Download Diversity of Amyloid Motifs in NLR Signaling in Fungi

biomolecules
Review
Diversity of Amyloid Motifs in NLR Signaling
in Fungi
Antoine Loquet 1 and Sven J. Saupe 2, *
1
2
*
Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR 5248 CBMN-CNRS
Université de Bordeaux, Allée Geoffroy Saint-Hillaire, 33600 Pessac, France; [email protected]
Non-Self Recognition in Fungi, Institut de Biochimie et de Génétique Cellulaire, UMR 5095 CNRS
Université de Bordeaux, 1 rue Camille Saint Saëns, 33077 Bordeaux CEDEX, France
Correspondence: [email protected]; Tel.: +33-556-99-90-27; Fax: +33-556-99-90-67
Academic Editors: Margaret Sunde, Matthew Chapman, Daniel Otzen and Sarah Perrett
Received: 2 March 2017; Accepted: 10 April 2017; Published: 13 April 2017
Abstract: Amyloid folds not only represent the underlying cause of a large class of human diseases
but also display a variety of functional roles both in prokaryote and eukaryote organisms. Among
these roles is a recently-described activity in signal transduction cascades functioning in host defense
and programmed cell death and involving Nod-like receptors (NLRs). In different fungal species,
prion amyloid folds convey activation signals from a receptor protein to an effector domain by an
amyloid templating and propagation mechanism. The discovery of these amyloid signaling motifs
derives from the study of [Het-s], a fungal prion of the species Podospora anserina. These signaling
pathways are typically composed of two basic components encoded by adjacent genes, the NLR
receptor bearing an amyloid motif at the N-terminal end and a cell death execution protein with a
HeLo pore-forming domain bearing a C-terminal amyloid motif. Activation of the NLR receptor
allows for amyloid folding of the N-terminal amyloid motifs which then template trans-conformation
of the homologous motif in the cell death execution protein. A variety of such motifs, which differ by
their sequence signature, have been described in fungi. Among them, the PP-motif bears resemblance
with the RHIM amyloid motif involved in the necroptosis pathway in mammals suggesting an
evolutionary conservation of amyloid signaling from fungi to mammals.
Keywords: amyloid; prion; programmed cell death; NLR; filamentous fungi
1. NLR in Animals and Plants and NLR-Like Receptors in Fungi
Recognition and adequate response to biotic interactions are critical in immune defense and for the
establishment of efficient symbioses. Immunity involves both cell surface and intracellular receptors.
Plants and animals display a repertoire of intracellular receptors of the NLR (nucleotide binding
domain, NBD and leucine-rich repeat, LRR) superfamily that is employed both to fight off pathogens
and to regulate symbiotic interactions with micro-organisms [1,2]. Although the acronym NLR is often
used as derived from the expression Nod-like receptors, originating from the animal immunity field,
some authors prefer to adhere to the nomenclature given above to take into account that this type of
receptors in found both in animals and in plants. NLRs display a typical tripartite domain architecture
with a central NBD domain flanked N-terminally with an effector domain and C-terminally with a
LRR domain. Two subclasses of NBDs are distinguished, NACHT domains are found in animals,
whereas plant NLRs display NB-ARC domains. In animal NLRs, the N-terminal domains most often
belong to the death-fold superfamily (including CARD and Pyrin domains) while in plants two
main subtypes can be distinguished as either TIR and CC (coiled-coil) domains (Figure 1A). NLRs
are activated by direct or indirect recognition of triggering cues and post-activation responses often
involve induction of programmed cell death (typically in the form of a hypersensitive response in
Biomolecules 2017, 7, 38; doi:10.3390/biom7020038
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plants) but
also production of pro-inflammatory cytokines in animals. The detailed mode of
activation
Biomolecules 2017, 7, 38 2 of 10 of NLRs remains poorly defined in particular in plant models. So far the APAF-1 inducer of the intrinsic
a hypersensitive response in plants) but also production of pro‐inflammatory cytokines in animals. apoptosis
pathway generally served as a paradigm from the activation mechanism of NLRs [3] but
The detailed mode of activation of NLRs remains poorly defined in particular in plant models. So far cryoelectron
tomography structures of animal NLRs start to emerge [4]. NLRs exist in an auto-inhibited
the APAF‐1 inducer of the intrinsic apoptosis pathway generally served as a paradigm from the form in which the LRR domain participates in the auto-inhibition, binding of a pathogen-derived
activation mechanism of NLRs [3] but cryoelectron tomography structures of animal NLRs start to ligand induces
formation
ofin anan oligomerisation
competent
state
that
leadsparticipates to the assembly
emerge [4]. NLRs exist auto‐inhibited form in which the LRR domain in the of an
active oligomer.
