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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

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Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites

Dan E McNamara1, Cole M Dovey2, Andrew T Hale3

  • 1Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Cell Chemical Biology
|April 30, 2019
PubMed

Insights

Inositol phosphates (IPs) activate mixed lineage kinase domain-like (MLKL) to trigger programmed cell death (necroptosis). Specific IPs bind MLKL

Area of Science:

  • Molecular Biology
  • Cell Death Research
  • Biochemistry

Background:

  • Necroptosis is an inflammatory programmed cell death pathway.
  • Mixed lineage kinase domain-like (MLKL) is the key executioner of necroptosis.
  • Inositol phosphates (IPs) were previously implicated in MLKL activation.

Purpose of the Study:

  • To elucidate the mechanism by which inositol phosphates activate MLKL.
  • To identify the specific inositol phosphates and binding sites involved in MLKL activation.
  • To investigate the role of inositol pentakisphosphate 2-kinase (IPPK) in necroptosis.

Main Methods:

  • Biochemical assays to study the interaction between MLKL and inositol phosphates.
  • Site-directed mutagenesis to identify critical residues in MLKL's auto-inhibitory region.
  • Genetic deletion of IPPK in cells to assess its role in necroptosis.

Main Results:

  • Specific inositol phosphates (I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6) directly bind MLKL's N-terminal executioner domain (NED).
  • Binding of these IPs destabilizes the auto-inhibitory region of MLKL, promoting its activation and membrane permeabilization.
  • IP6 and IPPK are essential for necroptosis, as IPPK deletion abolishes IP6 production and inhibits cell death.

Conclusions:

  • Inositol phosphates act as critical allosteric activators of MLKL.
  • The interaction between specific IPs and MLKL's NED is a key regulatory step in necroptosis.
  • This study reveals a novel mechanism for programmed cell death regulation involving IP metabolism.
Keywords:
CRISPR Cas9 gene editingMLKLNMR structure determinationcell deathinositol phosphateinositol phosphate kinasemembrane permeabilizationmetabolitenecroptosisplasma membrane rupture

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