Trends in Biochemical Sciences
ReviewThe Structural Basis of Necroptotic Cell Death Signaling
Section snippets
The Dying Code
In multicellular organisms orchestrated signaling pathways control the manner in which cells die and communicate information to the surrounding live cells as cues to dictate their responses. The best understood programmed cell death pathway, apoptosis (see Glossary), is generally immunologically silent, in keeping with its developmental roles, and is characterized by cell shrinking and plasma membrane blebbing without the release of cellular contents. By contrast, lytic cell death modes, such
The Protein Modules Controlling Necroptosis
At the molecular level, structural studies have played a crucial role in advancing our mechanistic understanding of the necroptosis pathway. The domains comprising the core components of the pathway – the protein kinases RIPK1 and RIPK3 and the pseudokinase MLKL – serve crucial functions in mediating protein–protein interactions in addition to the conventional catalytic activities mediated by the RIPK1 and RIPK3 protein kinase domains (Figure 2A).
Phospholipid Binding by MLKL
The MLKL 4HB has been established as a phosphatidylinositol phosphate (PIP)-binding domain, with binding interfaces mapped by NMR 45, 46 and mutational studies 35, 41, 49, 50 to clusters centered on the α1 and α2 helices and on the α3 and α4 helices, both of which are necessary for cell death 35, 41. While mutation of basic residues in the α4 helix abrogates PIP binding and cell killing, mutation of acidic and aliphatic residues in the α4 helix also perturbs cell killing 35, 41, suggesting that
Oligomerization of MLKL
The appearance of phosphorylated MLKL oligomers at the plasma membrane of a cell is typical of cells undergoing necroptosis. The structure of oligomeric MLKL has remained elusive and a point of contention in the literature, with trimers 34, 41, tetramers [35], hexamers [50], octamers [51], and polymers [52] reported. Biophysical analyses of recombinant MLKL using analytical ultracentrifugation, small-angle X-ray scattering, and native mass spectrometry indicate that mouse MLKL assembles into
Transition of MLKL from Dormant to Necroptotic Conformation
What governs the transition from a dormant monomeric MLKL form (Figure 3F) to a necroptotic oligomer (Figure 3G) in cells? Recent data suggest that divergent mechanisms are likely to exist between species (Figure 4). In the case of mouse MLKL, transient interaction with, and phosphorylation by, RIPK3 is sufficient to induce MLKL oligomerization and translocation to membranes and cell death 14, 31 (Figure 4A). Modification of mouse MLKL, or mutation of the pseudokinase domain to mimic
The Harbinger of Doom
The presence of phosphorylated MLKL oligomers at the plasma membrane is considered a hallmark of cells undergoing necroptosis. Nonetheless, several studies have revealed a time lag between when phosphorylated MLKL oligomers are detected at the plasma membrane and phosphatidylserine exposure occurs on the cell surface, to when plasma membrane integrity is lost to the point of cell death 48, 56, 57. One explanation for this lag can be accommodated by a number of proposed models (previously
Scrambling to Be ESCRTed from a Dying Cell
MLKL-directed lipid scrambling and cell-surface exposure of phosphatidylserine was recently linked to the formation of small bubbles on the plasma membrane, which are subsequently released as extracellular vesicles 56, 57, 61. The formation of these extracellular vesicles was linked to the endosomal sorting complex required for transport (ESCRT)-III machinery 56, 61, which was proposed to deliver activated MLKL forms from endosomes to the plasma membrane in a CHMP2A- and CHMP4B-dependent
Concluding Remarks and Future Perspectives
Fuelled by structural studies, our understanding of the molecular basis of necroptotic cell death executed by the kinases RIPK1 and RIPK3 and the pseudokinase MLKL has advanced enormously over the past 5 years. Each effector comprises modular signaling domains, with recent structures providing insights into how these domains mediate the formation of higher-order assemblies and the transmission of necroptotic signals and how MLKL can be triggered to kill cells. Nevertheless, the precise
Acknowledgments
We thank the National Health and Medical Research Council of Australia for their support of our studies via fellowships to P.E.C. (1079700) and J.M.M. (1105754), project grants on this subject (1124735, 1124737), and infrastructure funding (IRIISS 9000433). We are grateful to the Victorian State Government Operational Infrastructure Scheme and to the Australian Synchrotron for enabling structural studies reviewed herein. We apologize to our colleagues whose work we were unable to cite owing to
Glossary
- 4HB domain
- a helical domain at the N terminus of MLKL responsible for membrane permeabilization; predicted to resemble the HeLo-like domains of HET-s fungal and plant proteins.
- Apoptosis
- a caspase-dependent cell death pathway important for development in multicellular organisms.
- Autophosphorylation
- phosphorylation of the kinase itself in cis (to the same molecule) or in trans (to another copy of the same kinase).
- Brace helices
- a two-helix connector between the 4HB and pseudokinase domains of MLKL.
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