The
oligomerisation
step
is
thought
to
mediate
proximity-driven
activation
auto‐inhibition, binding of a pathogen‐derived ligand induces formation of an oligomerisation of the
competent state that leads to the assembly of an active oligomer. The oligomerisation step is thought N-terminal effector domains. For some animal NLRs it has been shown that the pathogen-derived
signal isto mediate proximity‐driven activation of the N‐terminal effector domains. For some animal NLRs it directly recognized (for instance, NAIP5/NLRC4 recognize bacterial flagellin) [5] but more
complexhas been shown that the pathogen‐derived signal is directly recognized (for instance, NAIP5/NLRC4 strategies of recognition have also been developed probably resulting from the intense arm
recognize bacterial flagellin) [5] but more complex strategies of recognition have also been race between
host and pathogen [6]. In plants, in the guard model it is the modification of a guardee
developed probably resulting from the intense arm race between host and pathogen [6]. In plants, in protein the by aguard pathogen-effector
triggersof activation.
Plants by also
employ decoys that
lure pathogen
model it is the that
modification a guardee protein a pathogen‐effector that triggers effectors
and
allow
their
detection.
Such
decoys
can
occur
as
independent
proteins
or
be present as
activation. Plants also employ decoys that lure pathogen effectors and allow their detection. Such decoys can occur as independent proteins or be present as integrated decoys in the C‐terminal integrated decoys in the C-terminal region of NLRs. In spite, of the common features of NLRs in
region of NLRs. In mechanistic
spite, of the common features of NLRs in mode
plants and
and animals, the mechanistic plants and
animals,
the
details in
the activation
the post-activation
responses
details in the activation mode and the post‐activation responses presumably are extremely diverse presumably are extremely diverse consistent with the role of these receptors in immune response, a
consistent with the role of these receptors in immune response, a biological activity in which biological
activity in which diversification is critical.
diversification is critical. Figure 1.
Domain organization of Nod-like receptor (NLR) and NLR-like proteins in animals, plants
Figure 1. Domain organization of Nod‐like receptor (NLR) and NLR‐like proteins in animals, plants and (A)
fungi. The common
most common domain organization Nod‐like NB‐leucine‐rich repeat
repeat (LRR))
and fungi.
The(A) most
domain
organization
for for Nod-like
(or(or NB-leucine-rich
proteins proteins
and related in plants
animals, plants and are
fungi are listed. animal group proteins(LRR)) and related
in proteins animals,
and
fungi
listed.
The The animal
group
includes
includes the APAF‐1 regulator of the intrinsic apoptosis pathway (bottom diagram). Only a selection the APAF-1 regulator of the intrinsic apoptosis pathway (bottom diagram). Only a selection of the
of the more common domain‐architectures is given. (B) Genome organization and domain diagram more common
domain-architectures is given. (B) Genome organization and domain diagram of the
of the nwd2/het‐s gene cluster of Podospora anserina. The star‐shaped figure represents the cognate nwd2/het-s
gene cluster of Podospora anserina. The star-shaped figure represents the cognate ligand of
ligand of the NWD2 repeat domain. the NWD2 repeat domain.
NLR have also been described in fungi adding an important piece to the comparative description of this type of receptors in the eukaryotic reign [7]. We chose to adhere to the NLR NLR
have also been described in fungi adding an important piece to the comparative description
designation for this class of proteins in fungi in using it as a derivation of Nod‐like receptors. But of this type
of receptors in the eukaryotic reign [7]. We chose to adhere to the NLR designation for this
they are also sometimes referred to as NLR‐like because in this branch LRR repeats are not found but class of are proteins
in fungi
in using
it as asuperstructure derivation offorming Nod-like
receptors.
are also sometimes
replaced by a variety of other repeats such as But
TPR they
(tetratricopeptide referredrepeats), WD repeats or ANK (ankyrin) repeats which makes the NLR designation (understood as to as NLR-like because in this branch LRR repeats are not found but are replaced by a variety
of otherNB‐LRR) unsuited. In contrast, to what is found in animals and plants, NBDs can be either of the superstructure forming repeats such as TPR (tetratricopeptide repeats), WD repeats or ANK
(ankyrin) repeats which makes the NLR designation (understood as NB-LRR) unsuited. In contrast,
to what is found in animals and plants, NBDs can be either of the NB-ARC or NACHT type (Figure 1A).
