BACKGROUND
malaria is a major threat to public health and economic development in endemic regions.
about 300- <dig> million cases are reported, and 1- <dig> million people die from malaria every year.
children and pregnant women are among the hardest hit of malaria victims.
five parasite species, p. falciparum, p. vivax, p. malariae, p. ovale, and p. knowlesi, cause human malaria.
p. falciparum is the most virulent and widespread one.
the continuous morbidity or mortality of malaria is largely due to the rapid development of parasite resistance to currently available drugs and the increasing insecticide resistance of mosquito vectors.
it is imperative to search for new lines of antimalarial drug and vaccine targets.
the complete genome sequencing of p. falciparum and its sibling species and strains  <cit> , the subsequent transcriptomic  <cit> , proteomic  <cit> , metabolic  <cit> , interactomic analyses  <cit>  and, most recently, next-generation sequencing  <cit>  efforts have set the stage for a quantum leap in our understanding of the fundamental processes of the parasite life cycle and mechanisms of drug resistance, immune evasion, and pathogenesis.
however, the paradigm of -omics driven target discovery has been significantly hampered by our limited knowledge of the cellular networks associated with parasite survival, development, transmission, invasion, and pathogenesis.
we propose to circumvent this limitation using a subnetwork alignment approach.
it has been shown that network alignment offers an effective means to elucidate network structure and predict protein orthologs  <cit> .
our approach extends the concept of network alignment to align subnetworks of proteins for measuring their functional relations in a network context.
it is particularly useful when the genome of interest suffers from poor annotation due to low or no sequence similarity to known proteins, a significant problem for p. falciparum, as over 60% of the predicted open reading frames  were annotated as "hypothetical" without functional assignment  <cit> .
previously, we developed a supervised learning algorithm for remote homology detection based on support vector machines  and profile kernels  <cit> , and predicted a group of novel proteases  <cit> , which were implicated in networks associated with signaling, stress response, cell cycle progression, metabolism, and invasion  <cit> .
in this study, we attempt to identify network components beyond sequence-similarity searches.
ppi network alignment algorithms are designed to match nodes in two ppi networks such that the conserved interactions between the orthologs in the networks are captured or maximized in counts.
the current network alignment algorithms are either local or global approaches.
local network alignment  <cit>  aims at detecting pairs of subnetwork modules with many functional orthologs.
typically, these algorithms start from conserved regions and expand the regions greedily in the two ppi networks.
global network alignment attempts to find the best consistent mapping of the proteins in the two ppi networks for maximizing the number of conserved interactions.
previous studies tackled the global network problem with markov random field   <cit> , combinatorial graph matching by optimization  <cit> , and random walk on a kronecker product graph of two ppi networks  <cit> .
since p. falciparum shares very few orthologous proteins with other species, the conserved interactions between p. falciparum ppi network and the ppi network of model organisms are too few to reveal meaningful alignments.
thus, network alignment is not directly applicable to the study of p. falciparum ppi network.
instead of focusing on detecting alignment, we propose to measure the functional relation between p. falciparum proteins and the annotated proteins in another species by aligning the neighborhood subnetworks of the two proteins.
the neighborhood subnetwork of a protein  contains the nearby neighbors reachable by the protein through a small number of hops in the ppi network.
our assumption is that the neighborhood subnetwork captures information on the functional role of the central protein.
based on this assumption, if two proteins are functional orthologs, their neighborhood subnetworks will share similar paths or other structural patterns.
our subgraph alignment approach is designed to summarize the structural similarity between neighborhood subnetworks for ortholog prediction.
as a proof-of-concept, we chose to predict the components in the transcriptional regulation network in p. falciparum.
it was chosen because:  parasite employs exquisite regulatory machinery on gene expression to assure timely and accurate coordination on parasite growth, development, infection, and virulence.
very little is known about the components, dynamics, and design principles of this network.
new discoveries of network components could significantly fill our knowledge gaps and possibly lead to new short lists of proteins that are poorly understood and poorly annotated for functional characterization.
the correspondent network used was from escherichia coli.
detection of network similarities among eukaryotes and among prokaryotes have been demonstrated  <cit> , but detection of similarities between these two groups is a more challenging problem.
the ability to make comparisons across such a wide phylogenetic gap means, firstly, that evolutionarily conserved  subnetworks can be detected and, secondly, that it is possible to search beyond more closely related strains.
this is especially significant in cases like p. falciparum, where the immediate relatives reveal comparatively little about its functional subsystems.
