Prion-Like Polymerization in Immunity and Inflammation

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Prion-Like Polymerization in Immunity and Inﬂammation

Xin Cai,1 Hui Xu,1 and Zhijian J. Chen1,2

1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148 2Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148 Correspondence: [email protected]; [email protected]

The innate immune system relies on receptors that sense common signs of infection to trigger a robust host-defense response. Receptors such as RIG-I and NLRP3 activate downstream adaptors mitochondrial antiviral signaling (MAVS) and apoptosis-associated speck-like protein (ASC), respectively, to propagate immune and inﬂammatory signaling. Recent studies have indicated that both MAVS and ASC form functional prion-like polymers to propagate immune signaling. Here, we summarize the biochemical, genetic, and structural studies that characterize the prion-like behavior of MAVSand ASC in their respective signaling pathways. We then discuss prion-like polymerization as an evolutionarily conserved mechanism of signal transduction in innate immunity in light of the similarity between the NLRP3–ASC, the NLRP3-ASC pathway in mammals, and the NWD2-HET-s pathway in fungi. We conclude by outlining the unique advantages to signaling through functional prions and potential future directions in the ﬁeld.

INNATE IMMUNE SIGNALING: SENSORS, mune sensors such as Toll-like receptors, reti- ADAPTORS, AND EFFECTORS noic acid-inducible gene I (RIG-I)-like receptors, nucleotide-binding oligomerization do- he innate immune system is an ancient and main (NOD)-like receptors (NLRs), and the Tconserved defense mechanism against path- recently identified cyclic GMP-AMP synthase ogen invasion. As the first line of host defense, it DNA sensor (Chen et al. 2009; Yoneyama and relies on numerous germline-encoded pattern Fujita 2009; Casanova et al. 2011; Sun et al. recognition receptors to detect signs of infec- 2013; Wu et al. 2013). Within minutes to hours tion or cellular danger to trigger a rapid, robust, of activation, the pattern recognition receptors and generic response for host protection (Ta- trigger a myriad of signaling cascades that cul- keuchi and Akira 2010). Conserved pathogen- minate in the production of protective chemo- or danger-associated molecular patterns, such kines and cytokines, which serve both as a rapid as bacterial lipopolysaccharide, foreign nucleic control of the insult and as a means of activating acids, and cytosolic DNA, activate innate im- the more specific and longer-lasting adaptive

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X. Cai et al.

immune response (Iwasaki and Medzhitov functions from Caenorhabditis elegans to mam- 2010). mals (Deddouche et al. 2008; Ashe et al. 2013). Following ligand recognition, each pattern In mammals, RIG-I is activated by binding recognition receptor activates a downstream to viral RNAs bearing 50 triphosphates (50-ppp) adaptor protein that forms an oligomeric sig- or disphosphates (50-pp), which distinguish nalosome for signal transduction. For instance, them from 50-capped cellular RNA (Hornung Toll-like receptors, RIG-I-like receptors, NLRs, et al. 2006; Goubau et al. 2014). Ligand binding and cyclic GMP-AMP synthase activate the to its C-terminal regulatory domain releases respective downstream adaptors MyD88/TRIF, RIG-I from an autoinhibited state and frees mitochondrial antiviral signaling (MAVS)(also its N-terminal tandem CARDs for binding known as VISA, IPS-1, or CARDIF), apopto- to lysine-63 (K63)-linked polyubiquitin (Ub) sis-associated speck-like protein (ASC), and chains (Zeng et al. 2010). K63-polyubiquitin STING (also known as MITA, MPYS, or ERIS) chain binding is essential for RIG-I activation, to propagate immune and inflammatory re- as it converts RIG-I into a signaling competent sponses. Each adaptor protein harbors distinct tetramer (Zeng et al. 2010; Jiang et al. 2012; protein interaction domains that are essential Peisley et al. 2014). The RIG-I:RNA:Ub complex for bridging the upstream receptors to their then activates the downstream adaptor MAVS, downstream effectors. In this regard, the death which also harbors an N-terminal CARD domain (DD) superfamily is one of the most (MAVSCARD). Interactions between the CARDs common protein domains observed in innate of RIG-I and MAVSconvert MAVSfrom an in- immunity, inflammation, and cell death (Park active, autoinhibited monomer into active pri- et al. 2007). Members of the DD superfamily are on-like filaments, the core of which is composed defined by their shared structural features, in- of polymerized MAVSCARD subunits (Hou et al. cluding a unique fold composed of six a-heli- 2011; Cai et al. 2014a; Shi et al. 2015). Function- ces. The caspase activation and recruitment do- al and active MAVS fibers then propagate down- main (CARD), PYRIN, death effector domain, stream signaling by recruiting the kinases TBK1 and DD subfamilies make up the DD superfam- and IKK, which in turn activate the transcrip- ily, and each subfamily mediates signal trans- tion factors IRF3 and NF-kB, respectively, to duction through homotypic interactions and induce the production of antiviral type-I inter- the formation of oligomeric complexes. ferons (Fig. 1) (Zeng et al. 2009; Liu et al. 2013, 2015).