There is a large diversity of effector domains, many of which still lack a functional annotation. Twelve
Biomolecules 2017, 7, 38
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main types have been identified, some of which correspond to domains with a predicted enzymatic
activity in contrast to TIR and death-fold family domains which function as adaptor domains. NB-ARC
and NACHT domains can be found in almost all combinations with the various types of N-terminal
effector domains and WD, ANK or TPR domains, suggesting that the different domain architectures
arise from combinatorial associations between elementary building blocks. WD, ANK and TPR repeats
of fungal NLR can display high levels of internal conservation and be subject to repeat copy number
polymorphism and concerted evolution between repeats. A fraction of the NLR repertoires found in
fungal genomes display an original gene and domain architecture. In this class of NLRs, a typical
effector domain is not found but instead is encoded by the gene immediately adjacent in the genome [8].
In place of the effector domain, a short amyloid sequence motif is found and the same motif is also
found as a C-terminal extension in the effector domain encoded by the adjacent gene. Activation
of the effector domain is not the direct result of the NLR oligomerization but rather is mediated
by transmission of the amyloid fold from the receptor to the effector protein. This mode of signal
transduction was first identified in the NWD2/HET-S system and further generalized to other NLR
and effector protein pairs [8–11] as will be described herein (Figure 1B). Description of this mode
of amyloid signaling stems from the study of a fungal non-self recognition phenomenon known as
heterokaryon incompatibility which was later found to derive from a NLR-based signaling cascade.
The description of this system offers a paradigm to frame the current understanding of other amyloid
signaling modules in fungi.
2. The HET-s/HET-S System
In fungi, when different isolates of the same species fuse, most often a cell death reaction
ensues. This phenomenon known as heterokaryon incompatibility is controlled by specific loci termed
heterokaryon incompatibility genes (het genes) [12]. In the species Podospora anserina, the het-s (small s)
and het-S (large S) genes form a pair of incompatibility genes. Fusion between a het-s and a het-S strain
leads to cell death [13]. The specific characteristic however of this incompatibility system is that the
het-s-encoded protein is a prion protein. The het-s strains exist in two distinct epigenetic states termed
[Het-s*] and [Het-s], in [Het-s*] strains the protein is soluble and monomeric while in [Het-s] strains in
assembles into prion aggregates. Importantly incompatibility occurs when the prion form of HET-s
interacts with HET-S. HET-s is a two domain protein, with a N-terminal globular domain termed
HeLo and a C-terminal prion forming domain (PFD). The C-terminal prion forming domain (HET-s
(218–289)) is necessary and sufficient for prion propagation and has been extensively used as a model
system for studying the structure-function relation in amyloid formation and prion propagation. HET-s
PFD fibrils show no evidence of structural polymorphism as they lead to very well-resolved solid state
nuclear magnetic resonance (NMR) spectra. The HET-s PFD prion fibrils form a β-solenoid structure
with two rungs of β-strands per monomer and comprising two 21 residue-long imperfect repeats
connected by a 15 amino acid flexible loop [14] (Figure 2). Each of pseudo-repeats forms one 4.7 Å
layer in the cross-β structure. Each repeat is composed of four β-strands delimiting a hydrophobic
core. At the C-terminal end, a semi-hydrophobic pocket is formed by an aromatic loop that folds back
into a groove delimited by the third and fourth β-strands. The structure is stabilized by two asparagine
ladders and three salt bridges resulting from the stacking of the two rungs of β-strands. The core is
composed of hydrophobic residues and of two hydroxyl residues forming a hydrogen bond between
the two layers inside the core (T233/S273). β-arc positions between the third and the fourth β-strand
in both layers are occupied by conserved glycine residues. These structural elements (N-ladders,
salt-bridges, hydrophobic core, buried polar residues and β-arc glycines) contribute to various extends
to the prion function, β-solenoid fold and fibril stability and fibril formation rate [15,16].
