BACKGROUND
amyloid precursor protein  is known as a pathological hallmark of alzheimer's disease .
nevertheless, app, a type i transmembrane glycoprotein consisting of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail, is expressed ubiquitously and given its receptor-like and adhesive characteristics may play important roles outside the nervous system.
in fact, we have previously showed that app is present in spermatozoa  <cit> .
the app superfamily includes app and app-like proteins   <dig> and  <dig>  alternative splicing of the app mrna produces eight isoforms, ranging in size from 677– <dig> amino acids  <cit> .
alternative splicing produces four aplp <dig> and two aplp <dig> protein isoforms.
although some isoforms may be cell type specific, app and aplp <dig> are ubiquitously expressed.
in contrast, aplp <dig> is expressed selectively in the nervous system  <cit> .
only app, but not aplp <dig> and  <dig>  contains a sequence encoding the beta-amyloid domain.
the transmembranar structure of app is consistent with a role of app as a receptor or a mediator of extracellular interactions.
it has been suggested that app may have cam  and sam  like activities  <cit> .
various lines of evidence implicate app and aplp <dig> in fertility.
app was shown to be expressed in rat testis and localized in the acrosome region and growing tail of spermatids in the seminiferous tubules  <cit> .
knock-out mice, homozygotes to either app or aplp <dig> were fertile, but mice with the deletion of both app and aplp <dig> were infertile   <cit> .
we previously characterized the subcellular distribution of the app superfamily members in spermatozoa using a variety of antibodies that either recognizes app-specific epitopes or the epitopes shared with other app family members  <cit> .
the presence of app superfamily members along the entire length of the tail may be related to signaling events involved in sperm motility, whereas their presence in the head and particularly in the equatorial region suggests their involvement in sperm-oocyte interaction  <cit> .
these results not only were consistent with the previous localization of aplp <dig> in mammalian sperm, but also prove the presence of app itself in human sperm.
app and aplps distribution only partially overlap suggesting that, besides a common role, they might also have distinct functions in spermatozoa.
a human sperm transmembrane protein initially termed ywk-ii  was shown to be involved in fertilization  <cit> .
the ywk-ii gene was shown to be expressed in germ cells at various stages of differentiation and in the plasma membrane enveloping the acrosome of mature spermatozoa  <cit> .
the discovery of tissue-specific interacting proteins can lead to the identification of pathways for the app family members associated with testis and sperm functions.
hence, we performed a yeast two-hybrid  screen of a human testis cdna library using app as bait.
a comprehensive bioinformatic analysis was also performed using the app interacting proteins identified in the yth in addition to the proteins selected from public protein-protein interactions  databases  and published app interactome data  <cit>  associated with testis/sperm.
aplp <dig> interacting partners were also included.
additionally, protein interaction maps were constructed allowing the visualization of ppi data as a connectivity graph and the data was subjected to a statistical analysis based on the complex network theory.
the advantage of this approach is that it allows the study not only of the local properties of proteins in the network, but also their global structural characteristics in the entire network of ppi.
we reveal that proteins with similar biological functions are tightly connected to each other and form dense groups  in the networks.
also, the function, cellular distribution and pathways were analyzed and significant features were classified.
RESULTS
in this study, we characterized the testis/spermatozoa interactome of app using a network-based approach.
identification of app interacting proteins in human testis by yeast two-hybrid screening
nowadays the yth methodology is a very robust technique to identify ppi  <cit> .
the method that we use has been highly improved and overcomes the initial problems of the yth, e.g.
the appearance of false positive or false negative interactions  <cit> , since, for instance, we use four reporter genes with different strength promoters  <cit> .
in order to identify app interacting proteins expressed in human testis, an yth screen of a human testis cdna library was carried out using full-length human app.
the screen yielded  <dig> positive clones from a total of 3× <dig> clones screened.
after partial or complete sequence analysis , in silico searches of the genbank db allowed their identification and classification into three separate groups.
table  <dig> corresponds to library inserts encoding known proteins identified as putative app interactors.
the second and third groups correspond to clones putatively encoding novel app interacting proteins with homology to genomic sequences and lists positives where the genbank sequence similarity did not correspond to an annotated gene and false positive hits, respectively.
table  <dig> lists only  <dig> positive encoding a previously identified app interacting protein  .
<dig> clones encoded  <dig> known proteins that were not previously associated with app .
only the clones in table  <dig> were included in the network and further functional analyses .table  <dig>
human testis cdna library inserts encoding known proteins identified as putative app interactors
genbank acession
uniprot id
gene symbol
protein name
chr
no.
of clonesexperimental methodology.
diagram for the number of proteins interacting with app from each dataset.
the numbers indicate testis/sperm-annotated proteins interacting with app from the different data sources; the numbers within parentheses represent the proteins enriched in testis and sperm.
self-connection for app was neglected.
analysis of the yth screen revealed that the most abundant interaction was detected with sec22c  .
this protein is involved in vesicle transport between the er and the golgi complex.
the  <dig> proteins identified as app interactors were classified into broad functional categories according to gene ontology annotation using the david bioinformatics resource .
regarding the biological process, the categories with the largest number of proteins were related to intracellular transport  and protein localization .
from the proteins involved in transport,  <dig> were linked with vesicle-mediated transport .