The RIG-I Antiviral Pathway Signals through The Inflammasomes: Model of PYRIN CARD Polymerization Domain Polymerization The RIG-I-like receptors include RIG-I, MDA5, The inflammasomes is another important DD- and LGP2, which detect cytosolic viral RNA to containing signaling complex, which is formed activate the downstream adaptor protein MAVS when NLRs are activated by pathogens and (Fig. 1). As the best-characterized RIG-I-like other noxious stimuli (Martinon et al. 2002; receptor, RIG-I contains N-terminal tandem Lamkanfi and Dixit 2014). Here, NLRs, such CARDs (RIG-I 2CARD), a middle helicase do- as NLRP3 and the DNA receptor AIM2, activate main, and a C-terminal regulatory domain. the adaptor protein ASC to propagate a proin- Bioinformatics analysis indicated that the flammatory response (Fig. 1). The exact trigger RIG-I helicase shares homology with the endo- for NLRP3 activation remains unclear. Both ribonuclease Dicer, which plays an essential role AIM2 and NLRP3 contain N-terminal PYRIN in RNA interference, a primordial form of an- domains that interact with the PYRIN domain tiviral immunity (Zou et al. 2009). Accumulat- of ASC (ASCPYD), thereby converting ASC into ing evidence suggests that RIG-I-like helicases functional prion-like filaments (Cai et al. 2014a; are evolutionarily conserved and serve antiviral Lu et al. 2014). In addition to an N-terminal

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Prion-Like Polymerization in Immunity and Inﬂammation

Antiviral Inflammasome

Sensors CARD CARD Helicase CTD PYD NACHT LRR RIG-I NLRP3

Adaptor CARD TM PYD CARD MAVS ASC

P Effectors IRF3 NF-κB CARD p20 p10 Caspase-1

Interferons IL-1β

Figure 1. Cartoon depictions of the sensors, adaptors, and effectors in the mitochondrial antiviral signaling (MAVS)-dependent antiviral and apoptosis-associated speck-like protein (ASC)-dependent inﬂammasome pathways. Both MAVSand ASC serve as key adaptor proteins that relay multiple upstream signals to downstream effectors. The cytosolic RNA sensor RIG-I activates the MAVSprotein to induce the production of interferon through the transcription factor IRF3 and NF-kB. Similarly, NLRP3 activates the adaptor ASC to produce proinﬂammatory cytokines such as IL-1b. Caspase-1 activation also leads to cell death (pyroptosis).

PYRIN that forms fibers, ASC also contains a C- MAVS was unique in two regards. First, it was terminal CARD that propagates downstream found to be tail-anchored to the outer mem- signaling by binding to the CARD of pro-cas- brane of the mitochondria through its C-termi- pase-1. Active ASC filaments then recruit mul- nal transmembrane domain (Seth et al. 2005). tiple pro-caspase-1 proteins, which are brought Second, activation of MAVSconverted the pro- into proximity to trigger their autoactivation. tein into a detergent-insoluble form that is re- Mature caspase-1 then converts pro-interleukin sistant to extraction by 0.5% Triton X-100 (Seth (IL)-1b into the mature IL-1b that is secreted by et al. 2005). the cell. MAVS provides a direct link between immune signaling and mitochondria, an organelle of bacterial origin. For reasons that remain MAVS IS A BENEFICIAL MAMMALIAN unclear, mitochondrial localization of MAVS PRION-LIKE PROTEIN is essential for its function, because deletion of its transmembrane domain completely abol- Initial Identification and Characterization ished MAVS signaling. For instance, viruses of MAVS such as hepatitis C have evolved proteases that Early studies of RIG-I revealed that it contains specifically cleave MAVS off the mitochon- tandem N-terminal CARDs, which are essential drial membrane to prevent a host immune re- for RIG-I signaling (Yoneyama et al. 2004). sponse (Li et al. 2005; Meylan et al. 2005). Mice MAVSalso contains an N-terminal CARD that deficient in MAVS were unable to produce interacts with RIG-I 2CARD (Kawai et al. 2005; type-I interferons (Sun et al. 2006). These ex- Meylan et al. 2005; Seth et al. 2005; Xu et al. amples highlight the essential role of MAVSand 2005). Initial studies revealed that unlike other its mitochondrial localization in antiviral im- innate immune-signaling proteins or adaptors, munity.

