BACKGROUND
in  <dig>  walter gilbert speculated that the presence of introns in eukaryotic genes would lead to non-homologous recombinations and the creation of new exon combinations  <cit> .
gilbert and others  <cit>  reasoned that exons could code for protein functional units, and proposed that exon shuffling could be an important mechanism for the evolution of genes in which these functional units are reused in new molecular contexts.
since then, abundant evidence has accumulated showing that exon shuffling has effectively occurred along evolution, playing a crucial role in the origin of numerous metazoan proteins, among which predominate extracellular matrix, immune system and membrane receptors  <cit> .
it has also become clear that exon shuffling is an important mechanism of protein domain shuffling  <cit> , in particular for some domain types that have expanded significantly in metazoans.
physical protein-protein interaction  is a critical phenomenon for the function of most proteins in living organisms, and the recent accumulation of ppi data from numerous small-scale and a few recent large-scale experiments now allow us to build proteome-wide ppi networks  <cit> .
they help us to understand more globally the multiple roles that a protein may play within a cell and the complex interdependence that functions of several of them may bear among themselves.
in the simplest and more usual form, these networks represent proteins by vertices, and a physical interaction between two proteins by an edge between the two corresponding vertices.
a fundamental property of each vertex is the number of edges, a feature known as vertex degree.
numerous recent works have analyzed the major topological properties of ppi networks, and a recurring theme has been the special properties of vertices with high degree.
they are called hubs and tend to be large multi-domain essential proteins  <cit> .
other investigations have shown that many domain-domain interactions are ubiquitous in ppi networks  <cit> , suggesting that the reuse of these interactions has been relevant for the evolution of ppi networks of extant species.
this last relationship, together with the role of exon shuffling as an evolutionary process to make possible the reuse of domains, led us to the natural hypothesis that exon shuffling may have contributed significantly to the evolution of ppi networks.
this present work is an effort to verify this hypothesis in several eukaryotic species and to discover what concrete and specific type of influence exon shuffling has had on ppi networks.
RESULTS
exon shuffling and network degree
with the purpose of generating ppi networks representative of the state-of-the-art knowledge, we compiled data from several public databases .
because of the incompleteness of data comparing interaction properties of several protein products of a gene we merged all data concerning proteins from a gene in just one vertex of the network, a strategy that has been adopted by others  <cit> .
given that we intended to focus on direct physical interactions between proteins, we excluded from our final network all mass spectrometry data, as also done by others  <cit> , since this technique does not discriminate between direct and indirect physical interactions.
we included the five species that we found to be more balanced concerning a reliable ppi network and a considerably sized intron density: human , mouse , the fruit fly drosophila melanogaster, the nematode worm caenorhabditis elegans, and the flowering plant arabidopsis thaliana.
we added to the analysis a sixth case: a predicted ppi network that was obtained by projecting the binary interactions of the exhaustively studied yeast saccharomyces cerevisiae  ppi network into the nearest ortholog genes in cryptococcus neoformans, a fungal species where exon shuffling analysis was possible because of less extensive intron loss  <cit> .
in several moments, however, the present work emphasizes the results obtained with the human ppi network, due to its higher completeness level, and to the abundance and conservation of human introns, which make exon shuffling identification more reliable.
in order to select a subset of genes enriched in exon shuffling events, we sought for the presence of homologous regions flanked by introns in coding sequences derived from non-homologous genes, as has been done by us and others  <cit> .
for the sake of simplicity, we just considered one of the coding sequences of maximum length for each gene.
taking into account the pervasive prevalence of intron loss  <cit>  and the antiquity of some exon shuffling cases  <cit> , we did not require bilateral flanking of both homologous regions, and the number of required introns was smaller in species for which a more extensive intron loss process was known to have taken place in their evolutionary histories.
although arbitrary, this less stringent requirement is convenient to include as many probable exon shuffling cases as possible in the exon shuffling gene sets and, at the same time, to deplete the complementary gene sets in these cases.
our strategy used local alignments to find such homologous regions resulting in a classification of protein-coding genes into three shuffling profile categories.
genes without any homologous regions were classified as without-shuffling  genes.
homologous regions were then analyzed in regard to the presence of introns in four windows adjacent to their extremities .
genes with at least one homologous region with a minimum number of windows containing introns were classified as exon shuffling  genes.
this minimum number was chosen between  <dig> and  <dig>  according to the abundance of introns in the respective species.
genes with homologous regions, but without the minimum number of windows with flanking introns, were classified as sequence shuffling  genes.