HET-S differs from HET-s by 13 residues and also comprises a HeLo domain and a C-terminal
prion forming domain (however as will be developed below, HET-S in contrast to HET-s cannot form
a prion) [17–20]. When HET-s in the prion form interacts with HET-S, the prion forming region is
templated into the β-solenoid fold and this structural transition induces in turn a conformational
Biomolecules 2017, 7, 38
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4 of 10 change in theBiomolecules 2017, 7, 38 HeLo domain which
becomes activated and acquires a pore-forming activity which
relies
on the formation
of
a
N-terminal
transmembrane
helix
[19].
In
vivo,
upon
interaction
with
[Het-s],
change in the HeLo domain which becomes activated and acquires a pore‐forming activity which the HET-S protein
relocates
to theof cell
membrane,
compromises
cell
integrity
triggers
cellwith death.
relies on the formation a N‐terminal transmembrane helix [19]. In vivo, and
upon interaction [Het‐s], the HET‐S protein relocates to the cell membrane, compromises cell integrity and triggers HET-s and HET-S
functionally differ in their HeLo domain, a substitution at position 33 deprives
cell death. HET‐s and HET‐S functionally differ in their HeLo domain, a substitution at position 33 HET-S of pore-forming
activity. In a sense HET-s can be viewed as a mutant form of HET-S devoid of
deprives HET‐S of pore‐forming activity. In a sense HET‐s can be viewed as a mutant form of HET‐S cell death inducing activity, it is that very property (lack of toxicity associated with the amyloid form)
devoid of cell death inducing activity, it is that very property (lack of toxicity associated with the that allows the protein to propagate as a prion in vivo [21].
amyloid form) that allows the protein to propagate as a prion in vivo [21]. Figure 2. Amyloid fold of the HET‐s prion forming domain. (A) Lateral and axial view of a trimer of Figure 2. Amyloid fold of the HET-s prion forming domain. (A) Lateral and axial view of a trimer
HET‐s (218–289) in its fibrillar amyloid conformation. Each monomer is given in a different color of HET-s (218–289)
in its fibrillar amyloid conformation. Each monomer is given in a different color
(PDB entry 2RMN). (B) Structure of the R1 and R2 repeats of HET‐s (218–289) given together with the (PDB entry 2RMN).
(B) Structure
ofsequence. the R1 and
repeats
HET-sin (218–289)
given together
corresponding amino acid Polar R2
residues are of
colored green, hydrophobic residues with
in the corresponding
amino acid sequence. Polar residues are colored in green, hydrophobic residues in
yellow, positively charged residues in red, negatively charged residues in blue, aromatic residues in magenta and glycine in white. yellow, positively
charged residues in red, negatively charged residues in blue, aromatic residues in
magenta and glycine in white.
3. The NWD2/HET‐S Paradigm In a search for sequences homologous to the HET‐s PFD, it was realized (with some surprise) 3. The NWD2/HET-S
Paradigm
that the protein encoded by the gene immediately adjacent to het‐S in the P. anserina genome In a search
for sequences
the HET-s
PFD, itto was
(with
some surprise)
that
contains at its very homologous
N‐terminus a to
region of homology the realized
elementary 21 amino acid repeat forming a single rung of the β‐solenoid [8]. This protein is termed NWD2 and corresponds to a NLR the protein encoded by the gene immediately adjacent to het-S in the P. anserina genome contains at its
with a a central domain toand C‐terminal WD repeat domain. It was then proposed very N-terminus
regionNACHT of homology
thea elementary
21 amino
acid repeat
forming
a singlethat rung
HET‐S in fact constitutes the effector protein activated by the NWD2 NLR (Figure 1B). Because the of the β-solenoid [8]. This protein is termed NWD2 and corresponds to a NLR with a central NACHT
cognate ligand of NWD2 is currently unknown chimeric variants of NWD2 responding to defined domain andligands were constructed. Such chimeric NWD2 variants efficiently induces [Het‐s] formation in a a C-terminal WD repeat domain. It was then proposed that HET-S in fact constitutes
the effector protein
activated by the NWD2 NLR (Figure 1B). Because the cognate ligand of NWD2
ligand‐dependent manner [10]. The N‐terminal region of NWD2 is both necessary and sufficient for this prion activity in vivo; this region was designated R0 by reference the R1 and R2 is currently unknowninducing chimeric
variants
of NWD2
responding
to defined
ligands to were
constructed.