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Biochemical Characterization of MAVS prion. Yeast contains more than a dozen prion- as a Prion like proteins and is arguably the best model Tofurther characterize the active form of MAVS, organism to study prions given the abundance subsequent biochemical studies found that fol- of well-established prion assays and genetic lowing virus infection, endogenous MAVS tools (Alberti et al. 2010). One of the best- formed very large particles (greater in size than characterized yeast prions is the SUP35 protein, the 26S proteasome), as analyzed by sucrose which contains an N-terminal prion domain NM density gradient ultracentrifugation (Hou et al. (NM or SUP35 ) and a C-terminal catalytic 2011). The large MAVSparticles were found to domain that carries out its translation termi- be resistant to 2% sodium dodecyl sulfate (SDS), nation function (Sup35C) (Tuite and Serio as revealed by high molecular weight smears 2010). Using the SUP35 yeast prion assay, in NM on the semi-denaturing detergent agarose gel which the prion domain of SUP35 (SUP35 ) CARD electrophoresis (SDD-AGE) assay, which is was replaced with MAVS , we showed that CARD commonly used for analyses of prions (Alberti MAVS faithfully recapitulated the key pri- NM et al. 2010). Only the SDS-resistant MAVSpoly- on properties of SUP35 in yeast (Cai et al. mers, but not soluble MAVS proteins, were 2014a). Specifically, transient expression of CARD CARD capable of activating the downstream transcrip- MAVS converts the MAVS –SUP35C tion factor IRF3 (Hou et al. 2011). Under the fusion protein into a prion that is dominant, electron microscope (EM), purified active cytoplasmically inherited, and transmissible MAVS particles formed homogenous fibers through numerous cell divisions. Mutagenesis with a striking resemblance to those formed by experiments suggest that MAVS prion conver- a fragment of the mammalian prion protein sion occurs through separable nucleation and (PrP). Proteinase-K digestion of MAVS fibers polymerization steps, much like other prions revealed a resistant fiber core composed of identified to date. MAVSCARD, indicating that MAVSCARD is the prion domain that mediates its assembly into RIG-I DIRECTLY NUCLEATES MAVS PRION filaments (Hou et al. 2011). CONVERSION One of the defining features of prions is their ability to form self-perpetuating structural Unlike other prion proteins that are stochasti- conformations that convert the native form of cally switched into the polymer forms, MAVS the protein into the prion form. Indeed, brief undergoes signal-dependent prion conversion incubation of a substoichiometric amount of regulated by its heterotypic upstream signaling MAVSCARD fibers converts endogenous MAVS protein, RIG-I. Central to our understanding into large, SDS-resistant polymers that are ca- of MAVSactivation is the mechanistic dissection pable of activating the IRF3 transcription fac- of how RIG-I nucleates MAVSprion conversion. tor, much like the biochemical switch observed Although the activation of full-length RIG-I in MAVS following viral infection (Hou et al. requires the sequential binding of both RNA 2011). High-resolution imaging of cells indi- (to the C-terminal regulatory domain) and cated that endogenous MAVS formed fibrous K63 poly-Ub (to RIG-I 2CARD), truncation polymers near the mitochondria following viral studies indicated that RIG-I 2CARD in com- infection (Xu et al. 2014). Together, these results plex with poly-Ub is sufficient to activate indicated that MAVSswitches into self-perpet- MAVS in vitro and in cells (Zeng et al. 2010). uating prion-like fibers to propagate innate im- Consistently, within minutes of incubation with mune signaling. crude mitochondria containing endogenous MAVS, a substoichiometric amount of active RIG-I 2CARD:polyUb complex induces nearly Genetic Validation of MAVS as a Prion complete conversion of MAVS into functional Subsequent genetic studies in yeast further sup- polymers that are capable of downstream signal- ported MAVS as a bona fide, gain-of-function ing (Hou et al. 2011).

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Prion-Like Polymerization in Immunity and Inﬂammation

In yeast that is not known to express a RIG- failed to stain with thioflavin T (ThT), which I-like pathway, transient expression of RIG-I typically binds to b-amyloids. 2CARD robustly induces MAVSCARD –SUP35C Although the structure of the full-length polymerization, indicating that RIG-I 2CARD RIG-I:RNA:Ub complex remains to be deter- is sufficient to directly nucleate MAVS prion mined, recent effort has revealed the structure conversion (Cai et al. 2014a). The frequency of of the RIG-I 2CARD and K63 di-ubiquitin the MAVSCARD prion switch induced by RIG-I complex, the minimal subunit that is necessary 2CARD expression was several orders of mag- and sufficient for MAVSactivation. X-ray crys- nitude greater than that induced by MAVSCARD tallography of the RIG-I 2CARD:Ub2 complex expression, suggesting that RIG-I 2CARD is a revealed a single-turn helical tetramer formed much more potent nucleation factor. Point by RIG-I 2CARD, stabilized by interactions mutations in MAVSCARD that disrupted RIG-I with K63 di-ubiquitin at the periphery (Peisley 2CARD-induced prion formation in yeast also et al. 2014). The active RIG-I 2CARD:Ub2 com- abolished its signaling activity in mammalian plex resembles a lock-washer that is displaced cells following viral infection. Together, these by half its thickness or one CARD subunit biochemical and genetic studies establish (RIG-I contains two CARDs) at the end of the MAVSas a bona fide gain-of-function mamma- complete turn, which is ideally positioned to lian prion essential for the propagation of RIG-I nucleate MAVSCARD polymerization. To char- antiviral signaling. acterize RIG-I 2CARD-mediated MAVSCARD nucleation, X-ray crystallography studies of a RIG-I 2CARD–MAVSCARD fusion protein indi- STRUCTURAL STUDIES OF MAVS FILAMENT cated that the MAVSCARD monomers polymer- REVEALED A SINGLE-TURN HELICAL FIBER ized following the trajectory set by the RIG-I NUCLEATED BY THE RIG-I 2CARD:K63-UB2 2CARD:Ub nucleus, which is consistent with COMPLEX the structures of active MAVSCARD filament Recent structural studies of MAVSCARD fila- made up of a single-stranded helical filament ments and active RIG-I 2CARD in complex (Wu et al. 2014). with K63 ubiquitin chains have begun to shed light on the mechanisms of MAVS activation Model of RIG-I–MAVS Signaling and polymerization. The crystal structure of inactive MAVSCARD monomer is consistent with The following proposed model of RIG-I–MAVS the characteristic six a-helical bundle fold that signaling summarizes the key findings de- is shared by members of the DD superfamily scribed above (Fig. 2). (1) Ligand binding: (Potter et al. 2008). Two recent cryo-electron RNA virus infection of cells generates 50-ppp microscopy (cryo-EM) structural models of RNA ligands that bind to the C-terminal regu- MAVSCARD fibers using proteins purified from latory domain of RIG-I, bringing multiple bacteria and mammalian cells have emerged. RIG-I proteins into proximity and releasing The two studies at 3.6- and 4.2-A˚ resolution RIG-I N-terminal CARDs from an autoinhib- revealed left-handed helical filaments with C1 ited state. (2) MAVS nucleation: The binding symmetry composed of individual MAVSCARD of K63 polyubiquitin chains to the freed RIG-I subunits stacked on top of one another (Wu 2CARD stabilizes the RIG-I:RNA complex into et al. 2014; Xu et al. 2014, 2015). Interestingly, a signaling-competent tetrameric complex that the transition from the soluble to the prion directly nucleates MAVS prion conversion form of MAVSdid not involve any a-helical to through CARD–CARD interactions. (3) MAVS b-sheet structural changes, as typically observed polymerization: Following MAVS nucleation, in other prion conversions. In addition, both irreversible and spontaneous polymerization cryo-EM models did not reveal any major struc- ensues such that an all-or-none response is gen- tural alterations of MAVSCARD in its monomeric erated as long as the RIG-I stimulation thresh- or polymerized form. Consistently, MAVSfibers old is reached. (4) Downstream signal transduc-