based on the assumption that exon shuffling may be an important mechanism for the evolution of new protein-protein interactions, we first compared genes in the three categories according to their presence in ppi networks, as shown in figure  <dig>  and additional file  <dig> .
we observe that, in mammalian intron-rich species, there is a higher fraction of es genes in ppi networks, compared to ss and ws genes.
interestingly, ss genes are also more frequently found in ppi networks than ws genes in all tested species, except worm.
we next performed pairwise comparisons of vertex degree among the three datasets.
we opted for using multiple statistical measures, as each one could give us a particular appreciation of the differences present among the distributions of the several gene sets.
boxplots of the distributions including these measures are in figure  <dig>  we evaluated the statistical significance of the differences among groups by means of both u test, a standard non-parametric test for comparison of two samples, and re-sampling procedures for arithmetic and geometric means .
as seen in table  <dig>  and in additional file  <dig> , the es gene set has higher degree than the other two groups, according to all above-mentioned statistical tests, in all four analyzed metazoan species, but neither in fungus nor in plant.
in addition, we observe that the ss set shows higher degree than the ws set in three cases: human, fungus and plant.
z indicates difference between real data and the resampling mean given in standard deviations of the resampling mean.
we interpret all these results as follows: exon shuffling events are associated to the acquisition of new interactions by proteins, although its relative importance seems to be distinct among different phylogenetic groups.
the resistance of this result to the use of both arithmetic and geometric mean in the re-sampling procedure, measures that give more or less weight to high-degree vertices, respectively, suggests additionally that this fact would apply to both low- and high-degree proteins.
ss genes probably represent a very heterogeneous set.
they may correspond to homologous genes with sequence conservation limited to the currently identified homologous stretch or, given the widespread intron loss suffered along evolution by all analyzed species  <cit> , to exon shuffling cases that lost too many introns and cannot be identified anymore.
they may be examples of exon shuffling-generated genes in which sequence divergence in certain regions of the shuffled region prevented the expected correlation between introns and regions of sequence similarity to be found.
finally, they may correspond to genes resulting from all types of recombination events that are not mediated by introns.
gene duplication is the other major evolutionary mechanism for the emergence of novelties, despite not being able to produce radically different genes in a short time span as exon shuffling can do.
is it known that eukaryotic genomes suffered both many single-gene and a few whole-genome duplications  <cit> .
this has two consequences for our study.
first, it is possible that a small number of extensively duplicated exon shuffling cases could explain all the degree differences observed among the shuffling profile categories.
in order to rule this out, a paralog confluence procedure was performed in which sets of network vertices corresponding to paralogs were exchanged in the network by single vertices with degree equal to the average degree of the removed sets.
after the confluence, in comparison to ss and ws sets, es set continue to present higher degree, whichever the statistical test, with or without re-sampling control for length, in all four considered metazoan species, as shown in additional file  <dig>
the second likely consequence of gene duplication for this study is the existence of a putative effect of gene duplications on ppi network vertex degree.
to test that, the relationship between existence of paralogs of a gene and its corresponding vertex degree was analyzed.
given the strong effect we already know exon shuffling has upon degree for metazoan species, we excluded the exon shuffling genes before making the analysis for those species.
as can be found in additional file  <dig>  we observed that genes with paralogs have a significantly higher degree in human, a significantly smaller degree in fly, and statistically non-significant differences in other organisms.
it must be noticed that the quality of these data, contrary to the shuffling profile category ones, is not affected by intron loss, and probably must depend basically on the completeness of ppi network knowledge in the species.
that knowledge is more extensive precisely in human and fly , suggesting these opposing trends in the two species may be real.
most importantly, all degree differences between genes with and without paralogs are numerically smaller than the degree differences found between es and non-es gene sets, suggesting that gene duplication is not per se particularly important for the evolution of degree in ppi networks.
it should be noted, however, that gene duplication may have an indirect importance as it is expected that exon shuffling would be less deleterious if involving duplicated copies of genes.
case analysis
a possible exon shuffling example promoting gain of a functionally relevant ppi may be furnished by amyloid precursor protein .
app is present in vertebrates and has several isoforms resulting from alternative splicing, some of them  including an exon containing a kunitz-type protease inhibitor  domain, which is absent from other isoforms .
app usually suffers one out of two major types of processing by proteases: either it is cleaved by an alpha-secretase , or is cleaved in succession by a beta- and a gamma-secretase  <cit> .
