MLKL (mixed-lineage kinase domain-like) protein is believed to be an effector of a type of regulated cell death, necroptosis. It consists of the pseudokinase domain and the four-helix bundle domain (4HB) that has a necroptosis executive function. It has been suggested that the first connecting helix between both domains inhibits 4HB. We used four variants of hMLKL1: full-length hMLKL1, the truncated variant of hMLKL1 consisting of the N-terminal 154 amino acid residues, and both variants with the D144K mutation, which is known to activate hMLKL1. Fluorescent tags are very useful tools in observing the cellular localization of proteins. EGFP (enhanced green fluorescent protein) is probably the most commonly used fluorescent tag, whereas the protein tag HaloTag7 is part of a new labelling technology, which is based on the binding of organic dyes with reactive chloroalkane linker into a modified version of the enzyme haloalkane dehalogenase. We investigated the influence of both fluorescent tags, HaloTag7 and EGFP, on the function of all hMLKL1 variants. We aimed to estimate which fluorescent tag is more suitable for observing hMLKL1 and its variants by confocal microscopy. We have generated stable HEK293 cell lines with inserted variants of hMLKL1 in combination with chosen fluorescent tags. We found that EGFP and HaloTag7 do not significantly affect the function of hMLKL1 and its variants but that in comparison the EGFP affects it less. We found that EGFP is better suited for observing hMLKL1 in HEK293 cell lines with confocal fluorescence microscopy. In hMLKL1 variants with said mutation, translocation to the membrane was observed using both fluorescent tags.
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