RESULTS
module-based subnetwork alignments predicted  <dig>  components in transcriptional regulation network in p. falciparum
it is a common belief that the malaria parasite possesses a complex and orchestrated transcriptional regulatory system  <cit> .
however, only a small number of transcriptional regulators have been identified, including a conserved set of basic transcription factors  <cit>  and those predicted based on parasite developmental microarray expression profiles and motif analysis  <cit> .
a recent study by bischoff and vaquero  <cit>  combining literature searches, motif finding, and transcriptomic, proteomic, and interactomic analyses expanded this list to include proteins related to chromatin functions and remodeling.
our functional module-based subnetwork alignments predicted that  <dig>  p. falciparum proteins were functional orthologs of known transcriptional regulators in the e. coli network .
37% of these predicted functional orthologs appeared to be "putative uncharacterized proteins" or "conserved plasmodium proteins" of unknown function.
this is in agreement with the fact that  <dig> years after the completion of genome sequencing, the proportion of orfs with no functional assignment has only been reduced to 45%  <cit> .
functional enrichment analysis  <cit>  revealed that  <dig> gene ontology  terms were over-represented , including those processes that are well known to be associated with transcriptional regulation such as proteolysis  <cit> , response to stimulus, and proteasome activity .
general transcriptional regulators
the predicted functional orthologs include several general transcriptional regulators  that are commonly present in a wide variety of species.
the first is basic transcription factor 3b .
it was found via yeast 2-hybrid  analysis  <cit>  to have a direct ppi with a nascent polypeptide associated complex α chain protein , the erythrocyte binding antigen- <dig>  and a putative coronin binding protein , suggesting that it may be involved in protein folding, immune evasion, and cellular actin dynamics .
the second is a putative ccaat-binding transcription factor .
a y2h assay  <cit>  showed that it had ppis with six proteins.
two of these proteins are likely involved in global transcription, including  a putative not <dig> protein .
proteins in the not <dig> family were shown to regulate the activity of general transcription factor tfiid  <cit> ; and  a conserved plasmodium protein  that has a functional domain rpc <dig> which comprises a subunit of the trna specific polymerase rna pol iii.
the third interacting protein is a merozoite surface protein  <dig>  precursor , which is a regulator of parasite growth and a surface antigen regarded as a potential vaccine target  <cit> .
the fourth protein associated with pf14_ <dig> is a conserved plasmodium membrane protein  that contains two fyve/phd zinc fingers for binding to potential target molecules.
the remaining two proteins associated with pf14_ <dig> are 40s ribosomal proteins s <dig>  and s20e , indicating the interactions between transcription and translation .
a putative yl <dig> nuclear protein  was predicted to be a transcriptional regulator.
it has two functional domains yl <dig>  and yl <dig> c-terminal domain , both of which are typical dna binding domains.
this protein may be related to chromatin remodeling.
in addition, a y2h assay using this protein as a bait pulled out a chloroquine resistance marker protein  .
apicomplexan-specific apiap <dig> transcriptional regulators
most interestingly, our subnetwork alignments also predicted  <dig> putative transcriptional regulators belonging to the apicomplexan-specific apiap <dig> family.
a characteristic feature of this family is the presence of the apetala <dig>  domain.
ap <dig> transcription factors play a pivotal role in floral development in plants  <cit> .
the recent discovery of ap <dig> in the apicomplexa, the phylum to which malaria parasites belong, suggested that the apiap <dig> proteins were derived from bacteria or the apicoplast progenitor via transponsons, followed by lineage-specific radiation  <cit> .
these apiap <dig> proteins, in addition to regulating heterochromatin formation and genome integrity, may develop novel parasite-specific functions such as antigenic variation, invasion, and sporozoite development  <cit> .
p. falciparum possesses  <dig> apiap <dig> members.
among the  <dig> apiap <dig> proteins predicted by our network alignments, five contain a single ap <dig> domain, four contain two ap <dig> domains, and two contain three ap <dig> domains .