both pathways produce a soluble app, although only the last one also leads to production of beta-amyloid peptide, which is an important component of amyloid plaques of alzheimer disease and whose physiological function appears to be regulation of cellular cholesterol and sphingomyelin levels  <cit> .
what determines the preferential cleavage pathway for app is the presence or absence of the kpi domain, which inhibits alpha-secretase activity by binding to its trypsin domain.
besides that, soluble app bearing the kpi domain also appears to favor beta amyloid accumulation in the cns by other mechanisms  <cit> .
the kpi domain of app has long ago been proposed to have been acquired by exon shuffling  <cit> .
analyzing more recent sequence data, we found several facts to support this hypothesis: it is closely flanked by introns; some metazoan putative acceptor proteins, in organisms as diverse as protostomes, echinoderms and cephalochordates, display similarity to app in regions both at 5' and 3' to the kpi domain, in a pattern that only requires a kpi domain insertion for production of a protein with domain structure very similar to that of app ; conversely, some putative donor proteins, also widely distributed among metazoans, present the kpi domain flanked by introns, but not by any sequence presenting any similarity to other regions of app.
exon shuffling and self-interactions
we next evaluated a special type of ppi, known as self-interaction, which refers to the capacity of a protein to interact with another copy of itself.
the self-interacting proteins are noteworthy because they usually present higher degrees than non-self-interacting ones  <cit> .
indeed, for all species analyzed in this study, the set of self-interacting proteins have on average higher degree than the set of non-self-interacting proteins .
besides that, self-interacting proteins are surprisingly abundant in the human and yeast ppi networks , and the duplication of self-interacting proteins probably has led to the evolution of many protein complexes  <cit> .
our three shuffling profile gene sets were compared in humans regarding their self-interaction properties.
es genes presented a higher frequency of self-interaction proteins than ss and ws genes .
this result resisted a statistical simulation with control for length as well as a paralog confluence procedure as described previously , suggesting that exon shuffling events might promote the acquisition of self-interaction capacity in proteins.
z indicates difference between real data and the resampling mean given in standard deviations of the resampling mean.
exon shuffling and interaction properties of domains
the observed relationship between exon shuffling and self-interactions led us to search for a mechanism by which exon shuffling might have promoted the emergence of self-interacting proteins.
the gain of a domain capable of interacting with another copy of itself may be the simplest, most economical and most effective evolutionary solution to generate homodimeric proteins.
we hypothesized that, if this mechanism had a significant participation in the evolution of the abundant self-interactions in the human ppi network, a stronger intron flanking signal  would be expected to exist around self-interacting domains.
a number of studies have tried to disclosure what pairs of domains establish physical interactions, either based on structural data  <cit> , or based on statistical methods applied to ppi networks enriched by domain data for each vertex  <cit> .
here, we simply analyzed the exon shuffling signal around domains that the literature usually classifies as self-interacting.
the relative frequency of intron flanking for self-interacting domains was compared to these relative frequencies for non-self-interacting domains that mediate ppis and for domains that are not known to mediate ppis.
again, in order to minimize the effect of gene duplication and concentrate on exon shuffling events, we used a paralog confluence procedure where the pondered contribution of each gene to the final average was given by the inverse of the number of domain-bearing paralogs of the gene.
as seen in tables  <dig> and  <dig>  we observed a significant excess of introns flanking self-interacting domains in comparison to other types of domains classes in metazoan species as diverse as human and the sea anemone nematostella vectensis.
in fact, we found a similar pattern for all other analyzed metazoan species .
in contrast, for the choanoflagellate monosiga brevicollis , as well as for more distant eukaryotic species such as c. neoformans and a. thaliana , this pattern was absent.
these findings suggest that in the metazoan lineage , self-interacting domains have suffered more exon shuffling when compared to non-self-interacting ones.
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
di = domain-interacting.
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
given the evidence that we found for the role of exon shuffling in the spread of self-interacting domains, we speculate about the evolutionary advantage that this type of exon shuffling might have.
the gain by a protein of a domain that mediates ppis might in principle make it possible for the acceptor protein to interact with proteins bearing the domains  that interacted with the shuffled domain in the donor protein.
we think that this kind of chemical affinity consequence is much more likely to have a functional consequence when the exon shuffling event involves self-interacting domains.
indeed, the abundance of self-interactions in humans and the widespread exon shuffling of self-interacting domains in metazoans may be molecular testimonies in favor of this hypothesis.
on the other hand, the same rationale would also predict that the gain of a domain by exon shuffling, whether self-interacting or not, would be more likely to have functional effects if the domain could interact with a higher number of other domains, leading us to expect a higher frequency of exon shuffling involving “promiscuous domain types”, that is, domain types that have the capacity to interact with many other domain types.
