Path2PPI - Tutorial, example and the algorithm
Introduction
Prediction of protein-protein interaction (PPI) networks is an
important approach to gain knowledge about protein interactions in model
organisms where only a small number of PPI information is available.
Current PPI databases, providing predicted interaction data, lack many
organisms or contain less reproducible information about the predicted
interactions. Currently available prediction approaches are mainly based
on biological data (functional annotation, co-expression etc.) which
often are not available for many “less established” organisms, for
example, where only sequence data is available. In addition, it is of
major interest to get knowledge about a certain pathway in such a
“less-studied” organism. To overcome these drawbacks
Path2PPI can be used to predict proteins and
interactions of a certain pathway of interest in a target
organism by using and combining the PPIs of other well established model
organisms.
To do so, it needs a list of proteins of interest from each reference species and the result files produced by the local NCBI BLAST (Camacho et al., 2009) tool (see next chapter). The relevant interactions based on the users’ protein lists are automatically extracted from the corresponding iRefIndex files (Razick et al., 2008).
Preparation of the data
In this tutorial, we make use of the test data set provided with the package. This data set consists of all data files necessary to predict the interactions of the induction step of autophagy in Podospora anserina by means of the corresponding PPIs in human and yeast. Hence, we first load the “autophagy induction” test data set:
## [1] "human.ai.irefindex" "human.ai.proteins" "pa2human.ai.homologs"
## [4] "pa2yeast.ai.homologs" "yeast.ai.irefindex" "yeast.ai.proteins"As stated by ls() the test data set contains six data
objects (three for each of the two reference species human and yeast).
First, the algorithm requires a list of proteins which define the
corresponding pathway for each reference species, defined in
“human.ai.proteins” and “yeast.ai.proteins” (see
section 2.1). Second, the algorithm requires the data frames which
contain the interactions of each reference species defined in
“human.ai.irefindex” and “yeast.ai.irefindex”
(described in more detail in section 2.1). Third, the algorithm needs to
know the homologous relations between the target species with each
reference species. These relations are defined in the data frames
“pa2human.ai.homologs” and “pa2yeast.ai.homologs” (we
describe this in more detail in section 2.2).
If you want to use Path2PPI for your own demands, you have to generate and prepare the necessary data files.
Proteins and interactions of pathways of interest
We list the proteins which are associated with a specific pathway of interest in a character vector for each reference species. To give you an example for such lists, we take a brief look into the loaded data set. Among others, we found the two named character vectors “human.ai.proteins” and “yeast.ai.proteins” which consist of the corresponding proteins for yeast and human, our two reference species:
## P42345 O75385 Q8IYT8 Q6PHR2 O75143
## "MTOR" "ULK1" "ULK2" "ULK3" "ATG13"## P35169 P32600 P53104 Q06410 Q12527 Q06628 P39968
## "TOR1" "TOR2" "ATG1" "ATG17" "ATG11" "ATG13" "VAC8"In this example, the values are the trivial names of the proteins and the names are the actual protein identifiers. Path2PPI also accepts simple character vectors where the values are the protein identifiers, if the trivial names of the proteins are not available. For example, this simple character vector, only consisting of the protein identifiers, would be also a valid protein list:
## [1] "P42345" "O75385" "Q8IYT8" "Q6PHR2" "O75143"The major advantage of using a named character vector with the trivial names, is that these names will be shown in the plots allowing for a more comfortable interpretation. You can use various accession formats for the protein identifiers which are supported by iRefIndex (e.g. UniProt, SwissProt, Ensembl). However, we urgently recommend to use UniProt identifiers, since those are the most established ones.
Use the default R functions to load your own protein lists of
interest into R (e.g. read.table).
These proteins of interest are applied to find relevant interactions in the corresponding species iRefIndex file. iRefIndex tables are available for the seven most established model organisms and can be found here: http://irefindex.org/wiki/index.php?title=iRefIndex. You can also use the corresponding iRefR-package to directly archive the iRefIndex data frames from this page. Unfortunately, the package is not updated as frequently as the web page and it may be that you do not get the latest release of a corresponding file.