analyzing the protein-protein association data from the string database  <cit> , in conjunction with the data from the y2h assays, temporal microarray experiments, proteomics, and literature, revealed that these  <dig> apiap <dig> proteins are associated with 1- <dig> proteins in the cellular networks .
at least four apiap <dig> proteins  have ppis, suggesting that they play central role in transcriptional regulation.
the apiap <dig> protein with highest connectivity is pfd0985w, which has  <dig> interaction partners .
it has direct physical interactions with two other apiap <dig> proteins .
it is associated with a nucleosome assembly protein  that is implicated in chromatin remodeling, and a putative ndc <dig> homolog  that may be a component of the mitotic spindle related to chromosome segregation.
it is also associated with three surface antigens including a reticulocyte binding protein  <dig> homologue a  which may play a role in determining host-cell invasion specificity  <cit> , an antigen  <dig>  in the duffy binding-like  protein family which may be related to parasite entry to the host, and an asparagine-rich antigen .
this apiap <dig> protein pfd0985w also appeared to be related to a number of secreted proteins including a putative secreted ookinete protein , and two proteins that are associated with maurer's clefts  <cit> , parasite-derived membranous structures within the host cell cytoplasm .
in addition, pfd0985w has direct ppis with the 26s proteasome aaa-atpase subunit rpt <dig> , which is a component in ubiquitin-proteasome system for protein degradation, and pyruvate kinase , an essential enzyme for glycolysis.
the apiap <dig> protein with second largest connectivity is pf07_ <dig>  it has  <dig> ppi partners  that can be divided into five categories:  transcriptional regulation.
it is associated with two otherapiap <dig> proteins , and a ccaat-box dna binding protein subunit b ;  epigenetic regulation.
it is associated with pfhmgb <dig> , which has a dna-binding domain: hmg-box .
the proteins in this family have been implicated in regulation of transcription, replication, repair, and chromatin remodeling;  signaling.
pf07_ <dig> is associated with at least three putative signaling proteins, including  pf13_ <dig>  which contains a forkhead-associated domain that is found in a variety of regulatory proteins involved in signaling.
a calcium/calmodulin-dependent protein kinase  that is implicated in signaling cascades.
a putative 14-3- <dig> protein .
proteins in 14-3- <dig> family include regulatory ligands to various signaling molecules such as kinases and receptors;  surface antigens for cell adhesion and entry to the host.
pf07_ <dig> is associated with a duffy binding-like antigen  <dig> , an erythrocyte membrane-associated antigen , and a qf <dig> antigen  with an rna-binding motif;  metabolism.
the glycolytic enzyme fructose-bisphosphate aldolase  is associated with the apiap <dig> protein pf07_ <dig>
the role of apiap <dig> proteins in transcriptional and epigenetic regulation is also indicated by a direct ppi between a putative apiap <dig> with  <dig> ap <dig> domains pf10_ <dig> and a histone acetyltransferase gcn <dig> , an enzyme for histone modification and chromatin remodeling  <cit> .
this apiap <dig> protein may also been involved in the regulation of genome integrity through a ppi with a dna repair protein rhp <dig> , and cytoskeleton organization of actin .
two of these  <dig> apiap <dig> proteins have been experimentally characterized to some extent:  the crystal structure of the ap <dig> domain of pf14_ <dig> has been determined, revealing a multiple-site binding pattern  <cit> , and gene disruption assays showed that its ortholog in the rodent parasite p. berghei was an indispensible regulator for sporozoite development in the mosquito stage  <cit> .
however, its regulatory roles and targets remain uncharacterized in p. falciparum.
as shown in figure  <dig>  it has only two direct ppis revealed by y2h assays  <cit> : the first is a ribosomal protein p <dig>  and the second protein ptex <dig>  is an important component in a translocon of exported proteins .
located in the vacuole membrane, ptex was recently discovered as a novel atp-dependent protein trafficking machinery  <cit> .
notably, ptex <dig> is only present in the plasmodium genus.
the ppi between ptex <dig> and apiap <dig> suggests that this export machinery may have parasite-specific regulation.
ptex is becoming an attractive therapeutic target due to its importance to virulence and parasite survival and its distant evolutionary relatedness to the human host.
pf11_ <dig>  its counterpart in p. berghei is a transcription factor that regulates ookinete-specific gene expression for parasite invasion of the mosquito midgut.
