The Evolution and Biological Activity of Metazoan Mixed Lineage Kinase Domain-Like Protein (MLKL)

Area of Science:

• Evolutionary biology
• Cellular biology
• Molecular mechanisms of cell death

Background:

• Mixed lineage kinase domain-like protein (MLKL) is the key effector of programmed necrosis (necroptosis) in mammals.
• MLKL's N-terminal domain (NTD) initiates necroptosis by forming plasma membrane pores, while the C-terminal domain regulates NTD activity.
• The evolutionary conservation and biological function of MLKL in invertebrates remain largely unexplored.

Purpose of the Study:

• To investigate the evolutionary distribution and functional activity of MLKL across diverse metazoan phyla.
• To characterize the phylogenetic relationships of MLKL, particularly focusing on invertebrate orthologs.
• To determine the necroptotic potential of invertebrate MLKL N-terminal domains when expressed in human cells.

Main Methods:

• Bioinformatic analysis of MLKL presence and conservation across 34 metazoan phyla.
• Phylogenetic analysis to construct evolutionary clades of MLKL.
• Functional assays involving the expression of invertebrate MLKL-NTDs in human cells to assess necroptosis induction.

Main Results:

• MLKL was detected in both Deuterostomia and Protostomia (Rotifera), expanding its known evolutionary range.
• Rotifera MLKL shows distinct evolutionary divergence, with higher similarity to non-metazoan MLKL than to other metazoan MLKL.
• MLKL-NTDs from Rotifera, Echinodermata, Urochordata, and Cephalochordata effectively induced necroptosis in human cells, highlighting conserved functional domains.

Conclusions:

• This study provides novel insights into the ancient evolutionary history of MLKL within Metazoa.
• It demonstrates that invertebrate MLKL, particularly from Rotifera, possesses functional necroptotic activity.
• The findings underscore the conserved role of the MLKL-NTD in executing necroptosis across a broad range of animal species.