Amyloid deposits are often thought of as junk—toxic waste products clogging up cells. A paper in today’s Cell, however, suggests that at least one type of amyloid plays a crucial role in physiologic signaling. Researchers led by Hao Wu, a structural biologist formerly at Weill Cornell Medical College in New York City and now at Children’s Hospital, Boston, Massachusetts, and Francis Chan, a cell death expert at the University of Massachusetts Medical School, Worcester, report that protein kinases involved in necrotic cell death form amyloid fibrils. In the absence of fibril formation, necrotic signaling fails, demonstrating that the structure is a key feature of the pathway. An unanswered but tantalizing question is whether necrotic amyloids may interact in some way with the β amyloid fibrils present in Alzheimer’s disease.

“This paper is among the first to show just how important amyloid formation is in normal cellular processes in mammalian cells,” said David Teplow at the University of California, Los Angeles. He praised the thoroughness of the work, noting, “In every possible way you could imagine from a physical biochemistry point of view, [the authors] showed that these things are classic amyloid structures. It is beautiful and impressive work.”

In the last few years, accumulating evidence indicates that amyloids can serve multiple physiologic functions, particularly in yeast and bacteria. To date, known roles mainly include structural tasks such as protein storage, scaffolding, cell adhesion, and biofilm formation (see ARF related news story; ARF news story; and Ramsook et al., 2010). Amyloids have not previously been connected to regulated cell death pathways.

Programmed cell death can be either apoptotic, in which cells shrivel neatly away, or necrotic, in which dying cells rupture messily (see Vandenabeele et al., 2010). Receptor interacting protein kinases 1 and 3 (RIP1 and RIP3) act as master switches that regulate cell fate. Depending on physiological conditions, these proteins can mediate cell survival, apoptosis, or necrosis. To initiate necrosis, RIP1 and RIP3 form a protein complex known as the necrosome. The kinases then phosphorylate and activate downstream proteins in the necrotic pathway (see Sun et al., 2012; Zhao et al., 2012). RIP1 and RIP3 are known to bind each other through regions called RIP homotypic interaction motifs (RHIMs), but the nature of the necrosome had never been elucidated.

To examine this structure, first author Jixi Li coexpressed tagged human RIP1 and RIP3 in cell cultures. Surprisingly, the proteins formed very large complexes. Li and colleagues used a wide variety of biochemical techniques, including electron microscopy, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy, to show that these complexes are amyloid fibrils. The fibrils display classic amyloidogenic properties. They bind amyloid dyes such as thioflavin T and Congo red, and are highly stable, dissolving only under harsh chemical conditions, the authors report. In addition, RIP1/RIP3 complexes can seed rapid polymerization in monomeric solutions of RIP kinases, another property of amyloid.

Structural analysis revealed that the fibrils consist of a buried amyloid core surrounded by long, flexible extensions. The RIP kinase domains are found in these extensions and likely maintain their globular shape and activity, the authors note. To define the amyloid-forming region, the researchers made short peptides from the RHIM domain of each protein. They found that fragments as short as 19 amino acids (RIP3) and 31 amino acids (RIP1) still bound each other. By mutating residues in these core regions one by one, the authors showed that only mutations in the central six or seven amino acids of each RHIM disrupted amyloid formation. This confirmed that the amyloid core is very small.

The authors then looked at function. Human cell cultures transfected with wild-type RIP kinases underwent necrosis when stimulated with the appropriate cell death signals. By contrast, cells transfected with amyloid-defective RIP mutants thrived, ignoring death signals. Likewise, treating cell cultures with amyloid dye inhibited the formation of the amyloid complex and prevented necrosis. These experiments indicate that the amyloid structure is essential for proper necrotic signaling. Intriguingly, the mutants that could not form amyloid also had little kinase activation, suggesting that amyloid formation helps turn on the kinases.

Based on these findings, the authors propose a model in which the long, disordered flanking sequences of RIP monomers hide the core RHIM sequences under normal cellular conditions, preventing the proteins from interacting with each other to form amyloid. Cell death signals switch on the kinases, and they autophosphorylate. The charged phosphate groups repel each other, opening up the proteins and exposing the RHIM regions. RIP1 and RIP3 can then bind to each other, which spurs further auto- and cross-phosphorylation, leading to even more aggregation in a feed-forward loop, the authors suggest. This model makes sense, Teplow agreed, because cells would need to control the formation of amyloid. Otherwise, the amyloid fibrils might choke the cell, or initiate unwanted cell death under normal conditions. Phosphorylation may act as a sensitive regulatory mechanism over this process.

What happens to necrotic amyloid fibrils after their original cells die? Wu does not know, but said she is investigating this question. She wonders whether these necrotic amyloid structures could cross-talk with β amyloid fibrils, perhaps helping to worsen Alzheimer’s disease. In future work, she and Chan will test this idea in vitro and in AD mouse models.