pf11_ <dig>  however, may play a role in the red blood cell  stage: it has one ppi partner, rhoptry-associated protein  <dig> .
rap <dig> is an escort protein required to localize rap <dig> to the rhoptries, apical organelles essential for rbc invasion  <cit> .
in summary, apiap <dig> proteins are a family of stage-specific transcriptional regulators for diverse processes ranging from epigenetic modification, chromosome organization and dynamics, invasion, protein sorting and trafficking, protein turnover, and metabolism.
other potentially important proteins that may be involved in transcriptional regulation
module based subnetwork alignments predicted additional proteins that are likely involved in transcriptional regulation .
two proteins  are members of the smc  superfamily; they both have a recf/recn/smc n terminal domain and may be involved in chromatin cohesion and dynamics.
a numbers of zinc-finger proteins were identified by network alignments as well.
they exhibit different types of domain configurations, including the classical dna-binding motif c2h <dig> found in transcription factors, the c3hc <dig> type  typically found in proteins mediating ubiquitination, the c-x8-c-x5-c-x3-h  type implicated in cell cycle regulation, the dhhc type found in proteins important for membrane association and trafficking, the dnl type implicated in protein translocation into mitochondria, and the cw type related to dna-binding and protein-protein interaction.
a putative transcriptional coactivator  has a zz-type zinc finger domain.
ada <dig> was shown, in baker's yeast and arabidopsis thaliana, to physically interact with gcn <dig>  a histone acetyltransferase and a potent transcriptional activator  <cit> .
the y2h assay in p. falciparum  <cit>  revealed that ada <dig> has direct physical interactions with proteins including a minichromosome maintenance  complex subunit , a pre-mrna splicing factor , a heat shock protein hsp <dig> interacting protein , a sodium-dependent phosphate transporter , a serine/threonine protein kinase in the fikk family , cathepsin c , and a mature parasite-infected erythrocyte surface antigen , suggesting its potential versatile roles in dna replication, splicing, transport, protein processing, signal transduction, and invasion.
other putative transcriptional regulators include pfe <dig> and pf14_ <dig>  pfe <dig> contains two functional domains: a fact complex subunit  domain which was reported to facilitate transcriptional initiation and interact with nucleosomes and histones  <cit> , and a histone chaperone rttp106-like domain.
this protein may be involved in heterochromatin silencing and epigenetic regulation.
pf14_ <dig> is a putative protein in the not global transcriptional regulator family.
y2h assays showed that it had direct physical interactions with ccaat-box dna binding protein subunit b , dna topoisomerase ii , and calcium dependent protein kinase  <dig> , emphasizing its involvement in general transcriptional control and chromosome topology and signaling processes.
it also has a ppi with a pf11- <dig> protein , which may play a role in protein trafficking processes associated with maurer's cleft.
CONCLUSIONS
a functional-module based alignment approach was used to predict system components in transcriptional regulatory networks in malaria parasite p. falciparum.
our results predicted general transcriptional regulators that may regulate gene expression in a global or pleiotropic mode.
our results also imply a group of parasite-specific transcriptional regulators in the apiap <dig> family that play roles in diverse cellular processes ranging from chromatin remodeling, protein sorting and secretion, signal transduction, and invasion.
finally, our analysis has identified other potentially important proteins involved in transcriptional regulation.
our present knowledge of the transcriptional machinery and its regulatory capacity is rudimentary.
the identification of network components in this machinery will open new avenues to the development of novel therapeutic targets and provide new insights into parasite biology, pathogenesis and virulence.
the premise of our subnetwork alignment approach is that functional annotations of the proteins can be transferred across species through conserved interactions in the aligned ppi networks.
under this framework, a priori information as to the identity or function of a gene is not necessary for the gene to be placed in a network.
thus genes identified only because of their key role in a network become potential targets.
furthermore, placement of the gene product in a systems context could, in itself, serve to identify the function of the gene product.
if successfully applied, a systems biology approach circumvents the limiting factor in comparative genomics - the difficulty in obtaining reliable functional assignments.