also,  <dig> proteins  were associated with cell adhesion.
cd <dig>  creb <dig> and fancm were annotated as being involved in reproduction.
the majority of app interactors identified in the yth  are intrinsic to membrane and  <dig> are specifically at the plasma membrane .
analysis of human proteomes  allowed the classification of dpep <dig> and tmprss <dig> as testicular proteins; itgb <dig> and cops <dig> as sperm-located testicular proteins also detected in epididymal fluid; and ftl as a non-sperm located epididymal fluid protein .
cd <dig>  cd <dig>  cops <dig> and fam <dig> were identified as testis/sperm-enriched in tissue-expression dbs  <cit> .
also, tmprss <dig> was reported in the unigene as a testicular/spermatozoa restricted protein.
to determine which proteins are known to be important for normal male reproductive function, the dataset was screened against the jackson laboratory mutant mouse db  <cit>  and phenopedia  <cit> .
from the app interactors identified in the yth screen,  <dig> were connected with reproductive phenotypes in gene knockout models .
from the comparison with the disease genes listed in phenopedia no results were obtained.
identification of literature curated interactions
in order to identify the potentially relevant interactors of app and aplp <dig> to male fertility, human ppi were collected from currently available public dbs, including apid  <cit> , biogrid  <cit> , dip  <cit> , hprd  <cit> , innatedb  <cit> , intact  <cit> , mint  <cit> , reactome  <cit> , topfind  <cit> , and string  <cit> .
only the interactions between both proteins associated with the terms “testis” and “sperm” in unigene, hprd  <cit>  and uniprot  <cit>  were selected.
then, the interactors characterized as highly specific to or strongly expressed in testis/sperm were identified from tissue-expression dbs .
.
besides the dbs, the tissue expression data was also retrieved from the published proteomes of reproductive tissues  <cit> .
this analysis allowed the classification of the app direct interactors into distinct but overlapped localizations .
first, we focused on local interactions of app/aplp <dig>  that is, the first direct interactors of app/aplp <dig> and interactions between them.
we identified  <dig> proteins connected to app  including the partners identified by yth .
all the proteins in the yth data were newly found as interactors of app except ranbp <dig>  which was previously published as an app interactor  <cit> .
the absence of protein overlapping may be due to the fact that the yth was performed using a library from human testis and the previous app interactors were mainly identified in neuronal tissues.
indeed, published data indicate that 4% of the mammalian genome  encodes genes specifically expressed in the male germ line during or after the completion of spermatogenesis  <cit> .
regarding aplp <dig>  we identified  <dig> proteins  as its interactors from the dbs which were highly specific to or strongly expressed in testis.
in total,  <dig>  interactions were identified between  <dig> proteins including app and aplp <dig>  only one protein  among the nearest neighbors of aplp <dig> was not directly connected to app which may reflect an isoform-specific role for aplp <dig> figure  <dig>
app protein-protein interaction networks.
local app/aplp <dig> network.
proteins with light orange  and dark orange  are mostly involved in g-protein coupled receptor protein signaling pathway  and located at the plasma membrane .
proteins with light green  and dark green  are mostly involved in regulation of apoptosis  and located at the cell surface .
subnetwork of app interactors involved in cell adhesion  extracted from the extended app/aplp <dig> protein-protein interaction network.
subnetwork of app interactors involved in vesicle-mediated transport  extracted from the extended network.
red colored nodes represent the proteins from the yth screen.
blue colored nodes indicate interactors extracted from the dbs.
node size represents relative degree of the nodes and the other interactions of nodes are neglected for the sake of simplicity.
dashed lines represent the interactions revealed by the yth screening in human testis and solid lines are interactions from dbs.
subnetwork of app interactors involved in g-protein-coupled singnal pathways.
second, we extended the local interaction network of app/aplp <dig> into the second nearest neighborhoods since the local network of app could limit an overview of the pathways in which this protein may be involved in testis and spermatozoa.
in this network, we had  <dig>  proteins and  <dig>  interactions between them .
topological analysis of app/aplp <dig> ppi network
the overall structural properties for the local and extended app/aplp <dig> network showed mostly linear relationship between degree  and betweenness centrality .
in our app/aplp <dig> local network, proteins with high connectivity also revealed high centrality which can be a significant indication of the relevant proteins in a biological network.
local app/aplp <dig> interaction network
in the testis/sperm related app/aplp <dig> network , most proteins were densely connected to each other.
average degree of this network was < q > =  <dig>  and global clustering coefficient was c =  <dig> .
the clustering coefficient reflects how neighbors of a node are connected to each other .
it is known that proteins with high connectivity  in ppi networks potentially have functional importance in biological systems and are likely to be critical proteins  <cit> .
the key proteins for disease are known to have low clustering coefficients in addition to high connectivities  <cit> .
in order to characterize the app network topology, the clustering coefficients of each protein were calculated.
betweenness centrality and closeness centrality of each protein in the app/aplp <dig> network were also measured to find the relevant proteins involved in pathways .
in biological networks, e.g.
signaling pathways and genetic interactions, the dysfunction of the proteins with high centrality may be crucial for the other biological functions due to missing of signal transference.
in yeast, the proteins with high betweenness centrality, but small number of degrees were found to be important links between well connected modules  <cit> .
proteins with high centrality rank in our network were represented in additional file 5: table s <dig> .
the top rated interactors included a calcium/phospholipid-binding protein which promotes membrane fusion and is involved in exocytosis .
pik3cg , plcb <dig> , lpar <dig> , rln <dig>  and adora <dig>  also composed the top rated proteins and were all related with the g-protein coupled receptor signaling pathway.
the proteins identified as app/aplp <dig> interactors were classified into functional categories according to gene ontology annotation using the david program  <cit> .
regarding the biological process, the results revealed that the categories with the largest number of proteins were related to cell surface receptor linked signal transduction  and g-protein coupled receptor protein signaling pathway .
