BACKGROUND
initiation of innate immune/inflammatory responses by pathogens results in the secretion of cytokines that recruit phagocytes, increase phagocyte microbicidal activity, promotes antigen presentation and the development of adaptive immunity  <cit> .
to initiate these responses, pathogens must be sensed through one or more host pattern recognition receptors .
prrs include the toll-like receptor , rig-i helicase-like receptor, or nucleotide-binding, leucine repeat  receptor families.
prr engagement by pathogen-associated molecular patterns activates receptor-mediated signaling via mapk, stat, and/or nf-κb .
activation of the mapk and nf-κb pathways cooperate to drive the gene expression of proinflammatory cytokines such as il-1β, il- <dig>  il- <dig>  and tnfα.
secretion of il-1β and the il-1β-related cytokine il- <dig>  requires processing of the respective pro-forms by caspase- <dig>  activation of caspase- <dig> occurs in the context of the dynamic multi-protein inflammasome complex through either direct or asc -mediated indirect recruitment via nlr proteins  <cit> .
while the molecular basis and regulation of nf-κb signal transduction downstream of prr family members is well-studied  <cit> , inflammasome function and regulation is poorly understood.
pyrin domain  and caspase recruitment domain  homodomain interactions are important for inflammasome formation, suggesting the potential for card-only proteins  and pyd-only proteins  to act as negative regulators.
cops  inhibit caspase- <dig> activation by preventing caspase- <dig> recruitment to the inflammasome complex  <cit> .
two mammalian pops have also been discovered.
pop <dig>  is highly similar to the pyd of asc , the adaptor molecule that bridges the pyd of nlrps to the card of caspase- <dig> to facilitate inflammasome assembly.
although a potential function, pop <dig> has not yet been shown to inhibit inflammasome formation/activation  <cit> .
pop <dig> is more similar to nlr pyds and effectively inhibits inflammasome activation by limiting the interaction of various nlrps with asc  <cit> .
importantly, the inflammasomes influenced by pop <dig> include nlrp <dig>  nlrp <dig>  and nlrp <dig> which have been linked to specific inflammatory diseases including atopic dermatitis  <cit> ; the cryopyrin-associated periodic syndromes  <cit> , and other hereditary periodic fevers  <cit> .
pop <dig> and pop <dig> are also capable of inhibiting nf-κb activation, although the mode of inhibition differs  <cit> .
thus pop <dig> has the potential to function as a dual regulator of innate immune/inflammatory responses by influencing both inflammasome function and prr signaling via nf-κb.
here we report that the genomes of mouse, rat, and a number of other domestic mammals with available complete genome sequence data lack pop <dig>  while the available genomes of catarrhine primates  contain pop <dig>  those of new world primates  do not, strongly supporting the recent evolution of pop <dig>  our data also reveals an increasing number of nlrp2-related sequences during mammalian evolution.
a functional analysis of macaque pop <dig> reveals a protein capable of both nf-κb and inflammasome inhibition, demonstrating that these functions likely coincide with the emergence of pop <dig> some time after the divergence of old world and new world primates approximately  <dig> mya.
the pattern of pop <dig> evolution and the significance of the recent emergence of both pop <dig> and pop <dig> as potential regulators of nf-κb signaling and inflammasome function are discussed.
RESULTS
pop <dig> is absent from the genomes of mice, rats, and other domestic mammals
to attempt to identify and isolate the mouse equivalent of human pop <dig>  we performed translated blast searches of the mouse genome.
curiously, although other pyds in nlr family members were detected, a sequence with high similarity to pop <dig> was not.
the completed genomes of a variety of other domestic mammals were also examined with similar results.
sequences with the highest homologies in these species were the pyds of the putative orthologs of nlrp <dig> or nlrp <dig>  the genes most closely related to pop <dig> in humans .
searches were also performed for human pop <dig>  which was also absent in these genomes.
as expected, the asc pyd had the greatest homology to pop <dig>  since pop <dig> is encoded by a single exon of  <dig> base pairs in humans adjacent to the gene for ccdc <dig>  and approximately  <dig> kbp upstream of the gene for fgf <dig>  <cit> , we examined the corresponding locus in mouse.
this locus is present in the mouse genome, has been completely sequenced, and is nearly identical to the human locus in size and gene arrangement with the exception that pop <dig> is absent .
these results suggest that both pop <dig> and pop <dig> may be recent developments in mammalian evolution and raise the possibility that both genes are unique to primates.
