proteins activated the cxcl8 promoter. Overexpression of individual Malt proteins resulted in gene- and stimulus-dependent modulation that generally attenuated the stimulus-induced expression of several pro-inflammatory genes. A notable finding, however, was the strong upregulation of mx1 transcripts in Malt-overexpressing CHSE-214 cells upon stimulation with poly(I:C). Co-expression experiments further suggested combinatorial effects, including enhanced suppression following zymosan stimulation and selective enhancement of il1b expression in response to flagellin, when all three paralogues were expressed together. Overall, these results provide an initial indication that Malt paralogues may contribute to context-dependent modulation of innate immune signalling; however, further studies in physiologically relevant systems are required.