repeats in the HET‐s PFD. A synthetic NWD2 (3–23) peptide corresponding to the R0 region Such chimeric NWD2 variants efficiently induces [Het-s] formation in a ligand-dependent manner
[10].
assembles into amyloids with prion infectivity and represents the smallest molecular entity with The N-terminal region of NWD2 is both necessary and sufficient for this prion inducing activity in vivo;
prion infectivity. Solid‐state NMR analyses of NWD2 (3–23) fibrils with residue‐specific labelling this region was designated R0 by reference to the R1 and R2 repeats in the HET-s PFD. A synthetic
support that R0 adopts a HET‐s‐like fold. It is proposed that ligand‐induced oligomerisation of NWD2 (3–23)
peptide
corresponding
to the R0
intoto amyloids
with
prion infectivity
NWD2 allows for spatial clustering of region
the R0 assembles
regions leading cooperative nucleation of the and represents
the
smallest
molecular
entity
with
prion
infectivity.
Solid-state
NMR
analyses
of NWD2
β‐solenoid fold that can then template HET‐S PFD transconformation and HET‐S activation. (3–23) fibrils with residue-specific labelling support that R0 adopts a HET-s-like fold. It is proposed
that ligand-induced oligomerisation of NWD2 allows for spatial clustering of the R0 regions leading to
cooperative nucleation of the β-solenoid fold that can then template HET-S PFD transconformation
and HET-S activation. NWD2/HET-S is thus a two-component system in which the signal transduction
process from NLR to effector domain involves propagation of an amyloid prion fold. Note that the
Biomolecules 2017, 7, 38
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incompatibility reaction between [Het-s] and HET-S occurs independently of NWD2. Incompatibility
is proposed to derive by exaptation from the NWD2/HET-S system, yet it does not require NWD2
to occur [21]. Remarkably, it was reported that the N-terminal region of NWD2 and the HET-s PFD
can functionally substitute for the Pyrin signaling domains in the human NLRP3 NLR and ASC, its
downstream effector. NWD2/HET-S can serve as a model for the understanding of other NLR/effector
pairs functioning through amyloid signaling [22].
4. Additional Het-s-Related Amyloid Motifs
Fungal genomes were screened for motifs occurring at the C-terminus of proteins with HeLo and
HeLo-like domains. In an ensemble of 579 proteins, 155 were found that harbored two repeats related
in sequence to the 21 amino acid elementary HET-s-motif. In most cases, the gene adjacent to the gene
encoding the HeLo (or Helo-like) domain protein did encode a NLR with at its very N-terminus a
further copy of the motif. Clustering analysis of the sequences revealed the existence of 5 subfamilies
that were termed HRAMs (for Het-s-related amyloid motifs) [9]. Each subfamily is defined by a specific
sequence signature and in spite of considerable variation among the motif signatures, features such
as presence of hydrophobic residues at positions 3, 6, 8 and 14 of the motif and presence of a G in
position 17 are common to all subfamilies (Figure 3). Co-variance analyses strongly suggest that the
additional HRAM subfamilies adopt a fold that is related to the P. anserina HET-s fold. In particular,
co-variance analyses predict in-register interactions between the R1 and R2 motifs. It appears that the
Het-s amyloid signaling motif has diversified into discrete subfamilies. Interestingly, in certain species
as for instance P. anserina, HRAMs belonging to different families co-exist, opening the possibility for
the occurrence of several parallel amyloid signaling pathways. Structural approaches are now required
to determine to which extend the fold of these distinct HRAM families resemble the canonical HET-s
fold and to which extend the different motifs do or do not cross-seed.
Figure 3. Sequences and motif signatures of the different Het-s-related-amyloid motifs (HRAM)
subfamilies. For each of the five HRAM subfamilies, for one representative example, sequences of
the R1, R2 and R0 sequences are given as well as the general motif signature (given as a consensus
sequence logo). The proteins chosen as examples are respectively: Fusarium graminearum I1S1J5
and I1S1J6 for HRAM1, Penicillium roqueforti W6Q0M1 and W6PZN6 for HRAM2, Cladophialophora
psammophila W9WR11 and W9XJW2 for HRAM3, Pestalotiopsis fici W3WR35 and W3WR59 for HRAM4
and P. anserina B2B1E9 and A0A090CH31 for HRAM5.