pop <dig> is an evolutionarily young gene present in hominid and old world primates
based on our observation that pop <dig> is absent from the genomes of all domestic mammal species examined, we performed the same screening of the completed genomes of simian non-human primates including pan troglodytes , macaca mulatta , and with the available whole genome shotgun  sequences of gorilla gorilla , pongo abelii , and callithrix jacchus .
pop <dig> related sequences were found in the genomes of all species examined .
sequence alignment shows that chimpanzee pop <dig> is identical to human pop <dig> at the protein level .
except for gorilla, the remaining hominid  and old world  primate species have a clear pop <dig> ortholog  with open reading frames, but marmosets  do not.
no pop <dig> orthologs were detected in the available genomic data from prosimian species.
pop <dig> was also examined with similar results.
maps of the pop <dig> and pop <dig> loci were constructed for those genomes having chromosomal map data .
the genes surrounding pop <dig> and pop <dig> in primate genomes are syntenic and have conserved sequences that have persisted since the emergence of the murine genomes .
collectively, these data strongly support the conclusion that pop <dig> is unique to primates and very likely unique to old world and hominid primates.
as old world primates are thought to have diverged from new world primates approximately  <dig> mya, and hominids from old world primates approximately  <dig> mya  <cit> , this observation suggests that pop <dig> emerged as a functional gene somewhere between  <dig> and  <dig> mya.
pop <dig> and pop <dig> thus appear to be among the youngest known human gene products.
pop <dig> likely resulted from a contraction of the nlrp2/ <dig> paralog pseudogene clrx/nod24
comparison of the identities of pop2-related proteins from all species examined reveals that the number of pop <dig> pyd-related sequences increases in number during mammalian evolution .
phylogenetic analysis of the pyd protein sequences indicates that in the most distant outgroup represented , the only pop2-related gene is nlrp <dig> .
in humans, based solely on the pyd protein sequence, the proteins most closely related to pop <dig> include nlrp <dig>  nlrp <dig>  and the predicted protein product of the clrx.1/nod <dig> locus, suggesting an evolutionary relationship between these proteins.
unlike nlrp <dig> and nlrp <dig>  the pseudogene clrx.
<dig>   <cit>  contains the stop-codon-rich remnants of its pyd, nucleotide binding domain, and leucine rich repeat coding regions.
like pop <dig>  clrx.
<dig> or clrx.1-like pseudogene sequences are present in all the primate species genomes examined including marmoset .
in our analysis based on pyd protein sequences alone, a number of sequences fail to cluster with either human nlrp <dig> or nlrp <dig>  suggesting that they represent intermediate forms of the gene .
to try to resolve whether these sequences represent evolutionary transitions we performed a phylogenetic analysis on the predicted full-length nucleotide and cdna sequences .
marmoset and orangutan sequences were excluded as the contigs from these databases were too small to ensure complete coding regions for individual sequences.
from this analysis, macaque xr  <dig>  appears to be macaque nlrp <dig>  macaque xr  <dig>  is most similar to clrx.
<dig>  mouse and rat nlrp <dig> and dog and horse nlrp2/-7-like are more closely related to human nlrp <dig> than nlrp <dig>  this indicates that nlrp <dig> in mouse and rat is currently a misleading designation.
accordingly, an nlrp2/ <dig> or nlrp2/7-like designation would more accurately reflect its orthologous, syntenic relationship with human nlrp <dig>  the presence of both nlrp <dig> and nlrp <dig> in human and non-human primate genomes and the observation that mouse and rat nlrp <dig> are more closely related to human nlrp <dig> than human nlrp <dig> suggests that an nlrp7-like gene is the common ancestor of the nlrp <dig>  nlrp <dig>  clrx.1/nod <dig>  and pop <dig> genes.
given the apparent synteny between mouse nlrp <dig> and human nlrp <dig>  it is highly likely that duplication of the ancestral nlrp <dig> gene  resulted in the pop <dig>  nlrp <dig>  and clrx.1/nod <dig> .
nlrp <dig>  and nlrp2/7-like  likely represent diversification of the ancestral locus.
in the human and chimp genomes, nlrp <dig> is adjacent to nlrp <dig> on chromosome  <dig>  further, the ccdc50/fgf <dig> locus which contains pop <dig> on human chromosome 3q <dig> is not in proximity to other nlrp-related genes suggesting the insertion of the pop <dig> sequence.
pop <dig> likely originated from a processed pseudogene
the marmoset  genome contains a pseudogene sequence representing a sister clade of pop <dig> .