<dig> % of the proteins were located at the plasma membrane  and  <dig> % in the extracellular region .
<dig> % were associated with vesicles .
these vesicle related proteins may participate in specialized vesicle activity in the testis, such as acrosome formation.
additionally,  <dig> % are annotated as part of a cell projection  .
metabolic pathways were analyzed using the kegg pathway  <cit> , which indicated that the top  <dig> significant categories were: neuroactive ligand-receptor interaction ; chemokine signaling pathway ; calcium signaling pathway ; and progesterone-mediated oocyte maturation .
in order to find the core and peripheral part of the local app/aplp <dig> network, k-core analysis was performed.
k-core is a subgraph of a graph in which all vertices have at least k-degree .
the core of this network has a connectivity, k =  <dig>  between them.
<dig> % of the proteins within this core shared the same biological process go category  and  <dig> % share the same subcellular localization .
four modules were identified by community detection analysis .
the nodes in a community are more tightly connected to each other than to nodes out of the community and may perform a common function.
our analysis shows that, in the local app/aplp <dig> network,  <dig> proteins are included in module  <dig> .
among them,  <dig> % were involved in the regulation of apoptosis  and the most significant represented cellular localization was the cell surface  was g-protein coupled receptor protein signaling pathway  and  <dig> % of the proteins in module  <dig> shared the same localization .
module  <dig> included  <dig> proteins , which were mainly involved in g-protein coupled receptor protein signaling pathway  and localized at the plasma membrane .
similarly to module  <dig>  the most significant category in module  <dig>  was regulation of apoptosis .
modules  <dig> and  <dig> also share a common biological function and cellular component.
in the local app/aplp <dig> network, the core part of the network, that is, the proteins with the highest k-core  which includes app, shared the same biological function .
the core part was included mostly in module  <dig>  which was also associated with the same function.
therefore, this result showed that app might be involved in g-protein coupled receptor protein signaling pathway in human testis/sperm.
in addition, app has high possibility that it is associated with regulation of apoptosis according to the results that the most interaction partners surrounding app share the same function within modular structure.
extended app/aplp <dig> network
the local app network only allows us to study relationships between app and its nearest neighboring proteins.
in order to study relationships with other proteins, we extended the network to the second nearest neighbors of app.
topological properties of the extended networks are analyzed in additional file 6: table s <dig>  from k-core analysis, a densely connected group with high k -core was found .
the proteins in the core were involved in cell cycle .
also, the majority of proteins were found in the cytosol  and the nucleoplasm .
based on the community structure analysis, app is located in module  <dig>  the most significant biological processes associated with this module were proteolysis  and cell adhesion .
additionally, the majority of proteins were located at the plasma membrane  and at the extracellular region .
table  <dig> represents the gene ontology analysis for the modules of the extended network in which at least 40% of proteins shared a biological function.table  <dig>
enriched go categories for each module of the extended network
m
most significant biological process
most significant cellular component
go term
p value
%
go term
p value
%
only modules with at least  <dig> % of the proteins sharing a biological function are represented.
m, modularity.
specific topological features of proteins from yth
based on the extended network structure analyses, cops <dig> has a large number of connections  and also a relatively high betweenness centrality  among our  <dig> yth proteins, contrasting with a low clustering coefficient .
cops <dig> is sperm-located testicular protein  <cit> .
cd <dig> , cd <dig>  and igtb <dig>  also revealed prominent topological properties .
topological role of app and aplp <dig> in the network
previous data has shown that the absence of both app and aplp <dig> lead to the abnormal developments of sexual organs, the reduction of synaptic vesicles, and even postnatal lethality in mice  <cit> .
on the other hand, the absence of either app or aplp <dig> does not affect viability and fertility.
based on these, one can imagine that these two proteins should co-exist for the mammalian life maintenance.
here, we focus on the role of app and aplp <dig> for the human male fertility.
based on experimental results of gene knock-outs in mice  <cit> , one can assume that app and aplp <dig> are simultaneously involved in important pathways.
some proteins or protein complexes cannot accomplish biological functions in the absence of app/aplp <dig>  because this blocks the functional routes between the proteins.
in order to find a conformity of the functional property within a structural property, we checked the local triangle structure between app, aplp <dig>  and the common interactors .
among the interactors of aplp <dig>  only brca <dig> is not connected to app.
these common interactors of app and aplp <dig> form a small modular structure .
therefore, one can guess the proteins in this module possibly share a biological function in testis.
discussion
biomolecular networks are now frameworks that facilitate many discoveries in molecular biology.