 malaria is a major threat to public health and economic development in endemic regions. about 300- million cases are reported, and 1- million people die from malaria every year. children and pregnant women are among the hardest hit of malaria victims. five parasite species, p. falciparum, p. vivax, p. malariae, p. ovale, and p. knowlesi, cause human malaria. p. falciparum is the most virulent and widespread one. the continuous morbidity or mortality of malaria is largely due to the rapid development of parasite resistance to currently available drugs and the increasing insecticide resistance of mosquito vectors. it is imperative to search for new lines of antimalarial drug and vaccine targets. the complete genome sequencing of p. falciparum and its sibling species and strains, the subsequent transcriptomic, proteomic, metabolic, interactomic analyses and, most recently, next-generation sequencing efforts have set the stage for a quantum leap in our understanding of the fundamental processes of the parasite life cycle and mechanisms of drug resistance, immune evasion, and pathogenesis. however, the paradigm of -omics driven target discovery has been significantly hampered by our limited knowledge of the cellular networks associated with parasite survival, development, transmission, invasion, and pathogenesis. we propose to circumvent this limitation using a subnetwork alignment approach. it has been shown that network alignment offers an effective means to elucidate network structure and predict protein orthologs. our approach extends the concept of network alignment to align subnetworks of proteins for measuring their functional relations in a network context. it is particularly useful when the genome of interest suffers from poor annotation due to low or no sequence similarity to known proteins, a significant problem for p. falciparum, as over 60% of the predicted open reading frames were annotated as "hypothetical" without functional assignment. previously, we developed a supervised learning algorithm for remote homology detection based on support vector machines and profile kernels, and predicted a group of novel proteases, which were implicated in networks associated with signaling, stress response, cell cycle progression, metabolism, and invasion. in this study, we attempt to identify network components beyond sequence-similarity searches. ppi network alignment algorithms are designed to match nodes in two ppi networks such that the conserved interactions between the orthologs in the networks are captured or maximized in counts. the current network alignment algorithms are either local or global approaches. local network alignment aims at detecting pairs of subnetwork modules with many functional orthologs. typically, these algorithms start from conserved regions and expand the regions greedily in the two ppi networks. global network alignment attempts to find the best consistent mapping of the proteins in the two ppi networks for maximizing the number of conserved interactions. previous studies tackled the global network problem with markov random field, combinatorial graph matching by optimization, and random walk on a kronecker product graph of two ppi networks. since p. falciparum shares very few orthologous proteins with other species, the conserved interactions between p. falciparum ppi network and the ppi network of model organisms are too few to reveal meaningful alignments. thus, network alignment is not directly applicable to the study of p. falciparum ppi network. instead of focusing on detecting alignment, we propose to measure the functional relation between p. falciparum proteins and the annotated proteins in another species by aligning the neighborhood subnetworks of the two proteins. the neighborhood subnetwork of a protein contains the nearby neighbors reachable by the protein through a small number of hops in the ppi network. our assumption is that the neighborhood subnetwork captures information on the functional role of the central protein. based on this assumption, if two proteins are functional orthologs, their neighborhood subnetworks will share similar paths or other structural patterns. our subgraph alignment approach is designed to summarize the structural similarity between neighborhood subnetworks for ortholog prediction. as a proof-of-concept, we chose to predict the components in the transcriptional regulation network in p. falciparum. it was chosen because: parasite employs exquisite regulatory machinery on gene expression to assure timely and accurate coordination on parasite growth, development, infection, and virulence. very little is known about the components, dynamics, and design principles of this network. new discoveries of network components could significantly fill our knowledge gaps and possibly lead to new short lists of proteins that are poorly understood and poorly annotated for functional characterization. the correspondent network used was from escherichia coli. detection of network similarities among eukaryotes and among prokaryotes have been demonstrated, but detection of similarities between these two groups is a more challenging problem. the ability to make comparisons across such a wide phylogenetic gap means, firstly, that evolutionarily conserved subnetworks can be detected and, secondly, that it is possible to search beyond more closely related strains. this is especially significant in cases like p. falciparum, where the immediate relatives reveal comparatively little about its functional subsystems.  