the cjΨpop <dig> pseudogene resides downstream of marmoset ccdc <dig> and upstream of marmoset fgf <dig> in same location and orientation as higher primate forms of pop <dig>  further inspection of cjΨpop <dig> reveals the presence not only of the pyd, but also a nucleotide binding domain, and evidence of leucine-rich repeat coding sequences.
introns between these domains are absent, strongly implicating an insertion of a retrogene copy  of one of the nlrs, likely the common ancestor of clrx.1/nlrp <dig>  the atg start codon is missing and numerous stop codons are present in-frame with the residual pyd sequence.
genes expressed in reproductive tissues are believed to be more likely to generate a heritable processed pseudogene  <cit>  and pop <dig> as well as a number of nlrs are expressed in testis and/or oocytes  <cit> .
all of these observations are consistent with our interpretation that in marmoset, cjΨpop <dig> is a processed pseudogene and suggest that in primate evolution, an older, functional nlr retrogene was acquired , modified, and rapidly selected in old world and hominid primates to produce pop <dig>
macaque pop <dig> is a functional intermediate between nlrp <dig> and pop2
like human and chimp pop <dig>  mqpop <dig> is a single exon gene, but unlike clrx.1/nod <dig>  none have discernable residual coding sequence downstream of their stop codons, suggesting that the loss of nbd and lrr encoding sequences was complete prior to the divergence of these species.
in agreement with this data and primate phylogeny  <cit> , mqpop <dig> likely retains features in common with the ancestral forms of nlrp <dig> and pop <dig> that diverged between approximately  <dig> and  <dig> mya.
both the pyd of human nlrp <dig> and pop <dig> inhibit nf-κb although their mode of action differs  <cit> , but while pop <dig> has been shown to inhibit inflammasome assembly  <cit> , the pyd of nlrp <dig> does not  <cit> .
to establish whether mqpop <dig> is more similar to nlrp <dig> or pop <dig> with respect to nf-κb and inflammasome inhibitory properties, we cloned mqpop <dig> and compared the inhibitory properties of the protein to that of hupop <dig>  comparison of the predicted amino acid sequence from two identical mqpop <dig> clones reveals four amino acid substitutions differing from the macaque genome reference sequence .
first, lysine  <dig> is substituted by threonine, a residue conserved in human and chimp pop <dig> and in human nlrp <dig>  residue  <dig>  a serine in the genome database, is a glycine in both clones and appears unique as pop <dig> , the macaque genome, and the nlrp <dig> pyd have either a serine or threonine at this position.
position  <dig> is not conserved between the various pop <dig> and related sequences, but our clones contain cysteine instead of the expected arginine.
mqpop <dig> differs from human/chimp pop <dig> most dramatically by the presence of a  <dig> amino acid c-terminal extension indicating that this region was likely lost at some point after the divergence of hominid and old world primates .
a cysteine residue at position  <dig> is a tyrosine in our clones; but this residue occurs in the additional c-terminal  <dig> amino acids absent in human and chimp pop <dig>  the differences between our clones and the reference sequence for mqpop <dig> may represent pop <dig> polymorphisms or diversification in macaques.
since human and chimp pop <dig> are identical, purifying selection may have occurred in, or prior to, these lineages.
however, a larger number of hupop <dig> cdnas will need to be examined to confirm that hupop <dig> is essentially invariant.
both pop <dig> and pop <dig> inhibit nf-κb signaling induced by tnfα.
while pop <dig> inhibits ikkα/ikkβ kinase activity upstream of iκbα phosphorylation and cannot inhibit transactivation by the active p <dig> subunit  <cit> , pop <dig> inhibits transactivation by p <dig>  <cit> .
the isolated pyd of nlrp <dig> acts similarly to pop <dig>  <cit> .
similar to hupop <dig>  mqpop <dig> reduces tnfα-mediated nf-κb activation .
mqpop <dig> also inhibits transactivation by nf-κb p <dig>  demonstrating that like hupop <dig>  mqpop <dig> is acting downstream of iκbα phosphorylation at the level of p <dig>  however, the extent of nf-κb inhibition by mqpop <dig> is less than that of hupop <dig>  this may result from the additional c-terminal sequence as its removal ) results in a version of the protein with p <dig> inhibitory activity similar to that of hupop <dig> .
neither pop <dig> nor nlrp <dig> have been demonstrated to prevent inflammasome activation, whereas pop <dig> is known to block nlr:asc interaction and inhibits a variety of inflammasomes  <cit> .
mqpop <dig> is less effective than human at inhibiting inflammasome activation mediated by asc-overexpression in hek <dig> cells .