In the “autophagy induction” test data set, only a very small part of the iRefIndex files for yeast and human are provided which contain the relevant interactions necessary for this tutorial. The complete files are much larger. The data frames “human.ai.irefindex” and “yeast.ai.irefindex” in the test data set contain these corresponding iRefIndex parts. See:
Get homology files using NCBI BLAST+
You also need the result files produced by the BLAST+ toolkit provided by the NCBI web page: http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download. The test data set already includes the necessary results of the BLAST searches of the proteoms of P. anserina against the proteoms of yeast and human (“pa2human.ai.homologs” and “pa2yeast.ai.homologs”):
## V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12
## 534 B2AX00 P53104 25.66 304 165 12 15 268 21 313 1e-15 79.0
## 960 B2AXW7 P53104 28.52 291 154 10 510 751 30 315 7e-24 106.0
## 1278 B2AUR5 P35169 26.74 288 154 10 602 837 2078 2360 2e-11 65.9
## 1279 B2AUR5 P32600 25.09 275 152 9 610 835 2090 2359 1e-09 60.5
## 1555 B2B7B1 P53104 25.99 177 99 7 807 963 149 313 5e-09 59.3
## 2469 B2ASL9 P53104 26.42 352 171 13 20 314 24 344 2e-19 87.8## V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12
## 2123 B2AX00 Q6PHR2 33.09 269 146 15 11 268 13 258 5e-24 106.0
## 2177 B2AX00 O75385 30.30 231 131 9 24 239 22 237 3e-22 103.0
## 2213 B2AX00 Q8IYT8 29.24 236 142 8 24 247 15 237 4e-21 99.4
## 4588 B2AXW7 Q6PHR2 32.96 267 159 12 499 754 6 263 9e-30 125.0
## 4649 B2AXW7 Q8IYT8 29.17 216 136 6 509 714 14 222 2e-23 107.0
## 4658 B2AXW7 O75385 29.95 217 133 9 509 714 21 229 5e-23 106.0The second column (V2) contains the protein identifiers of the corresponding reference species to which the protein of the target species (here: P. anserina) in the first column (V1) is homologous. Keep in mind that these protein identifiers are equal to those we used in the protein lists described above.
If you are unfamiliar with this toolkit, we refer to the BLAST+ user manual or to the broadly available tutorials in the web.
Nevertheless, we want to give you a very short description on how to use this toolkit. We assume that you already have loaded and installed the NCBI BLAST+ toolkit and you also have each proteome file in FASTA format of each species (target and reference species). You first have to create the databases for each reference species, here, for human and yeast:
makeblastdb -in human.fa -input_type fasta -dbtype prot -out human_proteins
-title human_proteins
makeblastdb -in yeast.fa -input_type fasta -dbtype prot -out yeast_proteins
-title yeast_proteinsSubsequently, you can start the comprehensive BLAST searches using the FASTA file of your target species (here: P. anserina):
blastp -query panserina.fa -db human_proteins -out human_panserina.out -evalue
0.0001 -outfmt 6
blastp -query panserina.fa -db yeast_proteins -out yeast_panserina.out -evalue
0.0001 -outfmt 6Please, make sure that you use as the output format the tab delimited
list indicated by the parameter -outfmt 6. The two
species-specific homology files which are now generated, can be imported
into your R session, using the function read.table, and
subsequently used as data frames for the
Path2PPI-package.
Predict PPI in target species
After the necessary data sets are generated or loaded, respectively, we can start with the prediction.
The Path2PPI object
An object of the class Path2PPI represents the major instance which is responsible for storing and managing of each data set and for each computation and prediction step. Hence, we first have to create a new instance of the class Path2PPI with the corresponding information:
The arguments are the title of the pathway we want to predict, the taxonomy name of the target species (“Podospora anserina”) and its corresponding taxonomy id (“5145”).
Add reference species
This new instance does not contain any reference species or a predicted PPI, yet:
## Autophagy induction in Podospora anserina (5145)
## -------------------------------------------------
## No reference species yet.
## -------------------------------------------------
## No predicted PPI yet.To add the reference species, for which we have collected the
necessary data, we make use of the method addReference.
ppi <- addReference(ppi, "Homo sapiens", "9606", human.ai.proteins,
human.ai.irefindex, pa2human.ai.homologs)## Search for all relevant interactions:## 0%--25%--50%--75%--100%
## Remove irrelevant homologs.Besides the taxonomy name and the taxonomy identifier, this method requires the list, containing the proteins of the pathway of interest, the corresponding iRefIndex-data frame or the file name of the corresponding iRefIndex file, and the species specific homology data set generated by the NCBI BLAST+ toolkit. This method searches for all relevant interactions in the iRefIndex data frame. There are different and often ambiguous protein identifiers defined in an iRefIndex file and the “major” identifiers are not necessarily those defined in the corresponding “major” columns “uidA” and “uidB”. Furthermore, iRefIndex also contains complexes. Hence, this method applies an advanced search algorithm to find automatically relevant interactions associated with the pathway or the proteins of interest, respectively. You do not have to predefine the identifiers’ types (UniProt, Swissprot, Ensembl etc.), since these types are often assigned ambiguously. The algorithm searches for each identifier in 10 columns where any type of identifier or accession number is defined, for example, “uidA”, “altA”, “OriginalReferenceA”, “FinalReferenceA”, “aliasA”, “uidB”, “altB”, “OriginalReferenceB”, “FinalReferenceB” and “aliasB”. Additionally, it searches for each complex to which one or more of the predefined proteins are associated. Subsequently, each homologous relationship which is not relevant for the previously found interactions is declined.
