Mechanisms of MLKL in necroptosis: from intramolecular rearrangements to isoform regulation

01/04/2019 to 31/03/2022

Necroptosis is a newly discovered form of regulated necrosis that is inflammatory and plays a
role in a number of diseases, like chronic inflammation and infection. It results in release of the
cellular contents after plasma membrane permeabilization dependent on the pseudokinase
mixed lineage kinase domain-like (MLKL) protein, which is the most terminal effector of
necroptosis known so far. Additionally, non-deadly roles of MLKL in intracellular vesicle
trafficking that seem to counterbalance necroptotic cell death have been recently described.
However, the molecular mechanism behind these MLKL functions remains obscure and is a
matter of debate. In preliminary work, we have discovered that the different isoforms of mouse
and human MLKL identified by transcriptome analysis exhibit distinct death-inducing potency.
While lack of the inhibitory pseudokinase domain renders MLKL intrinsically active, insertion of
just eight amino acids in the C-terminal helix abolishes its necroptotic activity. The main goal of
this project is to shed new light on the molecular mechanism how MLKL executes necroptosis.
To this aim, we will uncover the structural elements that regulate MLKL activity and
determine the contribution of the different MLKL isoforms to MLKL deadly and nondeadly
functions. Based on the effect of small differences in the sequence of the C-terminal
helix on MLKL activity, we plan to investigate how MLKL structure regulates function. We will
then use this information to discover new small molecules that regulate MLKL activity. Finally,
we will examine the role of the different MLKL isoforms on the regulation of necroptosis and of
additional non-deadly functions. This multidisciplinary project combines cell biology and
advanced microscopy with molecular dynamic simulations to disclose new molecular steps
involved in the coordination of necroptosis. The identification of new small molecules and
proteins implicated in necroptosis regulation will pave the way for the design of new drugs that
modulate MLKL activity for human health.