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CTD Helicase RIG-I CARD CARD

+ pppRNA PPP or dsRNA

CTD

CARD CARD

Helicase

+ K63polyUb

RIG-I CARD/Ub MAVS monomer MAVS filament complex Helical axis MAVS CARD CARD CARD

CARD CARD MAVS CARD CARD CARD CARD CARD RIG-I 2CARD

MAVS nucleation MAVS polymerization MAVS MAVS CARD MAVS CARD MAVS CARD CARD MAVS MAVS CARD CARD

MAVS

Mitochondrion

Figure 2. A proposed model for mitochondrial antiviral signaling (MAVS)activation by RIG-I-induced polymerization. Detection of viral 50-ppp RNA or double-stranded (ds) RNA by RIG-I triggers a conformational change that exposes the N-terminal tandem caspase activation and recruitment domains (CARDs) of RIG-I (RIG-I 2CARD). RIG-I 2CARD then binds to K63-linked polyubiquitin chains, which facilitate the formation of a RIG-I tetramer. The RIG-I 2CARD tetramer interacts with MAVSCARD, providing a template to initiate the nucleation of a MAVSCARD filament. The MAVSCARD filament recruits additional MAVSCARD to form long filaments on the surface of the mitochondrial outer membrane. The MAVSfilaments are competent in activating downstream signaling cascades to induce type-I interferons and other antiviral effectors. The models of RIG-I CARD/Ub/MAVS CARD complex and MAVS filaments are built based on PDB 4NQK, 4P4H, 2VGQ, and EMD-6428.

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Prion-Like Polymerization in Immunity and Inﬂammation

tion: MAVSfibers are then able to recruit down- When incubated with cell lysates containing stream signaling proteins to propagate a rapid soluble, inactive ASC–green fluorescent protein and robust immune response. (GFP), a substoichiometric amount of ASCPYD fibers converted the ASC–GFP protein into large polymers that sedimented to the bottom ASC MEDIATES INFLAMMATORY of a sucrose density gradient after ultracentri- SIGNALING THROUGH PRION fugation, indicating that ASCPYD fibers were CONVERSION able to convert soluble ASC into self-perpetuating polymers (Cai et al. 2014a). Only the high Identification of ASC as a Functional Prion molecular weight form of ASC–GFP was able In an effort to identify other DD-containing to recruit and activate downstream pro-cas- signaling proteins that may have prion-like pase-1. An ASC polymerization mutant re- properties, a candidate screen on an additional mained in the low molecular weight fractions 18 DDs was performed using the yeast SUP35- on sucrose gradient and was unable to recruit based prion assay (Cai et al. 2014a). Each DD pro-caspase-1. candidate was tested for its ability to functionally replace Sup35NM and was expressed as a Structural Studies of ASC Filaments DD–SUP35C fusion in place of endogenous SUP35 in an ade1-14 genetic background. Yeast The high-resolution cryo-EM structure of the strains expressing ASCPYD –SUP35C exhibited ASCPYD fiber at 3.8 A˚ revealed a three-start, a high frequency of spontaneous prion con- right-handed helical filament (Lu et al. 2014). version, as revealed by the formation of white Analogous to MAVSCARD filaments, no a-heli- colonies on complete (yeast extract peptone cal to b-sheet transition was observed between dextrose [YPD]) media. Transient expression the monomeric and polymerized form of of ASCPYD greatly enhanced the frequency of ASCPYD, and each subunit remained largely ASCPYD prion conversion in yeast, consistent unchanged during the prion switch. In vitro with properties of other prions (Cai et al. ASCPYD polymerization assay using gold- 2014a). Cytoduction and SDD-AGE analyses labeled AIM2PYD or NLRP3PYD-NBD showed in yeast confirmed that the ASCPYD prion par- that each receptor localized to the end of the ticles were cytoplasmically inherited and ASCPYD filament, consistent with their role as formed SDS-resistant polymers. nucleation factors (Lu et al. 2014). At times, Like MAVS,ASC is activated by heterotypic several gold-bound AIM2PYD proteins co-local- upstream proteins such as NLRP3 and AIM2. ized to one end of the filament, consistent with Indeed, transient expression of AIM2 or subsequent studies, and the model that limited NLRP3PYD induced robust ASCPYD –SUP35C oligomerization of upstream receptors precedes prion conversion in yeast at a frequency several the nucleation and polymerization of ASC hundred folds greater than that observed with (Morrone et al. 2015). expression of ASCPYD (Cai et al. 2014a). This again indicates that the physiologic upstream ASC Prion Particles Have “Infectious” signaling receptors more potently nucleate Properties ASC polymerization than overexpression of ASCPYD. Point mutations that inhibited ASC As further support for the prion model of ASC prion conversion in yeast also abolished its activation, cell-based and mouse studies indi- activation by NLRP3 or AIM2 in mammalian cate that activated ASC particles are released cells, indicating that its prion conversion is es- from dying cells and taken up by neighboring sential for its signaling ability. macrophages, leading to nucleation of soluble, Under the electron microscope, recombi- endogenous ASC proteins to perpetuate inflam- nant ASCPYD protein formed filaments resem- masome signaling (Baroja-Mazo et al. 2014; bling those of MAVSCARD (Cai et al. 2014a). Franklin et al. 2014). Intraperitoneal injection