module-based subnetwork alignments predicted components in transcriptional regulation network in p. falciparum it is a common belief that the malaria parasite possesses a complex and orchestrated transcriptional regulatory system. however, only a small number of transcriptional regulators have been identified, including a conserved set of basic transcription factors and those predicted based on parasite developmental microarray expression profiles and motif analysis. a recent study by bischoff and vaquero combining literature searches, motif finding, and transcriptomic, proteomic, and interactomic analyses expanded this list to include proteins related to chromatin functions and remodeling. our functional module-based subnetwork alignments predicted that p. falciparum proteins were functional orthologs of known transcriptional regulators in the e. coli network. 37% of these predicted functional orthologs appeared to be "putative uncharacterized proteins" or "conserved plasmodium proteins" of unknown function. this is in agreement with the fact that years after the completion of genome sequencing, the proportion of orfs with no functional assignment has only been reduced to 45%. functional enrichment analysis revealed that gene ontology terms were over-represented, including those processes that are well known to be associated with transcriptional regulation such as proteolysis, response to stimulus, and proteasome activity. general transcriptional regulators the predicted functional orthologs include several general transcriptional regulators that are commonly present in a wide variety of species. the first is basic transcription factor 3b. it was found via yeast 2-hybrid analysis to have a direct ppi with a nascent polypeptide associated complex α chain protein, the erythrocyte binding antigen- and a putative coronin binding protein, suggesting that it may be involved in protein folding, immune evasion, and cellular actin dynamics. the second is a putative ccaat-binding transcription factor. a y2h assay showed that it had ppis with six proteins. two of these proteins are likely involved in global transcription, including a putative not protein. proteins in the not family were shown to regulate the activity of general transcription factor tfiid; and a conserved plasmodium protein that has a functional domain rpc which comprises a subunit of the trna specific polymerase rna pol iii. the third interacting protein is a merozoite surface protein precursor, which is a regulator of parasite growth and a surface antigen regarded as a potential vaccine target. the fourth protein associated with pf14_ is a conserved plasmodium membrane protein that contains two fyve/phd zinc fingers for binding to potential target molecules. the remaining two proteins associated with pf14_ are 40s ribosomal proteins s and s20e, indicating the interactions between transcription and translation. a putative yl nuclear protein was predicted to be a transcriptional regulator. it has two functional domains yl and yl c-terminal domain, both of which are typical dna binding domains. this protein may be related to chromatin remodeling. in addition, a y2h assay using this protein as a bait pulled out a chloroquine resistance marker protein. apicomplexan-specific apiap transcriptional regulators most interestingly, our subnetwork alignments also predicted putative transcriptional regulators belonging to the apicomplexan-specific apiap family. a characteristic feature of this family is the presence of the apetala domain. ap transcription factors play a pivotal role in floral development in plants. the recent discovery of ap in the apicomplexa, the phylum to which malaria parasites belong, suggested that the apiap proteins were derived from bacteria or the apicoplast progenitor via transponsons, followed by lineage-specific radiation. these apiap proteins, in addition to regulating heterochromatin formation and genome integrity, may develop novel parasite-specific functions such as antigenic variation, invasion, and sporozoite development. p. falciparum possesses apiap members. among the apiap proteins predicted by our network alignments, five contain a single ap domain, four contain two ap domains, and two contain three ap domains. analyzing the protein-protein association data from the string database, in conjunction with the data from the y2h assays, temporal microarray experiments, proteomics, and literature, revealed that these apiap proteins are associated with 1- proteins in the cellular networks. at least four apiap proteins have ppis, suggesting that they play central role in transcriptional regulation. the apiap protein with highest connectivity is pfd0985w, which has interaction partners. it has direct physical interactions with two other apiap proteins. it is associated with a nucleosome assembly protein that is implicated in chromatin remodeling, and a putative ndc homolog that may be a component of the mitotic spindle related to chromosome segregation. it is also associated with three surface antigens including a reticulocyte binding protein homologue a which may play a role in determining host-cell invasion specificity, an antigen in the duffy binding-like protein family which may be related to parasite entry to the host, and an asparagine-rich antigen. this apiap protein pfd0985w also appeared to be related to a number of secreted proteins including a putative secreted ookinete protein, and two proteins that are associated with maurer's clefts, parasite-derived membranous structures within the host cell cytoplasm. in addition, pfd0985w has direct ppis