we observed this to be true in all metazoan species that we analyzed, with data for some selected eukaryotic species seen in tables  <dig> and  <dig>  in contrast to the strong statistical significances seen for metazoan species , trends below the  <dig>  significance level were also seen for choanoflagellate and plant, suggesting that similar phenomena might have taken place in non-metazoan eukaryotic species, although in much smaller scale, as attested by the rarity of domain intron flanking in those species.
the observation that the exon shuffling gene set is enriched with promiscuous domains provides an explanation for the observed association between vertex degree and exon shuffling.
an illustration of the effect upon the ppi network topology of an exon shuffling event leading to the gain of a domain with promiscuous property is seen in figure  <dig>  if a hypothetical exon shuffling event promotes the evolution of a new protein z, merging together domains of proteins x and y, this new protein z may be able to interact with several proteins through the interaction properties already available in proteins x and y. this suggests that exon shuffling events involving promiscuous domains could be responsible for the origin of hubs in ppi networks.
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
di = domain-interacting.
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
another domain property that seems to be correlated to exon shuffling events is abundance .
it is already known that abundant domains have on average stronger intron flanking signal  <cit> .
we observe this difference for metazoan species as diverse as sea anemone and human, but only marginally for choanoflagellate, and not at all for plant and fungus .
table  <dig> shows the number of human pfam domain types present in categories for the following attributes: self-interaction and promiscuity; self-interaction and abundance; promiscuity and abundance.
a significant association was found between all three attributes of domains, as can be seen in table  <dig>  we initially observed that self-interacting domains are more promiscuous than expected by chance.
similarly, both self-interacting and promiscuous domains tend to be abundant domains.
these associations were found to be strong for domains present in any analyzed eukaryotic species, despite the fact that the association of these characteristics to the intron flanking exon shuffling signal is strong only in metazoans.
this could be due to the dynamics of intron evolution and shuffling phenomena in different species.
cause and effect may be difficult to disentangle for the above-mentioned properties, and clearly more studies are necessary for this purpose.
we speculate that the interaction properties of a domain are important in determining the probabilities for that domain to be spread by exon shuffling.
in this sense, self-interaction and promiscuity would be properties to promote the abundance of domains along large evolutionary time frames.
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
h. sapiens
n. vectensis
m. brevicollis
c. neoformans
a. thaliana
CONCLUSIONS
in this work we have presented evidence for a relevant role of exon shuffling in the evolution of metazoan ppi networks.
we also showed that some interaction properties of domains might make them more likely to take part into exon shuffling phenomena that promote the evolution of new genes bearing novel ppis.
this idea, added to the fact that metazoan exon shuffling genes specifically show higher degree, suggests that the role of exon shuffling in ppi networks is primarily to promote the creation of novel ppis.
furthermore, the strong signal of exon shuffling in the set of promiscuous domains suggests a possible role of exon shuffling in the origin of hubs in ppi networks.
it is important to add a final remark concerning models normally used for ppi networks.
our findings suggest that an important evolutionary process contributing to the emergence of ppi networks of extant species has been overlooked in most graph growth models used for ppi network study in the literature.
it has been repeatedly proposed that duplication models can explain the degree distribution of ppi networks  <cit> , and even their subgraph distributions  <cit> .
however, gene duplication does not usually lead to the evolution of neither longer proteins nor genes with an increased number of domains.
this is in sharp contrast to exon shuffling, which has been shown to be commonly involved in the evolution of metazoan multidomain proteins  <cit> .
hence, any duplication model will not adequately predict the observed associations between degree and protein size, and between degree and number of domains  <cit> .
recently, a work has addressed the resilience of the powerlaw distribution produced by duplication models to shuffling phenomena interfering in the topology of the ppi network, and concluded that these phenomena did not significantly affect the powerlaw  <cit> .
however, the shuffling model used in the mentioned work did not present any trend for increasing the degree of shuffled vertices, in disagreement with what we found evaluating real biological data in the present work.
we think a natural next step in future investigations will be to define the relative contributions of duplication, exon shuffling and other shuffling events for the evolution of ppi networks in the several kingdoms of life, as well as to develop new graph growth models that better help us to understand the effects of these two evolutionary forces upon ppi network topology.