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of active ASC particles resulted in a proinflam- naling partner initiates their polymerization. In matory response only in mice that expressed other words, their prion conversion is regulated endogenous ASC but not in ASC deficient and can be quickly switched on by the appro- mice, suggesting that ASC particles possess in- priate stimulus. fectious properties that catalytically convert endogenous ASC into the prion form, much like mammalian PrP. In mice, ASC particles were A PARALLEL NLRP3–ASC INFLAMMASOME- also found to be stable in the extracellular space LIKE PATHWAY IN FUNGI for 96 hours, indicating that they are relatively MAVS and ASC Share Properties with resistant to protease inactivation, much like the Fungal Prion HET-s other prions. As gain-of-function prions, MAVS and ASC are similar in this regard to the fungal prion HET-s UNIQUE PROPERTIES OF PRIONS (Fig. 3). Similar to HET-s, MAVS and ASC do FUNCTIONING IN INNATE IMMUNITY not stain with ThTor require Hsp104 for prop- Although MAVS and ASC share the key bio- agation of their prions. HET-s triggers cell death chemical and genetic features of prions with by converting its allelic variant HET-S (big “S”) other known prion-like proteins, they differ in into a pore forming toxin (Saupe 2011; Seuring several important aspects. First, unlike most et al. 2012). HET-s and HET-S are both 289- prions that confer loss of function, MAVS and amino-acid proteins that differ at only 13 resi- ASC are both gain-of-function prions. Their dues (for review, see Riek and Saupe 2016). prion conversion is central to their function. Both proteins contain N-terminal HeLo do- For instance, cells containing MAVS mutants mains and interchangeable C-terminal prion defective in prion formation were phenotypi- domains (PrD) that polymerize into a b-sole- cally identical to mavs2/2 cells in response to noid. HET-s mediated HET-S activation has viral infection (Liu et al. 2013). Consistently, been proposed as a form of immune response MAVS and ASC polymerization mutants were that prevents the horizontal transfer of infec- unable to recruit essential downstream signal- tious pathogens during fungal cell fusion. Spe- ing proteins. Second, the monomeric forms of cifically, cell death occurs upon cell fusion only MAVSCARD and ASCPYD are well-folded globu- when one cell contains the prion form of HET-s, lar domains that are not enriched in glutamine which then nucleates the structural conversion and asparagine amino acids, which are often of soluble HET-S expressed by the other cell. found in prion domains. X-ray crystallography Following polymerization of its PrD, the HeLo and NMR studies revealed that both proteins domain of HET-S forms a membrane–perme- form a-helical bundles in their native states, able toxin to induce death of the fused cell (Ma- unlike the unstructured prion domains often thur et al. 2012; Seuring et al. 2012). Mutations found in other aggregation-prone proteins in the HET-s HeLo domain, however, have ren- (Liepinsh et al. 2003; Potter et al. 2008). Third, dered the protein inactive, leaving its PrD as the MAVS and ASC prion conversion lacked only functional domain. Although the two pro- b-amyloid transitions usually observed in other teins share a near identical prion domain (with prions. Both prion domains maintained their only a 3-amino-acid difference), the mecha- a-helical folds in their respective fibers, which nism of HET-s prion conversion had been large- are formed by the polymerization of individual ly unclear until recent studies. a-helical subunits. Consistently, neither filament stained with ThT, as would be expected NWD2 and HET-s Are Fungal Homologs for a b-amyloid. Fourth, unlike most prions of Mammalian NLRP3 and ASC that are formed stochastically, MAVS and ASC undergo regulated prion conversion, in which A recent bioinformatic search for fungal pro- activation of their corresponding upstream sig- teins that share a similar amino acid sequence

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Prion-Like Polymerization in Immunity and Inﬂammation