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5. PP and Sigma, Two Other Types of NLR-Associated Amyloid Signaling Motifs
Biomolecules 2017, 7, 38 6 of 10 In addition to the HET-s motif and the related HRAM subfamilies, at least two other types of
W9WR11 and W9XJW2 for in
HRAM3, Pestalotiopsis and W3WR59 NLR-associatedpsammophila amyloid signaling
motifs
occur
fungi [8,11].
First,fici a W3WR35 motif termed
PP wasfor found to
HRAM4 and P. anserina B2B1E9 and A0A090CH31 for HRAM5. regulate activation of a HeLo-like cell death inducing protein, termed HELLP and a putative lipase
termed SBP.5. PP and Sigma, Two Other Types of NLR‐Associated Amyloid Signaling Motifs The PP-motif is characterized by a pseudo-palindromic sequence signature centered on a
conserved Q residue (Figure 4A). The motif is about 20 amino acids in length. Importantly, this motif
In addition to the HET‐s motif and the related HRAM subfamilies, at least two other types of resembles the
RHIM amyloid motif (for instance by the presence of a common G×Q×G segment),
NLR‐associated amyloid signaling motifs occur in fungi [8,11]. First, a motif termed PP was found to found to control
assembly of the RIP1 and RIP3 kinases in the necroptosis pathway in mammals [23].
regulate activation of a HeLo‐like cell death inducing protein, termed HELLP and a putative lipase The similarity
between
the two motifs could either represent evidence for a long term conservation of
termed SBP. The PP‐motif is characterized by a pseudo‐palindromic sequence signature centered on a conserved Q residue (Figure 4A). The motif is about 20 amino acids in length. Importantly, this an amyloid signaling motif from fungi to mammals or alternatively result from convergent evolution of
motif from
resembles the RHIM amyloid motif (for instance by the presence of a common G×Q×G motifs unrelated
an evolutionary
point of
view.
The
PP-motif
allows
for activation
of the
HELLP
segment), found to control assembly of the RIP1 and RIP3 kinases in the necroptosis pathway in cell death inducing protein which shows homology to the N-terminal 4HB domain of MLKL, the
mammals [23]. The similarity between the two motifs could either represent evidence for a long term cell death execution
protein
mammalian
necroptosis.
The RHIM
motifor allows
for formation
of the
conservation of an in
amyloid signaling motif from fungi to mammals alternatively result from RIP1/RIP3 kinase
complex
which
phosphorylates
the
C-terminal
pseudo-kinase
domain
of
MLKL
convergent evolution of motifs unrelated from an evolutionary point of view. The PP‐motif allows [24].
Thus, whilefor activation of the HELLP cell death inducing protein which shows homology to the N‐terminal both the PP and RHIM amyloid motifs are involved in the signaling pathway leading to
necroptotic 4HB domain of MLKL, the cell death execution protein in mammalian necroptosis. The RHIM motif or necroptosis-like cell death, they do not occur at equivalent position in the pathway in
allows for formation of the RIP1/RIP3 kinase complex which phosphorylates the C‐terminal mammals and
fungi. Of note is the fact that a link between fungal cell death controlling amyloids and
pseudo‐kinase domain of MLKL [24]. Thus, while both the PP and RHIM amyloid motifs are the RHIM motif
had been previously proposed [25]. Kajava et al. reported a similarity between RHIM
involved in the signaling pathway leading to necroptotic or necroptosis‐like cell death, they do not motifs and occur at equivalent position in the pathway in mammals and fungi. Of note is the fact that a link the HET-s elementary repeats. This observation would suggest in turn that the PP and
the HET-s motifs
are also evolutionarily related. Future studies on the structural characterization of
between fungal cell death controlling amyloids and the RHIM motif had been previously proposed et al. help
reported a similarity between RHIM relations
motifs and the HET‐s elementary repeats. these motifs[25]. willKajava probably
clarifying
the evolutionary
between
these
different
amyloid
This observation would suggest in turn that the PP and the HET‐s motifs are also evolutionarily signaling motifs.