although it remains possible that mqpop <dig> may more profoundly inhibit other specific inflammasomes or exhibit species specific inflammasome inhibition, these results demonstrate that mqpop <dig> possesses the identified functions of its human ortholog.
given the high degree of similarity to the nlrp <dig> pyd, mqpop <dig> retains more sequence identity with the nlrp <dig> pyd than with human or chimp pop <dig> and likely represents a form of pop <dig> preceding refinements now fixed in the more broadly functional human pop <dig>  the high degree of conservation between human and chimp pop <dig> suggests that pop <dig> refined these functions at some point following the divergence of macaques with purifying selection acting at that point to completely conserve pop <dig>  prior to the divergence of humans and chimps .
the divergence of orangutans from other hominid primates occurred around  <dig> mya  <cit>  and orangutan pop <dig> is not identical to human and chimp pop <dig> revealing that as recently as  <dig> mya, this selection was still acting upon the pop <dig> locus.
the selective pressures driving the appearance of both nlrp <dig> and pop <dig> are unknown, however the recent emergence of pop <dig> suggests a very strong selective pressure impacting reproductive success.
CONCLUSIONS
of all extant species, chimpanzees and other primates are most similar to humans at the protein and genomic levels.
rodent species however, are widely used as models for biomedical research.
since arrival in the genomic age has accelerated the pace of discovery and increased our knowledge of comparative genomics, the differences between humans and those species used as disease models as well as the need to understand important differences has become increasingly apparent  <cit> .
it has become clear that humans possess greater diversity in protein families involved in inflammation than rodent models used to model inflammatory disease.
this is most evident in members of the il-1/il-1r family where both agonist and antagonist members are more abundant in humans than mice  <cit> .
not surprisingly, humans and mice also show differences in the number of nlr proteins potentially involved in inflammasome activity  <cit> .
in this report we have examined the evolutionary history of pyrin-only proteins  implicated in the regulation of inflammation and find that the emergence of pops in the mammalian genome is a very recent event occurring roughly at the divergence of old and new world primates.
further, our evidence suggests that pop <dig>  which can inhibit inflammasome activity, arose from gene duplication events that first gave rise to multiple paralogs of an ancestral nlrp2/7-like gene, followed by diversification of these genes, retrogene insertion of an nlrp2-like paralog, and rapid loss of the signature nbd and lrr encoding regions to yield a functional pop <dig> gene.
mqpop <dig> approximates the most distant intact ancestor of pop <dig>  predating the divergence of humans and chimps by approximately 5- <dig> million years, and possesses both the nf-κb p <dig> and inflammasome inhibitory properties reported for the human protein.
collectively, these data suggest a strong selective pressure driving the recent emergence of a small pyrin-only protein inhibiting both nf-κb signaling and the activation of multiple inflammasomes that corresponds with the emergence of hominid and old world primates.
the marmoset genome clearly reveals the prior insertion of a retrogene copy of an nlrp2-like transcript at the developing pop <dig> locus.
by the emergence old world primates, traces of the non-pyd coding portions of the retrogene are no longer apparent.
in humans, a functional polyadenylation sequence is present within the 3' utr of pop <dig>  <cit> .
similar sequences are present in the 3' utr of chimp and mqpop <dig>  beyond reflecting the selective pressures acting on the pop <dig> gene, these features lend additional support to the emerging hypothesis that retrogenes are frequently functional, add to the complexity of the genome and may confer important regulatory functions as recently demonstrated for fibroblast growth factor  <dig>  <cit> .
do the recent emergence of pop <dig> and other inflammatory regulators  reflect an increased need to control inflammation during the most recent stages of primate evolution?
this is a difficult question requiring further investigation.
however, considering that a strong selective pressure likely drove the evolutionary development of pop <dig>  it is reasonable that pop <dig> might act by offsetting inflammatory events that decrease reproductive success.
inflammation of the reproductive organs would be a direct example.
recently, mutations in nlrp <dig>  have been implicated in failed spermatogenesis and may dysregulate inflammation or promote apoptosis  <cit> .
interestingly, although inducible in monocytic cells, pop <dig> is expressed constitutively in the testis  <cit>  and could thus potentially have a role in modulating nlrp <dig> function.
as a more extreme example, nomid, one of the most severe autoinflammatory diseases, is one outcome of mutations in nlrp <dig>  an inflammasome-initiating protein modulated by pop <dig>  <cit> .
approximately 20% of individuals with nomid die before adulthood.
identification of mutations or deletions in pop <dig> correlating with male reproductive system failure would lend support to this idea.