Mammals Fungi

NLRP3 PYD NACHT LRR NWD2 NACHT WD40

NLRP3 PYD NACHT LRR NWD2 NACHT WD40 ASC PYD CARDHET-S/s HeLo PrD

PYD NACHT LRR NACHT WD40 PYD PYD PYD PYD

Antiviral defense Inflammation Cell death

Figure 3. Homologous mammalian and fungal host-defense pathways that signal through prion conversion. In mammals, receptors such as NLRP3 sense pathogen invasion or cell damage to rapidly convert its downstream apoptosis-associated speck-like protein (ASC) into its active prion form, which then leads to cytokine secretion and cell death to protect the host. A homologous pathway is present in ﬁlamentous fungi, where activated NWD2 receptor converts the HET-S/s protein into a prion to induce cell death. The fungal NWD2-HET-S/s pathway is remarkably similar to the mammalian NLRP3-ASC pathway in both function and domain organization, suggesting that signaling through self-perpetuating protein conformations for host defense and cell-fate determination is conserved from fungi to mammals.

as the HET-s PrD identified NWD2, a protein (where NLRP3PYD is replaced by NWD2N30), that strikingly resembles mammalian NLRP3 in but neither NLRP3 wild-type (WT) nor domain organization and is encoded by a gene NWDN30 alone converted HET-sPrD from a dif- immediately adjacent to het-S (Fig. 3) (Daska- fuse cytoplasmic pattern to a large, distinct fo- lov et al. 2012). Notably, the NWD2 N-terminus cus (Cai et al. 2014a). This result indicated that contains an approximate 30-amino-acid frag- NWD2N30 can indeed nucleate the polymeriza- ment (NWD2N30) that is predicted to take tion of HET-sPrD. However, the interaction be- on a similar structural fold as the HET-s prion tween NWD2 and HET-s depended on the oli- domain, much like the structural relationship gomerization of NWD2, which occurred when between NLRP3PYD and ASCPYD. In addition, NWD2N30 was fused to NLRP3DPYD (which both NWD2 and NLRP3 contain middle contains the NACHT domain) but not when NACHT domains that mediate protein oligo- expressed alone. Functionally, we found that merization. In its C-terminus, NWD2’s WD40 fungal NWD2N30 and HET-sPrD can replace repeat may serve as a ligand-binding domain, the PYRINs of NLRP3 and ASC, respectively, similar to the proposed function of NLRP3’s in mammalian inflammasome signaling (Cai leucine-rich repeat. et al. 2014a). Specifically, in response to niger- Immunofluorescence studies in mammali- icin, a chemical that activates the NLRP3 path- an cells indicated that transient expression of way, only NWD2N30 –NLRP3DPYD fusion, but the NWD2N30 –NLRP3DPYD fusion protein not WT NLRP3, was able to nucleate the poly-

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merization of HET-sPrD–ASCDPYD (where In mammals, ASC has evolved into a bipartite ASCPYD was replaced by HET-sPrD) to activate PYRIN–CARD adaptor and serves similar caspase-1. In contrast, WTASC polymerization functions as HET-s to bridge NLRP3 and pro- was induced by activation of NLRP3, but not caspase-1 activation. Like HET-S, pro-caspase- the NWD2N30 –NLRP3DPYD fusion protein. 1 harbors an N-terminal CARD for interaction These results indicate that the structure–activ- with ASC and a C-terminal executioner domain ity relationship between NWD2N30 and HET- that contains all of its catalytic function. In the sPrD is interchangeable with that between absence of HET-s, NWD2 might directly acti- NLRP3PYD and ASCPYD, where each distinct vate HET-S, which shares interchangeable PrDs prion domain can be nucleated only by the ef- with HET-s. Likewise, in mammalian inflam- fector domain of its cognate upstream receptor. masome signaling, a number of NLRs, such as Subsequent studies in the fungus Podospora NLRC4, can bypass ASC and directly activate anserina revealed that oligomerized NWD2 in- pro-caspase-1. Recent cryo-EM studies indicat- duced HET-s prion conversion (Daskalov et al. ed that NLRC4 is also activated through a self- 2015). Specifically, in fungi expressing chimeric catalytic conformational switch that results in NWD2 variants harboring foreign WD40 re- the formation of a helical wheel with its up- peats, HET-s prion formation was induced by stream activator, NLR apoptosis inhibitory pro- exposure to the respective ligand for each fused tein (NAIP) (Hu et al. 2015). The NLRC4 poly- WD40 repeat. NWDN30 was found to be re- mer can then either activate ASC or directly sponsible for its HET-s templating activity, recruit pro-caspase-1. because mutations predicted to affect its b- As further evidence for the evolutionary solenoid fold abolished its ability to nucleate conservation of the prion-like polymerization HET-s polymerization. In fungi, expression of as a signaling mechanism in cell death pathways, NWD2N30 –RFP induced the relocalization of a recent study found that mammalian necrosis HET-S to the cell periphery and activated the mediated by RIP1/RIP3 (upstream kinase) and pore-forming protein, providing evidence of MLKL (cell death effector) also shares features signaling interaction between NWD2 and with an NWD2/HET-S-like pathway in fungi HET-s/S in their original cellular context. (Daskalov et al. 2016). To characterize other The homology between the NWD2–HET-s NWD2 and HET-S analogs, Daskalov et al. and NLRP3–ASC pathways suggests that sig- identified another gene cluster that contains naling through prion-like polymerization is an NLR and an adjacent effector harboring a evolutionarily conserved, especially in path- prion domain—namely, PNT1 and HELLP, re- ways responsible for self-defense or fate switch spectively—in addition to a putative lipase, SBP. (Fig. 3). Specifically, both pathways rely on dis- PNT1, HELLP,and SBP each contains a PP mo- tinct domains in adaptor proteins that undergo tif, which is capable of amyloid conversion and transmissible structural changes nucleated by shares sequence features with the RIP homo- upstream receptors, leading to signal amplifi- typic interaction motif (RHIM) domains of cation that eventually results in cell death. In RIP1/RIP3 that had previously been observed addition to the similarities between NLRP3 to form amyloid fibrils (Li et al. 2012). As a and NWD2, there are several other reasons to HET-S homolog, HELLP is activated by PP- suggest that the NLRP3–ASC–pro-caspase-1 motif-mediated polymerization and contains a and other inflammasome pathways may have HeLo-like domain to execute cell death. Re- evolved from the NWD2–HET-s/HET-S path- markably, the HeLo-like domain of HELLP way. With a nonfunctional HeLo domain, was found to be homologous with the MLKL HET-s essentially serves as a self-perpetuating 4HB domain, which is responsible for mem- prion adaptor for activation of HET-S through brane permeation in mammalian necrosis interactions between their PrDs. Following ac- (Sun et al. 2012; Cai et al. 2014b; Chen et al. tivation by HET-s, HET-S is then able to execute 2014; Su et al. 2014). Consistently, prion con- cell death through its functional HeLo domain. version targets HELLP to the plasma membrane

