Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

The immune adaptor TRIF that signals for programmed cell death forms hybrid amyloids with other necroptosis-associated proteins (#139)

Max O. D. G. Baker 1 , Chi Le Lan Pham 1 , Nirukshan Shanmugam 1 , Megan Steain 2 , Margie Sunde 1
  1. Discipline of Pharmacology, University of Sydney, Camperdown, New South Wales, Australia
  2. Discipline of Infectious Diseases and Immunology, University of Sydney, Camperdown, New South Wales, Australia

TRIF is an innate immune protein that serves as an adaptor for several cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. An important outcome of TRIF-directed signalling is the activation of programmed cell death pathways, particularly necroptosis [1]. Necroptosis is a form of programmed cell death characterised by membrane rupture and systemic inflammation. It can result from the activation of three distinct initiating complexes, which converge on the oligomerisation and phosphorylation of the receptor interacting serine/threonine protein kinase 3 (RIPK3).

The three initiating complexes contain either TRIF, RIPK1, which acts downstream of the TNF receptor or Z-DNA binding protein 1 (ZBP1), which is a viral sensor. It has recently been established that the activation of RIPK3 by RIPK1 and ZBP1 occurs through formation of stable, heteromeric functional amyloid assemblies, wherein both RIPK3 and its initiating partner protein are incorporated into the same amyloid fibril, and in which the fibril structure is necessary for the activation and function of downstream cell death effectors [2, 3]. These interactions are driven by a conserved sequence termed the RIP Homotypic Interaction Motif (RHIM) present within these proteins [4].

Despite convincing cellular data indicating that TRIF can interact with RIPK3 to drive necroptotic cell death, there is a dearth of evidence describing the specific biophysical and structural mechanisms that allow it to do so. We show that, like RIPK1, RIPK3 and ZBP1, TRIF is capable of forming homomeric amyloid structures. We also show that mutation of the RHIM within TRIF alters the amyloid properties of these structures. We show that TRIF is able to interact with RIPK1 and RIPK3 to form heteromeric structures, and that the presence of TRIF alters the degree and stability of oligomerisation of its partner protein. In summary, we have identified that TRIF, like other RHIM-containing proteins, can form supramolecular amyloid assemblies that have unique structural properties, which may underlie their specific cellular functionality.

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  2. Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsaio YS, et al. Cell 159, 339-350 (2012).
  3. Pham CLL, Shanmugam N, Strange M, O’Carroll A, Brown JP, Sierecki E, et al. EMBO Rep 20, e46518 (2019).
  4. Baker MODG, Shanmugam N, Pham CLL, Strange M, Steain M, Sunde M. Semin Cell Dev Biol, https://doi.org/10.1016/j.semcdb.2018.05.004 (2018).