In the same manner we add yeast to our Path2PPI-instance:
ppi <- addReference(ppi, "Saccharomyces cerevisiae (S288c)", "559292",
yeast.ai.proteins, yeast.ai.irefindex,
pa2yeast.ai.homologs) ## Search for all relevant interactions:## 0%--25%--50%--75%--100%
## Remove irrelevant homologs.In this tutorial, we want to predict the PPIs in based on these two reference species. You can use other and/or more reference species for your demands.
Now, we can get all processed information about the added reference species using the method :
## Homo sapiens (TaxId: 9606)
## ---------------------------
## 5 proteins (0 not used)
## 894 interactions:
## - 6 interactions have both interactors in protein list.
## - 349 interactions have at least one interactor in protein list.
## - 660 interactions in 102 protein complexes.
##
##
## Saccharomyces cerevisiae (S288c) (TaxId: 559292)
## -------------------------------------------------
## 7 proteins (0 not used)
## 2910 interactions:
## - 15 interactions have both interactors in protein list.
## - 834 interactions have at least one interactor in protein list.
## - 2207 interactions in 102 protein complexes.If we want to know which interactions have been found or which interactions are associated with the proteins of interest in a specific reference species (e.g. human), we can use the method as follows:
## ref A.db A.accession A.in.prot.list B.db
## 1 287217 complex qx1eWqPyfshUfC/6x17AYjcT/3w FALSE replaced
## 7 287217 complex qx1eWqPyfshUfC/6x17AYjcT/3w FALSE uniprotkb
## 13 287217 complex qx1eWqPyfshUfC/6x17AYjcT/3w FALSE uniprotkb
## 19 436141 uniprotkb A0A090N900 FALSE replaced
## 32 502959 uniprotkb P62942 FALSE replaced
## 45 408315 uniprotkb Q8N122 FALSE replaced
## B.accession B.in.prot.list
## 1 P42345 TRUE
## 7 A0A0A0MR05 FALSE
## 13 Q6R327 FALSE
## 19 P42345 TRUE
## 32 P42345 TRUE
## 45 P42345 TRUEFor more information about the method we refer to the corresponding
manual page (?showReferences).
Predict PPI
After we added all reference species and all necessary data, we can
start with the prediction. To predict the PPI network in the target
species we use the method predictPPI:
## Begin with Homo sapiens## 6 interactions processed. These lead to 5 interactions in target species.## -------------------------------## Begin with Saccharomyces cerevisiae (S288c)## 15 interactions processed. These lead to 22 interactions in target species.## -------------------------------## Combine results to one single PPI.## A total of 13 putative interactions were predicted in target species.This method uses different arguments to influence the prediction
approach and to define the output of the PPI network. For a detailed
description of the various arguments we refer to the corresponding
manual (?predictPPI). Here, we only use the argument
h.range where the first value corresponds to the lower
bound and the second value to the upper bound of the homology range.
That means that each E-value which is equal or less the lower bound will
be scored with 1, and each E-value which is equal or larger than the
upper bound will be scored with 0 (see appendix for a detailed
description).
According to the reports generated by this method two species specific PPI networks led to a PPI network in the target species with 13 interactions. To achieve further information about the former prediction step, we just type:
## Autophagy induction in Podospora anserina (5145)
## -------------------------------------------------
## 2 reference species: 9606, 559292
## -------------------------------------------------
## Number of predicted proteins: 8
## Number of predicted interactions: 13
## Predicted PPI based on 2 reference species:
## 9606 (2 interactions and 4 homologous relations)
## 559292 (11 interactions and 11 homologous relations)
## -------------------------------------------------
## Settings:
## Homology threshold: 1e-05
## Homology range: [1e-60,1e-20]
## Interactions threshold: 0.7
## Consider complexes: FALSEResults of the prediction
After we predicted the PPI network of the “autophagy induction” pathway in P. anserina we now want to know how this network looks like. And we want to know which proteins and interactions actually are associated with this pathway in our target species.