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Prion-Like Polymerization in Immunity and Inﬂammation

to induce cell death. Taken together, these scale response will follow, which ensures a com- results suggest that mammalian necrosis and plete response in the early phase of viral infec- fungal cell death share homologous mecha- tion. In addition, the activation of MAVS not nisms mediated by evolutionarily conserved only leads to the production of type-I interfer- protein domains, where self-perpetuating prions, but also further amplifies the host response on-like polymerization governs programmed by inducing the expression of other self-defense cell death. genes such as RIG-I or NLRP3 and pro-IL-1b, which are usually expressed at low levels in un- stimulated cells. BENEFITS OF SIGNALING THROUGH PRION-LIKE POLYMERIZATION Altruistic Prions The finding that prion-like proteins mediate a protective innate immune response may be sur- Viral and bacterial infections frequently lead to prising at first. Until recently, the formation of cell death, which prevents further pathogen rep- prion-like aggregates in mammals had invari- lication and host damage. For instance, activa- ably been associated with pathologic processes, tion of the NLRP3 inflammasome triggers a in which the host’s homeostatic protective re- poorly understood form of cell death, known sponse had failed (Prusiner 1982; Jucker and as pyroptosis. The activation of cell death in Walker 2013; Ramaswami et al. 2013). Although conjunction with prion-like polymerization is the formation of large protein aggregates may unlikely to be a coincidence. These two mecha- be detrimental to individual cells, in the setting nisms likely evolved to accommodate each oth- of a noxious insult that threatens the fitness of er, allowing infected cells to secrete host-protec- an organism, the formation of irreversible pri- tive signals before undergoing senescence. ons offers the host numerous advantages, as Unlike in unicellular yeast, where prion forma- discussed in the following sections. tion has been proposed as a mechanism of phe- notypic diversification to increase the fitness of each cell, altruistic prions such as MAVS and Sensitive and Robust Signal Transduction ASC enhance overall host fitness at the expense Mechanism of individual cells. Interestingly, loss-of-func- Signal transduction plays an essential role in all tion phenotypes govern most if not all yeast aspects of an organism’s function. Although prions, whereas gain-of-function and benefi- certain signals are beneficial, others serve as cial prions have only been observed in multicel- warnings of imminent danger. As the first line lular organisms, such as filamentous fungi and of defense against pathogen invasion, innate mammals. immune signaling plays an essential role in the well-being of the host. In this regard, our Formation of Signalosomes cells must evolve mechanisms to detect minute signs of pathogen invasion. Signal propagation Formation of large polymers offers spatial and through self-perpetuating prions offers the host temporal control over signal transduction. Ac- a sensitive and robust response to minimal tivated MAVSand ASC form large signalosomes stimuli, in which the activation of a small num- that can be visualized as distinct puncta or rods ber of proteins leads to a self-catalytic, all-or- in cells (Broz et al. 2010; Xu et al. 2014). The none response. For instance, recent estimates formation of these large signalosomes is essen- suggest that the RIG-I/MAVS pathway can be tial to recruit and activate downstream signaling activated by 20 viral RNA molecules, much proteins. For example, mutations that abolish fewer than the number of RIG-I-like receptors MAVS or ASC polymerization also prevent the in the cell (Zeng et al. 2010). In this case, as long recruitment of downstream tumor necrosis fac- as a sufficient number of RIG-I-like receptors tor (TNF) receptor-associated factors (TRAFs) can form an oligomer to nucleate MAVS, a full- or pro-caspase-1, respectively. Here, the forma-

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X. Cai et al.