Cell death expert Peter Vandenabeele at VIB and Ghent University, Belgium, agreed, writing to ARF that there might be a link to AD: “[Because] amyloid structures may induce co-aggregation of other amyloidogenic proteins, it is conceivable that necrosis processes may contribute to AD pathogenesis.” He added that if so, it might be possible to delay AD onset with RIP kinase inhibitors (see full comment below).—Madolyn Bowman Rogers

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  1. Amyloid aggregation in programmed necrosis or necroptosis
    Our lab was one of the first describing necrotic cell death as a programmed form of cell death implicated in many inflammatory and degenerative pathologies (Vandenabeele et al., 2010). Until recently, the prototype cell death mechanism has been thought to be apoptosis, which involves activation of a cascade of caspases and the consecutive cleavage of proteins. Nowadays, regulated or programmed necrosis, also called necroptosis, is viewed as another major type of cell death. Necroptosis is mainly driven by the combined action of receptor interacting protein kinase 1 (RIPK1) and RIPK3 kinases (Declercq et al., 2009). Targeting these RIP kinases by small-molecule inhibitors (necrostatins) (Degterev et al., 2008) has been proven as a powerful tool to block necrotic cell death both in vitro and in vivo.

    Many stimuli can elicit necrotic cell death including pathogens, TNF family members, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs), and also some chemotherapeutics (Vanlangenakker et al., 2012). All these have been shown to activate or sensitize the formation of RIPK-containing complexes (called stressosome, ripoptosome, necrosome), which are implicated in the initiation of inflammatory and/or cell death signaling either by apoptosis or necroptosis (Vandenabeele et al., 2010). Which type of cell death ensues depends on the composition of these RIPK-containing complexes; for example, low levels of caspase 8 and the presence of RIPK3 kinase favor the necroptosis outcome.

    This recent paper by Li et al. represents a collaborative effort by the cell death expert group of Francis Chan (University of Massachusetts Medical School) and the structural biology expert group of Hao Wu (Weill Cornell Medical College and Harvard Medical School). They demonstrate elegantly how the cell death-inducing complex forms a large β amyloid-like structure of heterodimeric RIP1 and RIP3 in a 1:1 ratio. This filamentous fibril formation is initiated by necroptosis-inducing agents and depends on a core around the hydrophobic RHIM domain motif which allows heterotypic interaction between both kinases. The kinase activity of both RIP1 and RIP3 are required to induce this extremely stable amyloid-like structure, probably by causing an auto- and transphosphorylation-dependent exposure of the hydrophobic core of the RHIM domain. This aggregation forms seeds that can induce further polymerization, much like prions do. These RIP kinase-driven β amyloid-like structures may form a platform on which cell-death signaling molecules are recruited and propagated. Formation of higher-order scaffolds by death domain folds (Kersse et al., 2011), and now by RHIM domains, is an emerging theme in inflammation and cell-death signaling, and in cellular stress in general. It is still not clear what really initiates Alzheimer’s disease (Benilova et al., 2012); however, it is clear that the amyloid state of proteins may initiate or propagate toxic signaling pathways (Eisenberg and Jucker, 2012). Given that the core protein complex of necroptosis or programmed necrosis forms amyloid-like structures very similar to those observed in Alzheimer’s disease, and that amyloid structures may induce co-aggregation of other amyloidogenic proteins (Eisenberg and Jucker, 2012), it is conceivable that necroptosis processes may contribute to Alzheimer’s disease (AD) pathogenesis. The good news is that targeting necroptosis is recently possible thanks to RIPK1 kinase inhibitors such as necrostatins (Degterev et al., 2008). The big search for specific RIPK1 and RIPK3 kinase inhibitors has been started now, and if (though a big "if"!) necrotic cell death processes are implicated in AD pathogenesis, the use of these kinase inhibitors may retard the onset of the disease by interfering with the formation of RIPK1 and RIPK3 amyloid filamentous fibrils.

    References:

    . The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 2012 Mar;15(3):349-57. PubMed.

    . RIP kinases at the crossroads of cell death and survival. Cell. 2009 Jul 23;138(2):229-32. PubMed.

    . Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. PubMed.

    . The amyloid state of proteins in human diseases. Cell. 2012 Mar 16;148(6):1188-203. PubMed.

    . The death-fold superfamily of homotypic interaction motifs. Trends Biochem Sci. 2011 Oct;36(10):541-52. PubMed.

    . The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012 Jul 20;150(2):339-50. PubMed.

    . Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 2010 Oct;11(10):700-14. Epub 2010 Sep 8 PubMed.

    . Many stimuli pull the necrotic trigger, an overview. Cell Death Differ. 2012 Jan;19(1):75-86. PubMed.

References

News Citations

  1. Is It Good for You? Amyloid Shows New Side in Mammalian Cells
  2. Research Brief: Secretory Hormones—Example of Functional Amyloid?

Paper Citations

  1. . Yeast cell adhesion molecules have functional amyloid-forming sequences. Eukaryot Cell. 2010 Mar;9(3):393-404. PubMed.
  2. . Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 2010 Oct;11(10):700-14. Epub 2010 Sep 8 PubMed.
  3. . Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012 Jan 20;148(1-2):213-27. PubMed.
  4. . Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5322-7. PubMed.

Further Reading

Primary Papers

  1. . The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012 Jul 20;150(2):339-50. PubMed.