 in walter gilbert speculated that the presence of introns in eukaryotic genes would lead to non-homologous recombinations and the creation of new exon combinations. gilbert and others reasoned that exons could code for protein functional units, and proposed that exon shuffling could be an important mechanism for the evolution of genes in which these functional units are reused in new molecular contexts. since then, abundant evidence has accumulated showing that exon shuffling has effectively occurred along evolution, playing a crucial role in the origin of numerous metazoan proteins, among which predominate extracellular matrix, immune system and membrane receptors. it has also become clear that exon shuffling is an important mechanism of protein domain shuffling, in particular for some domain types that have expanded significantly in metazoans. physical protein-protein interaction is a critical phenomenon for the function of most proteins in living organisms, and the recent accumulation of ppi data from numerous small-scale and a few recent large-scale experiments now allow us to build proteome-wide ppi networks. they help us to understand more globally the multiple roles that a protein may play within a cell and the complex interdependence that functions of several of them may bear among themselves. in the simplest and more usual form, these networks represent proteins by vertices, and a physical interaction between two proteins by an edge between the two corresponding vertices. a fundamental property of each vertex is the number of edges, a feature known as vertex degree. numerous recent works have analyzed the major topological properties of ppi networks, and a recurring theme has been the special properties of vertices with high degree. they are called hubs and tend to be large multi-domain essential proteins. other investigations have shown that many domain-domain interactions are ubiquitous in ppi networks, suggesting that the reuse of these interactions has been relevant for the evolution of ppi networks of extant species. this last relationship, together with the role of exon shuffling as an evolutionary process to make possible the reuse of domains, led us to the natural hypothesis that exon shuffling may have contributed significantly to the evolution of ppi networks. this present work is an effort to verify this hypothesis in several eukaryotic species and to discover what concrete and specific type of influence exon shuffling has had on ppi networks.  exon shuffling and network degree with the purpose of generating ppi networks representative of the state-of-the-art knowledge, we compiled data from several public databases. because of the incompleteness of data comparing interaction properties of several protein products of a gene we merged all data concerning proteins from a gene in just one vertex of the network, a strategy that has been adopted by others. given that we intended to focus on direct physical interactions between proteins, we excluded from our final network all mass spectrometry data, as also done by others, since this technique does not discriminate between direct and indirect physical interactions. we included the five species that we found to be more balanced concerning a reliable ppi network and a considerably sized intron density: human, mouse, the fruit fly drosophila melanogaster, the nematode worm caenorhabditis elegans, and the flowering plant arabidopsis thaliana. we added to the analysis a sixth case: a predicted ppi network that was obtained by projecting the binary interactions of the exhaustively studied yeast saccharomyces cerevisiae ppi network into the nearest ortholog genes in cryptococcus neoformans, a fungal species where exon shuffling analysis was possible because of less extensive intron loss. in several moments, however, the present work emphasizes the results obtained with the human ppi network, due to its higher completeness level, and to the abundance and conservation of human introns, which make exon shuffling identification more reliable. in order to select a subset of genes enriched in exon shuffling events, we sought for the presence of homologous regions flanked by introns in coding sequences derived from non-homologous genes, as has been done by us and others. for the sake of simplicity, we just considered one of the coding sequences of maximum length for each gene. taking into account the pervasive prevalence of intron loss and the antiquity of some exon shuffling cases, we did not require bilateral flanking of both homologous regions, and the number of required introns was smaller in species for which a more extensive intron loss process was known to have taken place in their evolutionary histories. although arbitrary, this less stringent requirement is convenient to include as many probable exon shuffling cases as possible in the exon shuffling gene sets and, at the same time, to deplete the complementary gene sets in these cases. our strategy used local alignments to find such homologous regions resulting in a classification of protein-coding genes into three shuffling profile categories. genes without any homologous regions were classified as without-shuffling genes. homologous regions were then analyzed in regard to the presence of introns in four windows adjacent to their extremities. genes with at least one homologous region with a minimum number of windows containing introns were classified as exon shuffling genes. this minimum number was chosen between and according to the abundance of introns in the respective species. genes with homologous regions, but without the minimum number of windows with flanking introns, were classified as sequence shuffling genes. based on the assumption that exon shuffling may be an important mechanism for the evolution of new protein-protein interactions, we first compared genes in the three categories according to their presence in ppi networks, as shown in figure and additional file. we observe that, in mammalian intron-rich species, there is a higher fraction of es genes in ppi networks, compared to ss and ws genes. interestingly, ss genes are also more frequently found in ppi networks than ws genes in all tested species, except