the involvement of nlrp <dig> and nlrp <dig> in inflammasome inhibition  <cit>  and a connection between their presence among maternal rnas in oocytes and the formation of hydatiform moles  <cit> , a form of reproductive failure, further suggest that some nlrs may have roles in both inflammation and reproductive success.
nevertheless, as no specific disease associations have been identified for the pop <dig> locus, these ideas remain speculative and await the results of further studies exploring the role of pop <dig>
in summary, the recent emergence of the highly selected and functional pop <dig> gene in higher primates, apes, and humans suggests a strong selective pressure among these species for the functions of the pop <dig> protein.
although our understanding of the biological role of pop <dig> is in its infancy, its apparent biochemical roles in regulating nf-κb activity and inflammasome formation suggest a variety of possibilities that may shed light on important differences between higher primates and other mammalian species.

 initiation of innate immune/inflammatory responses by pathogens results in the secretion of cytokines that recruit phagocytes, increase phagocyte microbicidal activity, promotes antigen presentation and the development of adaptive immunity. to initiate these responses, pathogens must be sensed through one or more host pattern recognition receptors. prrs include the toll-like receptor, rig-i helicase-like receptor, or nucleotide-binding, leucine repeat receptor families. prr engagement by pathogen-associated molecular patterns activates receptor-mediated signaling via mapk, stat, and/or nf-κb. activation of the mapk and nf-κb pathways cooperate to drive the gene expression of proinflammatory cytokines such as il-1β, il- il- and tnfα. secretion of il-1β and the il-1β-related cytokine il- requires processing of the respective pro-forms by caspase- activation of caspase- occurs in the context of the dynamic multi-protein inflammasome complex through either direct or asc -mediated indirect recruitment via nlr proteins. while the molecular basis and regulation of nf-κb signal transduction downstream of prr family members is well-studied, inflammasome function and regulation is poorly understood. pyrin domain and caspase recruitment domain homodomain interactions are important for inflammasome formation, suggesting the potential for card-only proteins and pyd-only proteins to act as negative regulators. cops inhibit caspase- activation by preventing caspase- recruitment to the inflammasome complex. two mammalian pops have also been discovered. pop is highly similar to the pyd of asc, the adaptor molecule that bridges the pyd of nlrps to the card of caspase- to facilitate inflammasome assembly. although a potential function, pop has not yet been shown to inhibit inflammasome formation/activation. pop is more similar to nlr pyds and effectively inhibits inflammasome activation by limiting the interaction of various nlrps with asc. importantly, the inflammasomes influenced by pop include nlrp nlrp and nlrp which have been linked to specific inflammatory diseases including atopic dermatitis; the cryopyrin-associated periodic syndromes, and other hereditary periodic fevers. pop and pop are also capable of inhibiting nf-κb activation, although the mode of inhibition differs. thus pop has the potential to function as a dual regulator of innate immune/inflammatory responses by influencing both inflammasome function and prr signaling via nf-κb. here we report that the genomes of mouse, rat, and a number of other domestic mammals with available complete genome sequence data lack pop while the available genomes of catarrhine primates contain pop those of new world primates do not, strongly supporting the recent evolution of pop our data also reveals an increasing number of nlrp2-related sequences during mammalian evolution. a functional analysis of macaque pop reveals a protein capable of both nf-κb and inflammasome inhibition, demonstrating that these functions likely coincide with the emergence of pop some time after the divergence of old world and new world primates approximately mya. the pattern of pop evolution and the significance of the recent emergence of both pop and pop as potential regulators of nf-κb signaling and inflammasome function are discussed.  pop is absent from the genomes of mice, rats, and other domestic mammals to attempt to identify and isolate the mouse equivalent of human pop we performed translated blast searches of the mouse genome. curiously, although other pyds in nlr family members were detected, a sequence with high similarity to pop was not. the completed genomes of a variety of other domestic mammals were also examined with similar results. sequences with the highest homologies in these species were the pyds of the putative orthologs of nlrp or nlrp the genes most closely related to pop in humans. searches were also performed for human pop which was also absent in these genomes. as expected, the asc pyd had the greatest homology to pop since pop is encoded by a single exon of base pairs in humans adjacent to the gene for ccdc and approximately kbp upstream of the gene for fgf, we examined the corresponding locus in mouse. this locus is present in the mouse genome, has been completely sequenced, and is nearly identical to the human locus in size and gene arrangement with the exception that pop is absent. these results suggest that both pop and pop may be recent developments in mammalian evolution and raise the possibility that both genes are unique to primates. pop is an evolutionarily young gene present in hominid and old world primates based on