the theoretical advances in network science in parallel with high throughput efforts to map biological networks, offer an excellent opportunity to apply the principles of theoretical physics to the molecular biomedicine field.
the app network in testis/sperm was built first using an yth screen and then expanded by incorporating literature curated interactions.
since protein profiles of the different tissues are critical to understand the unique characteristics of the various human cell types, in this study, we took into account the tissue expression of the interactors in the network.
from the yth screen, we reported the identification of  <dig> novel app interacting proteins in human testis/sperm.
only  <dig> positive encoded a previously identified app interacting protein .
this may be explained by the fact of testis being a very peculiar organ which possesses specific patterns of transcription and expresses novel protein isoforms  <cit> .
aplp <dig> interacting partners were also included in this study.
to determine which ppi in our app/aplp <dig> network were biologically more relevant for male reproduction, we performed network structure analyses and bioinformatic analyses.
based on the community/modularity analysis of the ppi network along with gene ontology analysis, we confirmed that proteins involved in similar functions are group together and form modules.
the biological process go category more significantly represented in the app/aplp <dig> local network in human testis/sperm was cell surface receptor linked signal transduction with  <dig> % of the proteins annotated in this class.
these proteins may indicate how the male germ cells interact with the outside world.
among those proteins,  <dig> % carry the go functional tag for g-protein coupled receptor protein signaling pathway.
some studies indicate that full length app can function as a cell surface gpcr and show that app binds heterotrimeric g proteins   <cit> .
recently, deyts and colleagues discovered an interaction between app intracellular domain and the heterotrimeric g-protein subunit gαs  <cit> .
g protein-coupled receptors signalling pathways have been proposed to control several processes essential for sperm function and fertilization, namely in sperm capacitation and acrosome reaction  <cit> .
aplp2/ywk-ii also exhibits properties of a receptor and its extracellular domain was shown to interact with müllerian-inhibiting substance  <cit> .
müllerian-inhibiting substance increases the viability and longevity of human spermatozoa through binding the aplp2/ywk-ii component on the sperm membrane  <cit> .
huang and colleagues showed that aplp2/ywk-ii component binds to a gtp-binding protein .
the most abundant interaction detected in the yth was with sec22c.
this protein is involved in vesicle transport between the er and the golgi complex  <cit> .
vesicular membrane trafficking is an essential process during acrosome biogenesis  <cit> .
also, sec22c may control the app traffic through the secretory pathway.
besides sec22c, other four yth clones  are involved in vesicle-mediated transport .
this go term includes the regulation of the acrosomal vesicle exocytosis, an essential process for fertilization, which begins with the fusion of the outer acrosomal membrane with the sperm plasma membrane and ends with the exocytosis of the acrosomal contents into the oocyte.
the cellular component category most enriched in the go term analysis of the app/aplp local network was the plasma membrane.
fertilization is achieved through gamete interactions, specifically cell adhesion and then membrane fusion of the gamete plasma membranes.
the occurrence of  <dig> % of proteins in the plasma membrane may suggest their involvement in sperm-egg interaction.
additionally,  <dig> % of app interactors are involved in cell adhesion .
of these, cd <dig>  gpnmb, itgb <dig>  lypd <dig>  and sspn were identified in the yth screen performed using a testis library.
the app yeast mating efficiency in the yth was much higher than usual , which may be related to app cell adhesion properties.
this strengthens previous results suggesting app to be involved in cell-to-cell contact, a very important process in gamete fusion.
recent approaches to identify candidate proteins involved in sperm-egg interaction have been characterizing the sperm proteome and analyzing specific subpopulations of interest, for instance, glycoslylated proteins and integrins.
additionally, proteins with motifs or belonging to families of interest like transmembrane domains and the tetrasparin family should also be considered.
interestingly, some of the yth positive clones are included in those categories.
app interacts with itgb <dig>  identified in the yth screen, and itgb <dig>  <cit> , both belonging to the integrin beta chain family.
integrins on eggs became of interest with the discovery of an integrin ligand-like domain in adam <dig>  a sperm antigen essential for sperm-egg interaction.tspan <dig> and cd <dig> belong to the tetraspanin family.
the discovery that the knockout of cd <dig>  a member of the tetraspanin family, in mouse leads to healthy, but subfertile females due to defective sperm-egg interaction revolutionized the fertility field.
cd <dig> is 45% identical to cd <dig> and cd <dig> knockout mouse also presents defects in female fertility.
cd9−/−/cd81−/− female mice are completely infertile.
we found that, in local network, app, tspan <dig>  itgb <dig>  itgb <dig>  gpnmb, lypd <dig>  sspn, cd <dig> and cd <dig> were in the same module .
however, in extended network, app, tspan <dig>  gpnmb, lypd <dig>  sspn, and itgb <dig> were in module  <dig>  whereas itgb <dig>  cd <dig>  cd <dig> and cd <dig> were well connected in module  <dig> in which  <dig> % of the proteins share the biological function – cell adhesion .
we also identified tissue-specific app interacting proteins which can lead to the identification of pathways for the app family members associated with testis and sperm functions.
tmprss <dig>  a transmembrane serine protease, was identified in the yth screen and was reported in the unigene as testicular/spermatozoa restricted.
tmprss <dig> belongs to the same module from network community as app.
since this protein is connected to app only, it cannot have any route to the main network without app.
sperm-surface proteases were already shown to be required for fertilization  <cit> .
there is also evidence for the participation of serine proteolytic activities during spermatogenesis and sperm maturation  <cit> .
however, most of the specific proteases that are involved in these processes are unknown.
the exact localization of tmprss <dig> at sperm membrane has to be determined.