with the 26s proteasome aaa-atpase subunit rpt, which is a component in ubiquitin-proteasome system for protein degradation, and pyruvate kinase, an essential enzyme for glycolysis. the apiap protein with second largest connectivity is pf07_ it has ppi partners that can be divided into five categories: transcriptional regulation. it is associated with two otherapiap proteins, and a ccaat-box dna binding protein subunit b; epigenetic regulation. it is associated with pfhmgb, which has a dna-binding domain: hmg-box. the proteins in this family have been implicated in regulation of transcription, replication, repair, and chromatin remodeling; signaling. pf07_ is associated with at least three putative signaling proteins, including pf13_ which contains a forkhead-associated domain that is found in a variety of regulatory proteins involved in signaling. a calcium/calmodulin-dependent protein kinase that is implicated in signaling cascades. a putative 14-3- protein. proteins in 14-3- family include regulatory ligands to various signaling molecules such as kinases and receptors; surface antigens for cell adhesion and entry to the host. pf07_ is associated with a duffy binding-like antigen, an erythrocyte membrane-associated antigen, and a qf antigen with an rna-binding motif; metabolism. the glycolytic enzyme fructose-bisphosphate aldolase is associated with the apiap protein pf07_ the role of apiap proteins in transcriptional and epigenetic regulation is also indicated by a direct ppi between a putative apiap with ap domains pf10_ and a histone acetyltransferase gcn, an enzyme for histone modification and chromatin remodeling. this apiap protein may also been involved in the regulation of genome integrity through a ppi with a dna repair protein rhp, and cytoskeleton organization of actin. two of these apiap proteins have been experimentally characterized to some extent: the crystal structure of the ap domain of pf14_ has been determined, revealing a multiple-site binding pattern, and gene disruption assays showed that its ortholog in the rodent parasite p. berghei was an indispensible regulator for sporozoite development in the mosquito stage. however, its regulatory roles and targets remain uncharacterized in p. falciparum. as shown in figure it has only two direct ppis revealed by y2h assays: the first is a ribosomal protein p and the second protein ptex is an important component in a translocon of exported proteins. located in the vacuole membrane, ptex was recently discovered as a novel atp-dependent protein trafficking machinery. notably, ptex is only present in the plasmodium genus. the ppi between ptex and apiap suggests that this export machinery may have parasite-specific regulation. ptex is becoming an attractive therapeutic target due to its importance to virulence and parasite survival and its distant evolutionary relatedness to the human host. pf11_ its counterpart in p. berghei is a transcription factor that regulates ookinete-specific gene expression for parasite invasion of the mosquito midgut. pf11_ however, may play a role in the red blood cell stage: it has one ppi partner, rhoptry-associated protein. rap is an escort protein required to localize rap to the rhoptries, apical organelles essential for rbc invasion. in summary, apiap proteins are a family of stage-specific transcriptional regulators for diverse processes ranging from epigenetic modification, chromosome organization and dynamics, invasion, protein sorting and trafficking, protein turnover, and metabolism. other potentially important proteins that may be involved in transcriptional regulation module based subnetwork alignments predicted additional proteins that are likely involved in transcriptional regulation. two proteins are members of the smc superfamily; they both have a recf/recn/smc n terminal domain and may be involved in chromatin cohesion and dynamics. a numbers of zinc-finger proteins were identified by network alignments as well. they exhibit different types of domain configurations, including the classical dna-binding motif c2h found in transcription factors, the c3hc type typically found in proteins mediating ubiquitination, the c-x8-c-x5-c-x3-h type implicated in cell cycle regulation, the dhhc type found in proteins important for membrane association and trafficking, the dnl type implicated in protein translocation into mitochondria, and the cw type related to dna-binding and protein-protein interaction. a putative transcriptional coactivator has a zz-type zinc finger domain. ada was shown, in baker's yeast and arabidopsis thaliana, to physically interact with gcn a histone acetyltransferase and a potent transcriptional activator. the y2h assay in p. falciparum revealed that ada has direct physical interactions with proteins including a minichromosome maintenance complex subunit, a pre-mrna splicing factor, a heat shock protein hsp interacting protein, a sodium-dependent phosphate transporter, a serine/threonine protein kinase in the fikk family, cathepsin c, and a mature parasite-infected erythrocyte surface antigen, suggesting its potential versatile roles in dna replication, splicing, transport, protein processing, signal transduction, and invasion. other putative transcriptional regulators include pfe and pf14_ pfe contains two functional domains: a fact complex subunit domain which was reported to facilitate transcriptional initiation and interact with nucleosomes and histones, and a histone chaperone rttp106-like domain. this protein may be involved in heterochromatin silencing and epigenetic regulation. pf14_ is a putative protein in the not global transcriptional regulator family. y2h assays showed that it had direct physical interactions with ccaat-box dna binding protein subunit b, dna topoisomerase ii, and calcium dependent protein kinase, emphasizing its involvement in general transcriptional control and chromosome topology and signaling processes. it also has a ppi with a pf11- protein, which may play a role in protein trafficking processes associated with maurer's cleft.  