our observation that pop is absent from the genomes of all domestic mammal species examined, we performed the same screening of the completed genomes of simian non-human primates including pan troglodytes, macaca mulatta, and with the available whole genome shotgun sequences of gorilla gorilla, pongo abelii, and callithrix jacchus. pop related sequences were found in the genomes of all species examined. sequence alignment shows that chimpanzee pop is identical to human pop at the protein level. except for gorilla, the remaining hominid and old world primate species have a clear pop ortholog with open reading frames, but marmosets do not. no pop orthologs were detected in the available genomic data from prosimian species. pop was also examined with similar results. maps of the pop and pop loci were constructed for those genomes having chromosomal map data. the genes surrounding pop and pop in primate genomes are syntenic and have conserved sequences that have persisted since the emergence of the murine genomes. collectively, these data strongly support the conclusion that pop is unique to primates and very likely unique to old world and hominid primates. as old world primates are thought to have diverged from new world primates approximately mya, and hominids from old world primates approximately mya, this observation suggests that pop emerged as a functional gene somewhere between and mya. pop and pop thus appear to be among the youngest known human gene products. pop likely resulted from a contraction of the nlrp2/ paralog pseudogene clrx/nod24 comparison of the identities of pop2-related proteins from all species examined reveals that the number of pop pyd-related sequences increases in number during mammalian evolution. phylogenetic analysis of the pyd protein sequences indicates that in the most distant outgroup represented, the only pop2-related gene is nlrp. in humans, based solely on the pyd protein sequence, the proteins most closely related to pop include nlrp nlrp and the predicted protein product of the clrx.1/nod locus, suggesting an evolutionary relationship between these proteins. unlike nlrp and nlrp the pseudogene clrx. contains the stop-codon-rich remnants of its pyd, nucleotide binding domain, and leucine rich repeat coding regions. like pop clrx. or clrx.1-like pseudogene sequences are present in all the primate species genomes examined including marmoset. in our analysis based on pyd protein sequences alone, a number of sequences fail to cluster with either human nlrp or nlrp suggesting that they represent intermediate forms of the gene. to try to resolve whether these sequences represent evolutionary transitions we performed a phylogenetic analysis on the predicted full-length nucleotide and cdna sequences. marmoset and orangutan sequences were excluded as the contigs from these databases were too small to ensure complete coding regions for individual sequences. from this analysis, macaque xr appears to be macaque nlrp macaque xr is most similar to clrx. mouse and rat nlrp and dog and horse nlrp2/-7-like are more closely related to human nlrp than nlrp this indicates that nlrp in mouse and rat is currently a misleading designation. accordingly, an nlrp2/ or nlrp2/7-like designation would more accurately reflect its orthologous, syntenic relationship with human nlrp the presence of both nlrp and nlrp in human and non-human primate genomes and the observation that mouse and rat nlrp are more closely related to human nlrp than human nlrp suggests that an nlrp7-like gene is the common ancestor of the nlrp nlrp clrx.1/nod and pop genes. given the apparent synteny between mouse nlrp and human nlrp it is highly likely that duplication of the ancestral nlrp gene resulted in the pop nlrp and clrx.1/nod. nlrp and nlrp2/7-like likely represent diversification of the ancestral locus. in the human and chimp genomes, nlrp is adjacent to nlrp on chromosome further, the ccdc50/fgf locus which contains pop on human chromosome 3q is not in proximity to other nlrp-related genes suggesting the insertion of the pop sequence. pop likely originated from a processed pseudogene the marmoset genome contains a pseudogene sequence representing a sister clade of pop. the cjΨpop pseudogene resides downstream of marmoset ccdc and upstream of marmoset fgf in same location and orientation as higher primate forms of pop further inspection of cjΨpop reveals the presence not only of the pyd, but also a nucleotide binding domain, and evidence of leucine-rich repeat coding sequences. introns between these domains are absent, strongly implicating an insertion of a retrogene copy of one of the nlrs, likely the common ancestor of clrx.1/nlrp the atg start codon is missing and numerous stop codons are present in-frame with the residual pyd sequence. genes expressed in reproductive tissues are believed to be more likely to generate a heritable processed pseudogene and pop as well as a number of nlrs are expressed in testis and/or oocytes. all of these observations are consistent with our interpretation that in marmoset, cjΨpop is a processed pseudogene and suggest that in primate evolution, an older, functional nlr retrogene was acquired, modified, and rapidly selected in old world and hominid primates to produce pop macaque pop is a functional intermediate between nlrp and pop2 like human and chimp pop mqpop is a single exon gene, but unlike clrx.1/nod none have discernable residual coding sequence downstream of their stop codons, suggesting that the loss of nbd and lrr encoding sequences was complete prior to the divergence of these species. in agreement with this data and primate phylogeny, mqpop likely retains