worm. we next performed pairwise comparisons of vertex degree among the three datasets. we opted for using multiple statistical measures, as each one could give us a particular appreciation of the differences present among the distributions of the several gene sets. boxplots of the distributions including these measures are in figure we evaluated the statistical significance of the differences among groups by means of both u test, a standard non-parametric test for comparison of two samples, and re-sampling procedures for arithmetic and geometric means. as seen in table and in additional file, the es gene set has higher degree than the other two groups, according to all above-mentioned statistical tests, in all four analyzed metazoan species, but neither in fungus nor in plant. in addition, we observe that the ss set shows higher degree than the ws set in three cases: human, fungus and plant. z indicates difference between real data and the resampling mean given in standard deviations of the resampling mean. we interpret all these results as follows: exon shuffling events are associated to the acquisition of new interactions by proteins, although its relative importance seems to be distinct among different phylogenetic groups. the resistance of this result to the use of both arithmetic and geometric mean in the re-sampling procedure, measures that give more or less weight to high-degree vertices, respectively, suggests additionally that this fact would apply to both low- and high-degree proteins. ss genes probably represent a very heterogeneous set. they may correspond to homologous genes with sequence conservation limited to the currently identified homologous stretch or, given the widespread intron loss suffered along evolution by all analyzed species, to exon shuffling cases that lost too many introns and cannot be identified anymore. they may be examples of exon shuffling-generated genes in which sequence divergence in certain regions of the shuffled region prevented the expected correlation between introns and regions of sequence similarity to be found. finally, they may correspond to genes resulting from all types of recombination events that are not mediated by introns. gene duplication is the other major evolutionary mechanism for the emergence of novelties, despite not being able to produce radically different genes in a short time span as exon shuffling can do. is it known that eukaryotic genomes suffered both many single-gene and a few whole-genome duplications. this has two consequences for our study. first, it is possible that a small number of extensively duplicated exon shuffling cases could explain all the degree differences observed among the shuffling profile categories. in order to rule this out, a paralog confluence procedure was performed in which sets of network vertices corresponding to paralogs were exchanged in the network by single vertices with degree equal to the average degree of the removed sets. after the confluence, in comparison to ss and ws sets, es set continue to present higher degree, whichever the statistical test, with or without re-sampling control for length, in all four considered metazoan species, as shown in additional file the second likely consequence of gene duplication for this study is the existence of a putative effect of gene duplications on ppi network vertex degree. to test that, the relationship between existence of paralogs of a gene and its corresponding vertex degree was analyzed. given the strong effect we already know exon shuffling has upon degree for metazoan species, we excluded the exon shuffling genes before making the analysis for those species. as can be found in additional file we observed that genes with paralogs have a significantly higher degree in human, a significantly smaller degree in fly, and statistically non-significant differences in other organisms. it must be noticed that the quality of these data, contrary to the shuffling profile category ones, is not affected by intron loss, and probably must depend basically on the completeness of ppi network knowledge in the species. that knowledge is more extensive precisely in human and fly, suggesting these opposing trends in the two species may be real. most importantly, all degree differences between genes with and without paralogs are numerically smaller than the degree differences found between es and non-es gene sets, suggesting that gene duplication is not per se particularly important for the evolution of degree in ppi networks. it should be noted, however, that gene duplication may have an indirect importance as it is expected that exon shuffling would be less deleterious if involving duplicated copies of genes. case analysis a possible exon shuffling example promoting gain of a functionally relevant ppi may be furnished by amyloid precursor protein. app is present in vertebrates and has several isoforms resulting from alternative splicing, some of them including an exon containing a kunitz-type protease inhibitor domain, which is absent from other isoforms. app usually suffers one out of two major types of processing by proteases: either it is cleaved by an alpha-secretase, or is cleaved in succession by a beta- and a gamma-secretase. both pathways produce a soluble app, although only the last one also leads to production of beta-amyloid peptide, which is an important component of amyloid plaques of alzheimer disease and whose physiological function appears to be regulation of cellular cholesterol and sphingomyelin levels. what determines the preferential cleavage pathway for app is the presence or absence of the kpi domain, which inhibits alpha-secretase activity by binding to its trypsin domain. besides that, soluble app bearing the kpi domain also appears to favor beta amyloid accumulation in the cns by other mechanisms. the kpi domain of app has long ago been proposed to have been acquired by exon shuffling. analyzing more recent sequence data, we found several facts to support this hypothesis: it is closely flanked by introns; some metazoan putative acceptor proteins, in organisms as diverse as protostomes, echinoderms and cephalochordates, display similarity to app in regions both at 