The RIP1/RIP3 Necrosome Forms a Functional Amyloid Signaling Complex Required for Programmed Necrosis

Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065, USA.
Cell (Impact Factor: 32.24). 07/2012; 150(2):339-50. DOI: 10.1016/j.cell.2012.06.019
Source: PubMed


RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Download full-text


Available from: Francis Chan, Nov 17, 2014
  • Source
    • "Since RIPK1 is more highly expressed in activated microglia than RIPK3, targeting RIPK1 may be appropriate to attenuate microglial-mediated inflammatory signaling. Complex IIb, the critical signaling/executioner of necroptosis formed by the interaction of RIPK1 and RIPK3, exists in an amyloid-like conformation (Li et al., 2012). Consistent with the activation of necroptosis in MS, we obtained multiple lines of evidence for the formation of complex IIb in human MS samples . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8-deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as activation of RIPK1, RIPK3, and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNF-α and that inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a therapeutic strategy for MS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 03/2015; 10(11). DOI:10.1016/j.celrep.2015.02.051 · 8.36 Impact Factor
  • Source
    • "Consistent with a role for RHIM signaling in oligomerization, aggregated forms of RIP3 did not appear in the pellet when RHIM signaling was blocked (data not shown). D161N mutation or RIP3i binding therefore alters the conformation of RIP3 and triggers RHIM-dependent oligomerization reminiscent of aggregate, ''amyloid-like'' RIP1- RIP3 complexes (Li et al., 2012). "

  • Source
    • "These functional amyloid fibrils are used by organisms to perform diverse physiological functions such as biofilm formation [18], [19], cell adhesion [20], synaptic remodeling and learning [21], template to melanin biosynthesis [22], [23], and peptide hormones [24]. Other examples include mitochondrial protein MAVS [25], the necrosome proteins RIP1/RIP3 [26], and several dozen of proteins involved in RNA granule formation [27]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Amyloid fibrils are associated with many maladies, including Alzheimer's disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1-40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1-40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1-40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.
    PLoS ONE 08/2014; 9(8):e105433. DOI:10.1371/journal.pone.0105433 · 3.23 Impact Factor
Show more