tion of large polymers increases the local con- wise, mice infected with RNAvirus do not pro- centration of signaling molecules, thereby en- duce interferons indefinitely. Indeed, the phe- hancing an otherwise low affinity interaction nomenon of refractory immune tolerance has between MAVS and TRAFs or ASC and pro- been described. For instance, prior exposure to caspase-1. By forming large subcellular struc- endotoxin renders cells refractory to subsequent tures, MAVS and ASC fibers serve as hubs to endotoxin stimulation, pointing to potential recruit downstream proteins to the appropriate mechanisms that turn off innate immune sig- location for their activation. naling downstream from the adaptor proteins (Biswas and Lopez-Collazo 2009). Because un- regulated MAVSor ASC signaling would result CONCLUDING REMARKS AND FUTURE in autoimmune or autoinflammatory diseases, DIRECTION mechanisms must exist that appropriately shut Studies of innate immune signaling uncovered a off the signaling from these protein fibers. Al- surprisingly conserved mechanism of signal though MAVS and ASC represent the first ex- transduction that utilizes prion-like switches amples of functional mammalian prions, they to relay signals from divergent upstream sensors are unlikely to be the only ones. As prion-like to downstream effectors. Although these early polymerization offers the host many unique ad- studies have mostly focused on the activation of vantages, this mechanism is also likely to be MAVS and ASC, many important questions used by other biological pathways. remain. For instance, the mitochondrial localization of MAVS is essential for its prion-like activity. When expressed as a cytosolic protein, ACKNOWLEDGMENTS CARD MAVS is unable to form prion-like oligo- Research in our laboratory is supported by mers, suggesting that MAVS polymerization is grants from the National Institutes of Health either promoted by the mitochondria or inhib- (AI-93967), the Welch Foundation (I-1389), ited by the cytosol through unknown mecha- and the Cancer Research Prevention Institute nisms. Future studies on the contribution of of Texas (RP120718 and RP120718). Z.J.C. is mitochondria to the prion-like activity of a Howard Hughes Medical Institute (HHMI) MAVS are essential for understanding the link Investigator. between mitochondria and immunity. In addition, MAVSand ASC fibers must undergo cycles of seeding, polymerization, and fragmentation REFERENCES to stably propagate through numerous cell di- Reference is also in this collection. visions. Essential factors and chaperones for MAVSand ASC propagation remain to be iden- Alberti S, Halfmann R, Lindquist S. 2010. Biochemical, cell biological, and genetic assays to analyze amyloid and tified, because both proteins do not depend on prion aggregation in yeast. Methods Enzymol 470: 709– Hsp104, which normally governs the fragmen- 734. tation of most yeast prions. Understanding the Ashe A, Be´licard T,Le Pen J, Sarkies P,Fre´zal L, Lehrbach NJ, proteins that promote MAVS and ASC prion Fe´lix MA, Miska EA. 2013. A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral propagation may also yield pathogen virulence RNA dicing and antiviral immunity. eLife 2: e00994. targets against the immune system. Last, recent Baroja-Mazo A, Martıń-Sańchez F, Gomez AI, Martıńez studies suggest the existence of mechanisms CM, Amores-Iniesta J, Compan V, Barbera`-Cremades to prevent continuous signaling from MAVS M, Yaguë J, Ruiz-Ortiz E, Antoń J, et al. 2014. The NLRP3 inflammasome is released as a particulate danger prions. Specifically, we found that cell extracts signal that amplifies the inflammatory response. Nat Im- from virus-infected cells were refractory to ac- munol 15: 738–748. tivation by active MAVS prion particles, sug- Biswas SK, Lopez-Collazo E. 2009. Endotoxin tolerance: New mechanisms, molecules and clinical significance. gesting the existence of downstream mecha- Trends Immunol 30: 475–487. nisms to prevent continuous signaling from Broz P, Newton K, Lamkanfi M, Mariathasan S, Dixit VM, MAVS prion particles (Hou et al. 2011). Like- Monack DM. 2010. Redundant roles for inflammasome

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Prion-Like Polymerization in Immunity and Inflammation

Xin Cai, Hui Xu and Zhijian J. Chen

Cold Spring Harb Perspect Biol published online November 23, 2016

Subject Collection Prion Biology

Genetic PrP Prion Diseases Clinical Neurology and Epidemiology of the Major Mee-Ohk Kim, Leonel T. Takada, Katherine Wong, Neurodegenerative Diseases et al. Michael G. Erkkinen, Mee-Ohk Kim and Michael D. Geschwind Neurodegenerative Disease Transmission and Prion Properties of SOD1 in Amyotrophic Lateral Transgenesis in Mice Sclerosis and Potential Therapy Brittany N. Dugger, Daniel P. Perl and George A. Caroline Sibilla and Anne Bertolotti Carlson Toward the Atomic Structure of PrPSc Mapping Neurodegenerative Disease Onset and Jose A. Rodriguez, Lin Jiang and David S. Progression Eisenberg William W. Seeley Bioassays and Inactivation of Prions Erratum: Functional Prions in the Brain Kurt Giles, Amanda L. Woerman, David B. Berry, et Joseph B. Rayman and Eric R. Kandel al. Functional Prions in the Brain Pathology of Neurodegenerative Diseases Joseph B. Rayman and Eric R. Kandel Brittany N. Dugger and Dennis W. Dickson The Amyloid Phenomenon and Its Links with TIA-1 Is a Functional Prion-Like Protein Human Disease Joseph B. Rayman and Eric R. Kandel Christopher M. Dobson Tau Positron Emission Tomography Imaging Molecular Genetics of Neurodegenerative Hartmuth C. Kolb and José Ignacio Andrés Dementias Flora I. Hinz and Daniel H. Geschwind Prion-Like Polymerization in Immunity and Cross-β Polymerization of Low Complexity Inflammation Sequence Domains Xin Cai, Hui Xu and Zhijian J. Chen Masato Kato and Steven L. McKnight

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