CONCLUSIONS
the present work provided the first report on app interactome in human testis.
we identified several novel app interactions in human testis and incorporated yth data and ppi databases to construct the ppi network of app in human testis and spermatozoa.
the protein interaction network allowed the recognition of proteins complexes and modules crucial for several biological functions, such as cell adhesion.

 amyloid precursor protein is known as a pathological hallmark of alzheimer's disease. nevertheless, app, a type i transmembrane glycoprotein consisting of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail, is expressed ubiquitously and given its receptor-like and adhesive characteristics may play important roles outside the nervous system. in fact, we have previously showed that app is present in spermatozoa. the app superfamily includes app and app-like proteins and alternative splicing of the app mrna produces eight isoforms, ranging in size from 677– amino acids. alternative splicing produces four aplp and two aplp protein isoforms. although some isoforms may be cell type specific, app and aplp are ubiquitously expressed. in contrast, aplp is expressed selectively in the nervous system. only app, but not aplp and contains a sequence encoding the beta-amyloid domain. the transmembranar structure of app is consistent with a role of app as a receptor or a mediator of extracellular interactions. it has been suggested that app may have cam and sam like activities. various lines of evidence implicate app and aplp in fertility. app was shown to be expressed in rat testis and localized in the acrosome region and growing tail of spermatids in the seminiferous tubules. knock-out mice, homozygotes to either app or aplp were fertile, but mice with the deletion of both app and aplp were infertile. we previously characterized the subcellular distribution of the app superfamily members in spermatozoa using a variety of antibodies that either recognizes app-specific epitopes or the epitopes shared with other app family members. the presence of app superfamily members along the entire length of the tail may be related to signaling events involved in sperm motility, whereas their presence in the head and particularly in the equatorial region suggests their involvement in sperm-oocyte interaction. these results not only were consistent with the previous localization of aplp in mammalian sperm, but also prove the presence of app itself in human sperm. app and aplps distribution only partially overlap suggesting that, besides a common role, they might also have distinct functions in spermatozoa. a human sperm transmembrane protein initially termed ywk-ii was shown to be involved in fertilization. the ywk-ii gene was shown to be expressed in germ cells at various stages of differentiation and in the plasma membrane enveloping the acrosome of mature spermatozoa. the discovery of tissue-specific interacting proteins can lead to the identification of pathways for the app family members associated with testis and sperm functions. hence, we performed a yeast two-hybrid screen of a human testis cdna library using app as bait. a comprehensive bioinformatic analysis was also performed using the app interacting proteins identified in the yth in addition to the proteins selected from public protein-protein interactions databases and published app interactome data associated with testis/sperm. aplp interacting partners were also included. additionally, protein interaction maps were constructed allowing the visualization of ppi data as a connectivity graph and the data was subjected to a statistical analysis based on the complex network theory. the advantage of this approach is that it allows the study not only of the local properties of proteins in the network, but also their global structural characteristics in the entire network of ppi. we reveal that proteins with similar biological functions are tightly connected to each other and form dense groups in the networks. also, the function, cellular distribution and pathways were analyzed and significant features were classified.  in this study, we characterized the testis/spermatozoa interactome of app using a network-based approach. identification of app interacting proteins in human testis by yeast two-hybrid screening nowadays the yth methodology is a very robust technique to identify ppi. the method that we use has been highly improved and overcomes the initial problems of the yth, e.g. the appearance of false positive or false negative interactions, since, for instance, we use four reporter genes with different strength promoters. in order to identify app interacting proteins expressed in human testis, an yth screen of a human testis cdna library was carried out using full-length human app. the screen yielded positive clones from a total of 3× clones screened. after partial or complete sequence analysis, in silico searches of the genbank db allowed their identification and classification into three separate groups. table  corresponds to library inserts encoding known proteins identified as putative app interactors. the second and third groups correspond to clones putatively encoding novel app interacting proteins with homology to genomic sequences and lists positives where the genbank sequence similarity did not correspond to an annotated gene and false positive hits, respectively. table  lists only positive encoding a previously identified app interacting protein. clones encoded known proteins that were not previously associated with app. only the clones in table  were included in the network and further functional analyses.table human testis cdna library inserts encoding known proteins identified as putative app interactors genbank acession uniprot id gene symbol protein name chr no. of clonesexperimental methodology. diagram for the number of proteins interacting with app from each dataset. the numbers indicate testis/sperm-annotated proteins interacting with app from the different data sources; the numbers within parentheses represent the proteins enriched in testis and sperm. self-connection for app was neglected. analysis of the yth screen revealed that the most abundant interaction was detected with sec22c. this protein is involved in vesicle transport between the er and the golgi complex. the proteins identified as app interactors were classified into broad functional categories according to gene ontology annotation using the david bioinformatics resource. regarding the biological process, the categories with the largest number of proteins were related to intracellular transport and protein localization. from the proteins involved in transport, were linked with vesicle-mediated transport. also, proteins were associated with cell adhesion. cd creb and fancm were annotated as being involved in reproduction. the majority of app interactors identified in the yth are intrinsic to membrane and are specifically at the plasma membrane. analysis of human proteomes allowed the classification of dpep and tmprss as testicular proteins; itgb and