related. Future studies on the structural characterization of these motifs will probably help clarifying PNT1, the NLR with PP-motif appears to control simultaneous activation of two distinct effector
the evolutionary relations between these different amyloid signaling motifs. proteins insteadPNT1, of a single
one
as PP‐motif in the case
of the
The same
situation
occurs
the NLR with appears to HET-s-motif.
control simultaneous activation of two also
distinct for the sigma
motif originally identified in Nectria haematococca [8,26]. The motif was identified in a
effector proteins instead of a single one as in the case of the HET‐s‐motif. The same situation also for the formation
sigma motif identified in Nectria haematococca [8,26]. The motif in
was gene clusteroccurs controlling
oforiginally a cytoplasmic
infectious
element
termed
σ and
found
three
identified in a gene cluster controlling formation of a cytoplasmic infectious element termed σ and proteins, a putative
NLR termed Het-eN, a predicted lipase (SESB) and a protein termed SESA (which
found in three proteins, putative termed domains)
Het‐eN, a (Figure
predicted lipase and a (σ)
protein shows a remote
homology
to botha HeLo
andNLR Helo-like
4B)
[7]. (SESB) The sigma
motif is
termed SESA (which shows a remote homology to both HeLo and Helo‐like domains) (Figure 4B) about 40 amino acids in length and was found to exist as different variants composed of the three-fold
[7]. The sigma (σ) motif is about 40 amino acids in length and was found to exist as different variants repetition ofcomposed of the three‐fold repetition of two submotifs [8]. two submotifs [8].
Figure 4. PP and sigma amyloid signaling motifs. (A) The gene architecture of the hellp locus of Figure 4. PP
and sigma amyloid signaling motifs. (A) The gene architecture of the hellp locus of
Chaetomium globosum is given as well as the domain architecture of the corresponding proteins. The Chaetomium globosum is given as well as the domain architecture of the corresponding proteins. The
sequence of the PP regions of HELLP, SBP and the PNT1 NLR is given. (B) The gene architecture of
the ses locus of Nectria haematococca is given as well as the domain architecture of the corresponding
proteins. The sequence of the sigma regions of SESA, SESB and the HET-eN NLR is given.
Biomolecules 2017, 7, 38
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6. Potential Advantages of Amyloid Signaling
The formation of higher-order signaling machines appears to be a common scheme in cellular
pathways controlling cell fate and immunity [27,28]. Among such complexes are the apoptosome [3],
the Myddosome [29], the MAVS CARD filaments [30], the RIP1/RIP3 necrosome [23] and the
ASC-dependent inflammasomes [31]. In many cases, these signaling complexes form large filamentous
assemblies. In these macromolecular complexes, different domains or motifs that have the ability
to self-assemble into higher-order oligomers are employed such as the death domain fold family
(DD), CARD and PYD domains, the TIR domain or an amyloid motif in the case of RHIM. Due
to this capacity to self-assemble, the CARD and PYD domains were found to be able to behave
as prion-forming domains in yeast [22,30]. Thus, the prion-like polymerization and propagation
process in these signaling machines involves in some instances, but not in others, amyloid structures
and is conserved from fungi to humans. Such higher-order assemblies whether they are based on
amyloids or folded domains function by nucleated polymerization, a principle that entitles signal
transduction cascades with specific properties such as signal amplification, threshold response and
noise reduction [27]. The remarkable similarity between the CARD and PFD mediated signaling in
NLRs has been illustrated by the fact that the HET-s prion forming motif can functionally replace
the PYD domains in mammalian NLRP3 and ASC [22,28]. Self-assembly of folded globular domains
or amyloid templating appear as two possible solutions for the same task of mediating an efficient
transmission of a cell fate signal in an all-or-none fashion. The strictly cooperative nature of amyloid
folding and the robustness of its templating and propagative properties, as well as extreme compactness
make amyloid motifs well suited for such signal transduction tasks. These amyloid motifs are small
and typically shorter than segments that permit formation of folded globular domains. The fact
that several different motifs have been identified in fungi suggests that this mechanism shows an
evolutionary success in this branch. Apparently, this mode of signal transduction has been diversified
into various classes allowing for creation of versatile templating modules that can co-exist in the same
cell much in the same way as diversification of the CARD domain allows several parallel signaling
cascades to employ self-assembly of variants this domain. It should be noted that for each combination
of effector domain and NLR-type that has been characterized to date in fungi, the same domain
association can also be found in a single NLR. For instance, while in P. anserina the HeLo/NACHT–WD
organization is found in the HET-S/NWD2 pair, in many other species display NLRs with an all-in-one
HeLo–NACHT–WD architecture. The converse is so far not true as certain domain associations (such
as Patatin–NB–ARC–TPR) have not been found yet in the two-gene architecture. What then could be
the potential advantage of the two-gene architecture as opposed to the simpler all-in-one structure?
