Journal article

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Emma J Petrie, Richard W Birkinshaw, Akiko Koide, Eric Denbaum, Joanne M Hildebrand, Sarah E Garnish, Katherine A Davies, Jarrod J Sandow, Andre L Samson, Xavier Gavin, Cheree Fitzgibbon, Samuel N Young, Patrick J Hennessy, Phoebe PC Smith, Andrew I Webb, Peter E Czabotar, Shohei Koide, James M Murphy

Proceedings of the National Academy of Sciences | NATL ACAD SCIENCES | Published : 2020

Abstract

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not p..

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Grants

Awarded by National Health and Medical Research Council


Funding Acknowledgements

We thank the Australian Synchrotron MX beamline staff for assistance with data collection and the CSIRO C3 Facility for assistance with crystallization. We are grateful to the National Health and Medical Research Council for fellowship (541951 to J.M.H.; 1079700 to P.E.C.; 1105754 and 1172929 to J.M.M.), project grant (1124735, 1124737), and infrastructure (9000587) support. We thank the Victorian State Government for Operational Infrastructure Support and acknowledge Australian Government Research Training Program Stipend PhD scholarship support for S.E.G. and K.A.D., and an Australian Institute of Nuclear Science and Engineering Postgraduate Research Award for K.A.D. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organization, and made use of the Australian Cancer Research Foundation (ACRF) detector.