cops as sperm-located testicular proteins also detected in epididymal fluid; and ftl as a non-sperm located epididymal fluid protein. cd cd cops and fam were identified as testis/sperm-enriched in tissue-expression dbs. also, tmprss was reported in the unigene as a testicular/spermatozoa restricted protein. to determine which proteins are known to be important for normal male reproductive function, the dataset was screened against the jackson laboratory mutant mouse db and phenopedia. from the app interactors identified in the yth screen, were connected with reproductive phenotypes in gene knockout models. from the comparison with the disease genes listed in phenopedia no results were obtained. identification of literature curated interactions in order to identify the potentially relevant interactors of app and aplp to male fertility, human ppi were collected from currently available public dbs, including apid, biogrid, dip, hprd, innatedb, intact, mint, reactome, topfind, and string. only the interactions between both proteins associated with the terms “testis” and “sperm” in unigene, hprd and uniprot were selected. then, the interactors characterized as highly specific to or strongly expressed in testis/sperm were identified from tissue-expression dbs.. besides the dbs, the tissue expression data was also retrieved from the published proteomes of reproductive tissues. this analysis allowed the classification of the app direct interactors into distinct but overlapped localizations. first, we focused on local interactions of app/aplp that is, the first direct interactors of app/aplp and interactions between them. we identified proteins connected to app including the partners identified by yth. all the proteins in the yth data were newly found as interactors of app except ranbp which was previously published as an app interactor. the absence of protein overlapping may be due to the fact that the yth was performed using a library from human testis and the previous app interactors were mainly identified in neuronal tissues. indeed, published data indicate that 4% of the mammalian genome encodes genes specifically expressed in the male germ line during or after the completion of spermatogenesis. regarding aplp we identified proteins as its interactors from the dbs which were highly specific to or strongly expressed in testis. in total, interactions were identified between proteins including app and aplp only one protein among the nearest neighbors of aplp was not directly connected to app which may reflect an isoform-specific role for aplp figure app protein-protein interaction networks. local app/aplp network. proteins with light orange and dark orange are mostly involved in g-protein coupled receptor protein signaling pathway and located at the plasma membrane. proteins with light green and dark green are mostly involved in regulation of apoptosis and located at the cell surface. subnetwork of app interactors involved in cell adhesion extracted from the extended app/aplp protein-protein interaction network. subnetwork of app interactors involved in vesicle-mediated transport extracted from the extended network. red colored nodes represent the proteins from the yth screen. blue colored nodes indicate interactors extracted from the dbs. node size represents relative degree of the nodes and the other interactions of nodes are neglected for the sake of simplicity. dashed lines represent the interactions revealed by the yth screening in human testis and solid lines are interactions from dbs. subnetwork of app interactors involved in g-protein-coupled singnal pathways. second, we extended the local interaction network of app/aplp into the second nearest neighborhoods since the local network of app could limit an overview of the pathways in which this protein may be involved in testis and spermatozoa. in this network, we had proteins and interactions between them. topological analysis of app/aplp ppi network the overall structural properties for the local and extended app/aplp network showed mostly linear relationship between degree and betweenness centrality. in our app/aplp local network, proteins with high connectivity also revealed high centrality which can be a significant indication of the relevant proteins in a biological network. local app/aplp interaction network in the testis/sperm related app/aplp network, most proteins were densely connected to each other. average degree of this network was < q > = and global clustering coefficient was c = . the clustering coefficient reflects how neighbors of a node are connected to each other. it is known that proteins with high connectivity in ppi networks potentially have functional importance in biological systems and are likely to be critical proteins. the key proteins for disease are known to have low clustering coefficients in addition to high connectivities. in order to characterize the app network topology, the clustering coefficients of each protein were calculated. betweenness centrality and closeness centrality of each protein in the app/aplp network were also measured to find the relevant proteins involved in pathways. in biological networks, e.g. signaling pathways and genetic interactions, the dysfunction of the proteins with high centrality may be crucial for the other biological functions due to missing of signal transference. in yeast, the proteins with high betweenness centrality, but small number of degrees were found to be important links between well connected modules. proteins with high centrality rank in our network were represented in additional file 5: table s. the top rated interactors included a calcium/phospholipid-binding protein which promotes membrane fusion and is involved in exocytosis. pik3cg, plcb, lpar, rln and adora also composed the top rated proteins and were all related with the g-protein coupled receptor signaling pathway. the proteins identified as app/aplp interactors were classified into functional categories according to gene ontology annotation using the david program. regarding the biological process, the results revealed that the categories with the largest number of proteins were related to cell surface receptor linked signal transduction and g-protein coupled receptor protein signaling pathway. % of the proteins were located at the plasma membrane and % in the extracellular region. % were associated with vesicles. these vesicle related proteins may participate in specialized vesicle activity in the testis, such as acrosome formation. additionally, % are annotated as part of a cell projection. metabolic pathways were analyzed using the kegg pathway, which indicated that the top significant categories were: neuroactive ligand-receptor interaction; chemokine signaling pathway; calcium signaling pathway; and progesterone-mediated oocyte maturation. in order to find the core and peripheral part of the local app/aplp network, k-core analysis was performed. k-core is a subgraph of a graph in which all vertices have at least k-degree. the core of this network has a connectivity, k =  between them. % of the