One obvious possibility as discussed above is that the amyloid-mediated signaling allows for signal
amplification. Another possible advantage, is that this mode of activation allows a single NLR to
activate two distinct effector domains (as occurs for instance with the PP motif in HELLP and SBP) [11]
or a single effector to be activated by different NLRs (Figure 5).
Biomolecules 2017, 7, 38
Biomolecules 2017, 7, 38 8 of 10
8 of 10 Figure 5. The different effector domain activation modes in fungal NLRs. different domain Figure
5. The
different
effector
domain
activation
modes
in fungal
NLRs.
TheThe different
domain
architecture found in fungal NLR are given. The repeated boxes represent architecture found in fungal NLR are given. The repeated boxes represent the superstructure-forming the superstructure‐forming repeat domains (either ANK, TPR or WD), the central oval, the NBD (either repeat
domains (either ANK, TPR or WD), the central oval, the NBD (either NB-ARC or NACHT), the
NB‐ARC or NACHT), the circle the effector domains (HeLo, HeLo‐like, sesA, circle
the effector
domains (HeLo,
HeLo-like,
sesA, sesB,
PNP-UDP
phosphorylase,
etc.).sesB, PNP‐UDP phosphorylase, etc.). 7. Conclusions and Future Prospects
7. Conclusions and Future Prospects Fungi display large repertoires of proteins resembling plant and animal NLRs some of which have
Fungi display large repertoires of proteins resembling plant and animal NLRs some of which been found
to be involved in non-self recognition and control of cell fate [10,11,32–34]. Rather than
have been found to be involved in non‐self recognition and control of cell fate [10,11,32‐34]. Rather displaying LRR repeats, fungal NLR-related protein display other types of super-structure forming
than such
displaying LRR repeats, fungal NLR‐related other types of super‐structure repeats
as ANK,
TPR
or WD-repeats.
A fractionprotein of thesedisplay NLRs induce
activation
of effector
forming repeats such as ANK, TPR or WD‐repeats. A fraction of these NLRs induce activation domains by an amyloid templating mechanism. The nwd2/het-S gene pair of P. anserina represents
the of effector domains by an amyloid templating mechanism. The nwd2/het‐S gene pair of P. anserina paradigm of this type of amyloid signaling mechanism but several additional amyloid signaling motifs
represents the paradigm of this type of amyloid signaling mechanism but several additional amyloid such
as the diversified HRAM family, the PP and the sigma motif have been described. These motifs
signaling motifs as amyloid
the diversified HRAM family, the PP and
and the sigma the
motif have been appear
related
to thesuch RHIM
motif described
in mammals
controlling
necroptosis
described. These motifs appear related to the RHIM amyloid these
motif described mammals pathway.
It will
now be
of great
interest
to structurally
characterize
novel
motifs in to see
to whichand controlling the necroptosis pathway. It will now be of great interest to structurally characterize these extent they are structurally related to the Het-s prion motif and to get a better understanding of the
novel motifs to see to which extent they are structurally related to the Het‐s prion motif and to get a structural
diversification of this evolutionarily successful mode of signal transduction.
better understanding of the structural diversification of this evolutionarily successful mode of signal Acknowledgments:
transduction. A.L. acknowledges financial support from the European Research Council (ERC)
(ERC-2015-StGA No. 105945).
Acknowledgments: A.L. acknowledges financial support from the European Research Council (ERC) Author
Contributions: A.L. and S.J.S. contributed to the writing of this review.
(ERC‐2015‐StGA No. 105945). Conflicts of Interest: The authors declare no conflict of interest
Author Contributions: A.L. and S.J.S. contributed to the writing of this review. Conflicts of Interest: The authors declare no conflict of interest References
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