5' and 3' to the kpi domain, in a pattern that only requires a kpi domain insertion for production of a protein with domain structure very similar to that of app; conversely, some putative donor proteins, also widely distributed among metazoans, present the kpi domain flanked by introns, but not by any sequence presenting any similarity to other regions of app. exon shuffling and self-interactions we next evaluated a special type of ppi, known as self-interaction, which refers to the capacity of a protein to interact with another copy of itself. the self-interacting proteins are noteworthy because they usually present higher degrees than non-self-interacting ones. indeed, for all species analyzed in this study, the set of self-interacting proteins have on average higher degree than the set of non-self-interacting proteins. besides that, self-interacting proteins are surprisingly abundant in the human and yeast ppi networks, and the duplication of self-interacting proteins probably has led to the evolution of many protein complexes. our three shuffling profile gene sets were compared in humans regarding their self-interaction properties. es genes presented a higher frequency of self-interaction proteins than ss and ws genes. this result resisted a statistical simulation with control for length as well as a paralog confluence procedure as described previously, suggesting that exon shuffling events might promote the acquisition of self-interaction capacity in proteins. z indicates difference between real data and the resampling mean given in standard deviations of the resampling mean. exon shuffling and interaction properties of domains the observed relationship between exon shuffling and self-interactions led us to search for a mechanism by which exon shuffling might have promoted the emergence of self-interacting proteins. the gain of a domain capable of interacting with another copy of itself may be the simplest, most economical and most effective evolutionary solution to generate homodimeric proteins. we hypothesized that, if this mechanism had a significant participation in the evolution of the abundant self-interactions in the human ppi network, a stronger intron flanking signal would be expected to exist around self-interacting domains. a number of studies have tried to disclosure what pairs of domains establish physical interactions, either based on structural data, or based on statistical methods applied to ppi networks enriched by domain data for each vertex. here, we simply analyzed the exon shuffling signal around domains that the literature usually classifies as self-interacting. the relative frequency of intron flanking for self-interacting domains was compared to these relative frequencies for non-self-interacting domains that mediate ppis and for domains that are not known to mediate ppis. again, in order to minimize the effect of gene duplication and concentrate on exon shuffling events, we used a paralog confluence procedure where the pondered contribution of each gene to the final average was given by the inverse of the number of domain-bearing paralogs of the gene. as seen in tables and we observed a significant excess of introns flanking self-interacting domains in comparison to other types of domains classes in metazoan species as diverse as human and the sea anemone nematostella vectensis. in fact, we found a similar pattern for all other analyzed metazoan species. in contrast, for the choanoflagellate monosiga brevicollis, as well as for more distant eukaryotic species such as c. neoformans and a. thaliana, this pattern was absent. these findings suggest that in the metazoan lineage, self-interacting domains have suffered more exon shuffling when compared to non-self-interacting ones. h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana di = domain-interacting. h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana given the evidence that we found for the role of exon shuffling in the spread of self-interacting domains, we speculate about the evolutionary advantage that this type of exon shuffling might have. the gain by a protein of a domain that mediates ppis might in principle make it possible for the acceptor protein to interact with proteins bearing the domains that interacted with the shuffled domain in the donor protein. we think that this kind of chemical affinity consequence is much more likely to have a functional consequence when the exon shuffling event involves self-interacting domains. indeed, the abundance of self-interactions in humans and the widespread exon shuffling of self-interacting domains in metazoans may be molecular testimonies in favor of this hypothesis. on the other hand, the same rationale would also predict that the gain of a domain by exon shuffling, whether self-interacting or not, would be more likely to have functional effects if the domain could interact with a higher number of other domains, leading us to expect a higher frequency of exon shuffling involving “promiscuous domain types”, that is, domain types that have the capacity to interact with many other domain types. we observed this to be true in all metazoan species that we analyzed, with data for some selected eukaryotic species seen in tables and in contrast to the strong statistical significances seen for metazoan species, trends below the significance level were also seen for choanoflagellate and plant, suggesting that similar phenomena might have taken place in non-metazoan eukaryotic species, although in much smaller scale, as attested by the rarity of domain intron flanking in those species. the observation that the exon shuffling gene set is enriched with promiscuous domains provides an explanation for the observed association between vertex degree and exon shuffling. an illustration of the effect upon the ppi network topology of an exon shuffling event leading to the gain of a domain with promiscuous property is seen in figure if a hypothetical exon shuffling event promotes the evolution of a new protein z, merging together domains of proteins x and y, this new protein z may be able to interact with several proteins through the interaction properties already available in proteins x and y. this