proteins within this core shared the same biological process go category and % share the same subcellular localization. four modules were identified by community detection analysis. the nodes in a community are more tightly connected to each other than to nodes out of the community and may perform a common function. our analysis shows that, in the local app/aplp network, proteins are included in module. among them, % were involved in the regulation of apoptosis and the most significant represented cellular localization was the cell surface was g-protein coupled receptor protein signaling pathway and % of the proteins in module shared the same localization. module included proteins, which were mainly involved in g-protein coupled receptor protein signaling pathway and localized at the plasma membrane. similarly to module the most significant category in module was regulation of apoptosis. modules and also share a common biological function and cellular component. in the local app/aplp network, the core part of the network, that is, the proteins with the highest k-core which includes app, shared the same biological function. the core part was included mostly in module which was also associated with the same function. therefore, this result showed that app might be involved in g-protein coupled receptor protein signaling pathway in human testis/sperm. in addition, app has high possibility that it is associated with regulation of apoptosis according to the results that the most interaction partners surrounding app share the same function within modular structure. extended app/aplp network the local app network only allows us to study relationships between app and its nearest neighboring proteins. in order to study relationships with other proteins, we extended the network to the second nearest neighbors of app. topological properties of the extended networks are analyzed in additional file 6: table s from k-core analysis, a densely connected group with high k -core was found. the proteins in the core were involved in cell cycle. also, the majority of proteins were found in the cytosol and the nucleoplasm. based on the community structure analysis, app is located in module the most significant biological processes associated with this module were proteolysis and cell adhesion. additionally, the majority of proteins were located at the plasma membrane and at the extracellular region. table  represents the gene ontology analysis for the modules of the extended network in which at least 40% of proteins shared a biological function.table enriched go categories for each module of the extended network m most significant biological process most significant cellular component go term p value % go term p value % only modules with at least % of the proteins sharing a biological function are represented. m, modularity. specific topological features of proteins from yth based on the extended network structure analyses, cops has a large number of connections and also a relatively high betweenness centrality among our yth proteins, contrasting with a low clustering coefficient. cops is sperm-located testicular protein. cd, cd and igtb also revealed prominent topological properties. topological role of app and aplp in the network previous data has shown that the absence of both app and aplp lead to the abnormal developments of sexual organs, the reduction of synaptic vesicles, and even postnatal lethality in mice. on the other hand, the absence of either app or aplp does not affect viability and fertility. based on these, one can imagine that these two proteins should co-exist for the mammalian life maintenance. here, we focus on the role of app and aplp for the human male fertility. based on experimental results of gene knock-outs in mice, one can assume that app and aplp are simultaneously involved in important pathways. some proteins or protein complexes cannot accomplish biological functions in the absence of app/aplp because this blocks the functional routes between the proteins. in order to find a conformity of the functional property within a structural property, we checked the local triangle structure between app, aplp and the common interactors. among the interactors of aplp only brca is not connected to app. these common interactors of app and aplp form a small modular structure. therefore, one can guess the proteins in this module possibly share a biological function in testis. discussion biomolecular networks are now frameworks that facilitate many discoveries in molecular biology. the theoretical advances in network science in parallel with high throughput efforts to map biological networks, offer an excellent opportunity to apply the principles of theoretical physics to the molecular biomedicine field. the app network in testis/sperm was built first using an yth screen and then expanded by incorporating literature curated interactions. since protein profiles of the different tissues are critical to understand the unique characteristics of the various human cell types, in this study, we took into account the tissue expression of the interactors in the network. from the yth screen, we reported the identification of novel app interacting proteins in human testis/sperm. only positive encoded a previously identified app interacting protein. this may be explained by the fact of testis being a very peculiar organ which possesses specific patterns of transcription and expresses novel protein isoforms. aplp interacting partners were also included in this study. to determine which ppi in our app/aplp network were biologically more relevant for male reproduction, we performed network structure analyses and bioinformatic analyses. based on the community/modularity analysis of the ppi network along with gene ontology analysis, we confirmed that proteins involved in similar functions are group together and form modules. the biological process go category more significantly represented in the app/aplp local network in human testis/sperm was cell surface receptor linked signal transduction with % of the proteins annotated in this class. these proteins may indicate how the male germ cells interact with the outside world. among those proteins, % carry the go functional tag for g-protein coupled receptor protein signaling pathway. some studies indicate that full length app can function as a cell surface gpcr and show that app binds heterotrimeric g proteins. recently, deyts and colleagues discovered an interaction between app intracellular domain and the heterotrimeric g-protein subunit gαs. g protein-coupled receptors signalling pathways have been proposed to control several processes essential for sperm function and fertilization, namely in sperm capacitation and acrosome reaction. aplp2/ywk-ii also exhibits properties of a receptor and its extracellular domain was shown to interact with müllerian-inhibiting substance. müllerian-inhibiting substance increases the viability and longevity of human spermatozoa through binding the aplp2/ywk-ii component on the sperm membrane. huang and colleagues showed that