features in common with the ancestral forms of nlrp and pop that diverged between approximately and mya. both the pyd of human nlrp and pop inhibit nf-κb although their mode of action differs, but while pop has been shown to inhibit inflammasome assembly, the pyd of nlrp does not. to establish whether mqpop is more similar to nlrp or pop with respect to nf-κb and inflammasome inhibitory properties, we cloned mqpop and compared the inhibitory properties of the protein to that of hupop comparison of the predicted amino acid sequence from two identical mqpop clones reveals four amino acid substitutions differing from the macaque genome reference sequence. first, lysine is substituted by threonine, a residue conserved in human and chimp pop and in human nlrp residue a serine in the genome database, is a glycine in both clones and appears unique as pop, the macaque genome, and the nlrp pyd have either a serine or threonine at this position. position is not conserved between the various pop and related sequences, but our clones contain cysteine instead of the expected arginine. mqpop differs from human/chimp pop most dramatically by the presence of a amino acid c-terminal extension indicating that this region was likely lost at some point after the divergence of hominid and old world primates. a cysteine residue at position is a tyrosine in our clones; but this residue occurs in the additional c-terminal amino acids absent in human and chimp pop the differences between our clones and the reference sequence for mqpop may represent pop polymorphisms or diversification in macaques. since human and chimp pop are identical, purifying selection may have occurred in, or prior to, these lineages. however, a larger number of hupop cdnas will need to be examined to confirm that hupop is essentially invariant. both pop and pop inhibit nf-κb signaling induced by tnfα. while pop inhibits ikkα/ikkβ kinase activity upstream of iκbα phosphorylation and cannot inhibit transactivation by the active p subunit, pop inhibits transactivation by p. the isolated pyd of nlrp acts similarly to pop. similar to hupop mqpop reduces tnfα-mediated nf-κb activation. mqpop also inhibits transactivation by nf-κb p demonstrating that like hupop mqpop is acting downstream of iκbα phosphorylation at the level of p however, the extent of nf-κb inhibition by mqpop is less than that of hupop this may result from the additional c-terminal sequence as its removal ) results in a version of the protein with p inhibitory activity similar to that of hupop. neither pop nor nlrp have been demonstrated to prevent inflammasome activation, whereas pop is known to block nlr:asc interaction and inhibits a variety of inflammasomes. mqpop is less effective than human at inhibiting inflammasome activation mediated by asc-overexpression in hek cells. although it remains possible that mqpop may more profoundly inhibit other specific inflammasomes or exhibit species specific inflammasome inhibition, these results demonstrate that mqpop possesses the identified functions of its human ortholog. given the high degree of similarity to the nlrp pyd, mqpop retains more sequence identity with the nlrp pyd than with human or chimp pop and likely represents a form of pop preceding refinements now fixed in the more broadly functional human pop the high degree of conservation between human and chimp pop suggests that pop refined these functions at some point following the divergence of macaques with purifying selection acting at that point to completely conserve pop prior to the divergence of humans and chimps. the divergence of orangutans from other hominid primates occurred around mya and orangutan pop is not identical to human and chimp pop revealing that as recently as mya, this selection was still acting upon the pop locus. the selective pressures driving the appearance of both nlrp and pop are unknown, however the recent emergence of pop suggests a very strong selective pressure impacting reproductive success.  of all extant species, chimpanzees and other primates are most similar to humans at the protein and genomic levels. rodent species however, are widely used as models for biomedical research. since arrival in the genomic age has accelerated the pace of discovery and increased our knowledge of comparative genomics, the differences between humans and those species used as disease models as well as the need to understand important differences has become increasingly apparent. it has become clear that humans possess greater diversity in protein families involved in inflammation than rodent models used to model inflammatory disease. this is most evident in members of the il-1/il-1r family where both agonist and antagonist members are more abundant in humans than mice. not surprisingly, humans and mice also show differences in the number of nlr proteins potentially involved in inflammasome activity. in this report we have examined the evolutionary history of pyrin-only proteins implicated in the regulation of inflammation and find that the emergence of pops in the mammalian genome is a very recent event occurring roughly at the divergence of old and new world primates. further, our evidence suggests that pop which can inhibit inflammasome activity, arose from gene duplication events that first gave rise to multiple paralogs of an ancestral nlrp2/7-like gene, followed by diversification of these genes, retrogene insertion of an nlrp2-like paralog, and rapid loss of the signature nbd and lrr encoding regions to yield a functional pop gene. mqpop approximates the most distant intact ancestor of pop predating the divergence of humans and chimps by approximately 5- million