a functional-module based alignment approach was used to predict system components in transcriptional regulatory networks in malaria parasite p. falciparum. our results predicted general transcriptional regulators that may regulate gene expression in a global or pleiotropic mode. our results also imply a group of parasite-specific transcriptional regulators in the apiap family that play roles in diverse cellular processes ranging from chromatin remodeling, protein sorting and secretion, signal transduction, and invasion. finally, our analysis has identified other potentially important proteins involved in transcriptional regulation. our present knowledge of the transcriptional machinery and its regulatory capacity is rudimentary. the identification of network components in this machinery will open new avenues to the development of novel therapeutic targets and provide new insights into parasite biology, pathogenesis and virulence. the premise of our subnetwork alignment approach is that functional annotations of the proteins can be transferred across species through conserved interactions in the aligned ppi networks. under this framework, a priori information as to the identity or function of a gene is not necessary for the gene to be placed in a network. thus genes identified only because of their key role in a network become potential targets. furthermore, placement of the gene product in a systems context could, in itself, serve to identify the function of the gene product. if successfully applied, a systems biology approach circumvents the limiting factor in comparative genomics - the difficulty in obtaining reliable functional assignments. 
in this proof-of-concept study, we demonstrate that a subnetwork alignment approach can reveal previously uncharacterized members of the subnetworks, which opens new opportunities to identify potential therapeutic targets and provide new insights into parasite biology, pathogenesis and virulence. instead of only relying on sequence similarities to detect functional orthologs, our approach measures the conservation between the neighborhood subnetworks in protein-protein interaction networks in two species, p. falciparum and e. coli. malaria causes over one million deaths annually, posing an enormous health and economic burden in endemic regions. p. falciparum proteins were predicted as functional orthologs of known transcriptional regulators in the e. coli network, including general transcriptional regulators, parasite-specific transcriptional regulators in the apiap protein family, and other potential regulatory proteins. they are implicated in a variety of cellular processes involving chromatin remodeling, genome integrity, secretion, invasion, protein processing, and metabolism. in this paper, we propose an approach based on aligning neighborhood ppi subnetworks across species to identify network components in the malaria parasite p. falciparum. however, genomics-driven target discovery has been significantly hampered by our limited knowledge of the cellular networks associated with parasite development and pathogenesis. this approach can be extended to other systems, especially those with poor genome annotation and a paucity of knowledge about cellular networks.
 malaria causes over one million deaths annually, posing an enormous health and economic burden in endemic regions. the completion of genome sequencing of the causative agents, a group of parasites in the genus plasmodium, revealed potential drug and vaccine candidates. however, genomics-driven target discovery has been significantly hampered by our limited knowledge of the cellular networks associated with parasite development and pathogenesis. in this paper, we propose an approach based on aligning neighborhood ppi subnetworks across species to identify network components in the malaria parasite p. falciparum.  instead of only relying on sequence similarities to detect functional orthologs, our approach measures the conservation between the neighborhood subnetworks in protein-protein interaction networks in two species, p. falciparum and e. coli. p. falciparum proteins were predicted as functional orthologs of known transcriptional regulators in the e. coli network, including general transcriptional regulators, parasite-specific transcriptional regulators in the apiap protein family, and other potential regulatory proteins. they are implicated in a variety of cellular processes involving chromatin remodeling, genome integrity, secretion, invasion, protein processing, and metabolism.  in this proof-of-concept study, we demonstrate that a subnetwork alignment approach can reveal previously uncharacterized members of the subnetworks, which opens new opportunities to identify potential therapeutic targets and provide new insights into parasite biology, pathogenesis and virulence. this approach can be extended to other systems, especially those with poor genome annotation and a paucity of knowledge about cellular networks. 22- april the international conference on intelligent biology and medicine nashville, tn, usa 