aplp2/ywk-ii component binds to a gtp-binding protein. the most abundant interaction detected in the yth was with sec22c. this protein is involved in vesicle transport between the er and the golgi complex. vesicular membrane trafficking is an essential process during acrosome biogenesis. also, sec22c may control the app traffic through the secretory pathway. besides sec22c, other four yth clones are involved in vesicle-mediated transport. this go term includes the regulation of the acrosomal vesicle exocytosis, an essential process for fertilization, which begins with the fusion of the outer acrosomal membrane with the sperm plasma membrane and ends with the exocytosis of the acrosomal contents into the oocyte. the cellular component category most enriched in the go term analysis of the app/aplp local network was the plasma membrane. fertilization is achieved through gamete interactions, specifically cell adhesion and then membrane fusion of the gamete plasma membranes. the occurrence of % of proteins in the plasma membrane may suggest their involvement in sperm-egg interaction. additionally, % of app interactors are involved in cell adhesion. of these, cd gpnmb, itgb lypd and sspn were identified in the yth screen performed using a testis library. the app yeast mating efficiency in the yth was much higher than usual, which may be related to app cell adhesion properties. this strengthens previous results suggesting app to be involved in cell-to-cell contact, a very important process in gamete fusion. recent approaches to identify candidate proteins involved in sperm-egg interaction have been characterizing the sperm proteome and analyzing specific subpopulations of interest, for instance, glycoslylated proteins and integrins. additionally, proteins with motifs or belonging to families of interest like transmembrane domains and the tetrasparin family should also be considered. interestingly, some of the yth positive clones are included in those categories. app interacts with itgb identified in the yth screen, and itgb, both belonging to the integrin beta chain family. integrins on eggs became of interest with the discovery of an integrin ligand-like domain in adam a sperm antigen essential for sperm-egg interaction.tspan and cd belong to the tetraspanin family. the discovery that the knockout of cd a member of the tetraspanin family, in mouse leads to healthy, but subfertile females due to defective sperm-egg interaction revolutionized the fertility field. cd is 45% identical to cd and cd knockout mouse also presents defects in female fertility. cd9−/−/cd81−/− female mice are completely infertile. we found that, in local network, app, tspan itgb itgb gpnmb, lypd sspn, cd and cd were in the same module. however, in extended network, app, tspan gpnmb, lypd sspn, and itgb were in module whereas itgb cd cd and cd were well connected in module in which % of the proteins share the biological function – cell adhesion. we also identified tissue-specific app interacting proteins which can lead to the identification of pathways for the app family members associated with testis and sperm functions. tmprss a transmembrane serine protease, was identified in the yth screen and was reported in the unigene as testicular/spermatozoa restricted. tmprss belongs to the same module from network community as app. since this protein is connected to app only, it cannot have any route to the main network without app. sperm-surface proteases were already shown to be required for fertilization. there is also evidence for the participation of serine proteolytic activities during spermatogenesis and sperm maturation. however, most of the specific proteases that are involved in these processes are unknown. the exact localization of tmprss at sperm membrane has to be determined.  the present work provided the first report on app interactome in human testis. we identified several novel app interactions in human testis and incorporated yth data and ppi databases to construct the ppi network of app in human testis and spermatozoa. the protein interaction network allowed the recognition of proteins complexes and modules crucial for several biological functions, such as cell adhesion. 
the present work provides the first report on the app interactome in human testis. app is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. we classified significant features related to the human male reproduction for the app interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility. amyloid precursor protein is widely recognized for playing a central role in alzheimer's disease pathogenesis. amyloid precursor proteinhuman testismale reproductionprotein-protein interaction networkyeast two-hybridissue-copyright-statement© the author 2015 knowledge on the protein networks of app in human testis and spermatozoa will shed light on the function of app in the male reproductive system. we performed a yeast two-hybrid screen and a database search to study the interaction network of app in human testis and sperm. our approach allowed the identification of novel interactions and recognition of key app interacting proteins for male reproduction, particularly in sperm-oocyte interaction. we analyzed several topological properties of the app interaction network and the biological and physiological properties of the proteins in the app interaction network were also specified by gene ontologyand pathways analyses.
 amyloid precursor protein is widely recognized for playing a central role in alzheimer's disease pathogenesis. although app is expressed in several tissues outside the human central nervous system, the functions of app and its family members in other tissues are still poorly understood. app is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. furthermore, app superfamily members are known to be associated with fertility. knowledge on the protein networks of app in human testis and spermatozoa will shed light on the function of app in the male reproductive system.  we performed a yeast two-hybrid screen and a database search to study the interaction network of app in human testis and sperm. to gain insights into the role of app superfamily members in fertility, the study was extended to app-like protein. we analyzed several topological properties of the app interaction network and the biological and physiological properties of the proteins in the app interaction network were also specified by gene ontologyand pathways analyses. we classified significant features related to the human male reproduction for the app interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility.  the present work provides the first report on the app interactome in human testis. our approach allowed the identification of novel interactions and recognition of key app interacting proteins for male reproduction, particularly in sperm-oocyte interaction. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords amyloid precursor proteinhuman testismale reproductionprotein-protein interaction networkyeast two-hybridissue-copyright-statement© the author 2015 