years, and possesses both the nf-κb p and inflammasome inhibitory properties reported for the human protein. collectively, these data suggest a strong selective pressure driving the recent emergence of a small pyrin-only protein inhibiting both nf-κb signaling and the activation of multiple inflammasomes that corresponds with the emergence of hominid and old world primates. the marmoset genome clearly reveals the prior insertion of a retrogene copy of an nlrp2-like transcript at the developing pop locus. by the emergence old world primates, traces of the non-pyd coding portions of the retrogene are no longer apparent. in humans, a functional polyadenylation sequence is present within the 3' utr of pop. similar sequences are present in the 3' utr of chimp and mqpop beyond reflecting the selective pressures acting on the pop gene, these features lend additional support to the emerging hypothesis that retrogenes are frequently functional, add to the complexity of the genome and may confer important regulatory functions as recently demonstrated for fibroblast growth factor. do the recent emergence of pop and other inflammatory regulators reflect an increased need to control inflammation during the most recent stages of primate evolution? this is a difficult question requiring further investigation. however, considering that a strong selective pressure likely drove the evolutionary development of pop it is reasonable that pop might act by offsetting inflammatory events that decrease reproductive success. inflammation of the reproductive organs would be a direct example. recently, mutations in nlrp have been implicated in failed spermatogenesis and may dysregulate inflammation or promote apoptosis. interestingly, although inducible in monocytic cells, pop is expressed constitutively in the testis and could thus potentially have a role in modulating nlrp function. as a more extreme example, nomid, one of the most severe autoinflammatory diseases, is one outcome of mutations in nlrp an inflammasome-initiating protein modulated by pop. approximately 20% of individuals with nomid die before adulthood. identification of mutations or deletions in pop correlating with male reproductive system failure would lend support to this idea. the involvement of nlrp and nlrp in inflammasome inhibition and a connection between their presence among maternal rnas in oocytes and the formation of hydatiform moles, a form of reproductive failure, further suggest that some nlrs may have roles in both inflammation and reproductive success. nevertheless, as no specific disease associations have been identified for the pop locus, these ideas remain speculative and await the results of further studies exploring the role of pop in summary, the recent emergence of the highly selected and functional pop gene in higher primates, apes, and humans suggests a strong selective pressure among these species for the functions of the pop protein. although our understanding of the biological role of pop is in its infancy, its apparent biochemical roles in regulating nf-κb activity and inflammasome formation suggest a variety of possibilities that may shed light on important differences between higher primates and other mammalian species. 
pyrin-only protein is a small human protein comprised solely of a pyrin domain that inhibits nf-κb p65/rela and blocks the formation of functional il-1β processing inflammasomes. further, pop appears to have arisen in the mammalian genome relatively recently and likely derived from retrogene insertion of nlrp blast searches revealed that pop is absent from the available genomes of not only mice and rats, but those of other domestic mammals and new world monkeys as well. pop is however present in the genome of the primate species most closely related to humans including pan troglodytes, macaca mulatta and others. macaque pop, although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. because pop knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether pop is common in the mammalian lineage. since pop is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and old world primates further suggests that additional regulation of these signals is important in these species. interestingly, chimpanzee pop is identical to human pop at both the dna and protein level.
 pyrin-only protein is a small human protein comprised solely of a pyrin domain that inhibits nf-κb p65/rela and blocks the formation of functional il-1β processing inflammasomes. pyrin proteins are abundant in mammals and several, like pop have been linked to activation or regulation of inflammatory processes. because pop knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether pop is common in the mammalian lineage.  blast searches revealed that pop is absent from the available genomes of not only mice and rats, but those of other domestic mammals and new world monkeys as well. pop is however present in the genome of the primate species most closely related to humans including pan troglodytes, macaca mulatta and others. interestingly, chimpanzee pop is identical to human pop at both the dna and protein level. macaque pop, although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. further, pop appears to have arisen in the mammalian genome relatively recently and likely derived from retrogene insertion of nlrp  our findings support the hypothesis that the nlr loci of mammals, encoding proteins involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures. since pop is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and old world primates further suggests that additional regulation of these signals is important in these species. 