suggests that exon shuffling events involving promiscuous domains could be responsible for the origin of hubs in ppi networks. h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana di = domain-interacting. h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana another domain property that seems to be correlated to exon shuffling events is abundance. it is already known that abundant domains have on average stronger intron flanking signal. we observe this difference for metazoan species as diverse as sea anemone and human, but only marginally for choanoflagellate, and not at all for plant and fungus. table shows the number of human pfam domain types present in categories for the following attributes: self-interaction and promiscuity; self-interaction and abundance; promiscuity and abundance. a significant association was found between all three attributes of domains, as can be seen in table we initially observed that self-interacting domains are more promiscuous than expected by chance. similarly, both self-interacting and promiscuous domains tend to be abundant domains. these associations were found to be strong for domains present in any analyzed eukaryotic species, despite the fact that the association of these characteristics to the intron flanking exon shuffling signal is strong only in metazoans. this could be due to the dynamics of intron evolution and shuffling phenomena in different species. cause and effect may be difficult to disentangle for the above-mentioned properties, and clearly more studies are necessary for this purpose. we speculate that the interaction properties of a domain are important in determining the probabilities for that domain to be spread by exon shuffling. in this sense, self-interaction and promiscuity would be properties to promote the abundance of domains along large evolutionary time frames. h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana h. sapiens n. vectensis m. brevicollis c. neoformans a. thaliana  in this work we have presented evidence for a relevant role of exon shuffling in the evolution of metazoan ppi networks. we also showed that some interaction properties of domains might make them more likely to take part into exon shuffling phenomena that promote the evolution of new genes bearing novel ppis. this idea, added to the fact that metazoan exon shuffling genes specifically show higher degree, suggests that the role of exon shuffling in ppi networks is primarily to promote the creation of novel ppis. furthermore, the strong signal of exon shuffling in the set of promiscuous domains suggests a possible role of exon shuffling in the origin of hubs in ppi networks. it is important to add a final remark concerning models normally used for ppi networks. our findings suggest that an important evolutionary process contributing to the emergence of ppi networks of extant species has been overlooked in most graph growth models used for ppi network study in the literature. it has been repeatedly proposed that duplication models can explain the degree distribution of ppi networks, and even their subgraph distributions. however, gene duplication does not usually lead to the evolution of neither longer proteins nor genes with an increased number of domains. this is in sharp contrast to exon shuffling, which has been shown to be commonly involved in the evolution of metazoan multidomain proteins. hence, any duplication model will not adequately predict the observed associations between degree and protein size, and between degree and number of domains. recently, a work has addressed the resilience of the powerlaw distribution produced by duplication models to shuffling phenomena interfering in the topology of the ppi network, and concluded that these phenomena did not significantly affect the powerlaw. however, the shuffling model used in the mentioned work did not present any trend for increasing the degree of shuffled vertices, in disagreement with what we found evaluating real biological data in the present work. we think a natural next step in future investigations will be to define the relative contributions of duplication, exon shuffling and other shuffling events for the evolution of ppi networks in the several kingdoms of life, as well as to develop new graph growth models that better help us to understand the effects of these two evolutionary forces upon ppi network topology. 
physical protein-protein interaction is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of ppis are the result of domain-domain interactions. we tested our hypothesis by compiling exon shuffling and ppi data from six eukaryotic species: homo sapiens, mus musculus, drosophila melanogaster, caenorhabditis elegans, cryptococcus neoformans and arabidopsis thaliana. for all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree in ppi networks. thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of ppi networks.
 physical protein-protein interaction is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of ppis are the result of domain-domain interactions. exon shuffling, intron-mediated recombination of exons from existing genes, is known to have been a major mechanism of domain shuffling in metazoans. thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of ppi networks.  we tested our hypothesis by compiling exon shuffling and ppi data from six eukaryotic species: homo sapiens, mus musculus, drosophila melanogaster, caenorhabditis elegans, cryptococcus neoformans and arabidopsis thaliana. for all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree in ppi networks. furthermore, we verified that a set of protein domains that are simultaneously promiscuous, self-interacting and abundant in the genomes presents a stronger signal for exon shuffling.  exon shuffling appears to have been a recurrent mechanism for the emergence of new ppis along metazoan evolution. in metazoan genomes, exon shuffling also promoted the expansion of some protein domains. we speculate that their promiscuous and self-interacting properties may have been decisive for that expansion. 18– october 5th international conference of the brazilian association for bioinformatics and computational biology angra dos reis, rj, brazil 