Plotting the results
To get a graphical representation of the predicted PPI network, Path2PPI provides three different plotting types. First, to get only the predicted PPI, we use the plot function of the Path2PPI-object, which is based on the igraph plotting function (Csardi and Nepusz, 2006):
There are various arguments provided with this method (see
?plot.Path2PPI). Here, we initially use the
return.coordinates argument since we want to save the
coordinates of the vertices for the next plotting approach.
In the second approach, we want to know from which reference species the different predicted interactions originated. We assign the previously computed coordinates to the plotting function since we want to compare both networks:
The different colors of the edges correspond to the species, see the
taxonomy identifiers in the legend: 5154 for P. anserina, 9606
for human, and 559292 for yeast) from which the interaction was deduced.
For example, we can see that the edge between the proteins “B2AWL” and
“B2AE79” in the upper network is thicker than the others. This indicates
that the interaction was found in more than one reference species. In
the second plot, we see that this interaction is based on six
interactions found in yeast and two interactions found in human.
Next, we want to plot the so-called hybrid PPI network, where we additionally can see the underlying reference interactions or the underlying reference PPI networks, respectively, and each homologous relationship. We also want to set the vertex labels, since we know the trivial names of the target species proteins. You can set the label for each protein of each species. Additionally, we want to change the species colors:
set.seed(40)
target.labels<-c("B2AE79"="PaTOR","B2AXK6"="PaATG1",
"B2AUW3"="PaATG17","B2AM44"="PaATG11",
"B2AQV0"="PaATG13","B2B5M3"="PaVAC8")
species.colors <- c("5145"="red","9606"="blue","559292"="green")
plot(ppi,type="hybrid",species.colors=species.colors,
protein.labels=target.labels)
The dotted edges correspond to an homologous relationship between a
protein of the target species and a reference species. Only those
proteins and interactions of the reference species are shown which
actually led to an interaction in the target species.
Get detailed information about each interaction
After we know how the predicted PPI network of this pathway may look
like in the target species we want to know more about the predicted
interactions. For this purpose we make use of the method
showInteraction which requires the protein identifiers of
the interaction:
## Score: 1.989472
## 8 reference interactions: 559292 (6) 9606 (2)For further details about the underlying reference interactions we
can use the additional argument mode:
## source.id target.id score h.scoreA h.scoreB r.species r.species.s r.species.t
## 1 B2AT71 B2AE79 0.855 0.71 1.00 9606 P42345 P42345
## 2 B2AT71 B2AE79 0.820 0.64 1.00 559292 P35169 P32600
## 3 B2AT71 B2AE79 0.825 0.65 1.00 559292 P32600 P32600
## 4 B2AT71 B2AE79 0.820 0.64 1.00 559292 P35169 P35169
## 5 B2AE79 B2AT71 0.855 1.00 0.71 9606 P42345 P42345
## 6 B2AE79 B2AT71 0.825 1.00 0.65 559292 P35169 P32600
## 7 B2AE79 B2AT71 0.825 1.00 0.65 559292 P32600 P32600
## 8 B2AE79 B2AT71 0.820 1.00 0.64 559292 P35169 P35169
## pos.edges used.edges ref
## 1 6 4 742389
## 2 6 4 522660
## 3 6 4 858136
## 4 6 4 105672
## 5 6 4 742389
## 6 6 4 522660
## 7 6 4 858136
## 8 6 4 105672This data frame contains each single predicted interaction of the current interaction and all corresponding reference interactions. For the interaction of the proteins in columns one and two, the third column gives the prediction score. The fourth and the fifth columns show the homology score of the source protein (A) or the target protein (B), respectively, in the target species to its equivalent (column seven and eight) in the reference species (column six). The column “pos.edges” (possible edges) indicates how many interactions could be derived from this interaction in the reference species, in contrast, to the number of interactions in “used.edges” which actually were adopted. The last column gives the identifier of this interaction in the iRefIndex data set.
To get the corresponding iRefIndex entries for these reference interactions we can use this method as follows:
ref.interaction <- showInteraction(ppi,interaction=c("B2AT71","B2AE79"),
mode="references.detailed",verbose=FALSE)The data frame now stored in the variable
ref.interaction is part of the iRefIndex table
which contains all information about the current reference interactions.
We can use this data frame with default R functionality to search for
specific information of the reference interactions. For example, if we
want to know from which study (author and publication) and from which
database the interaction of the proteins “P42345” and “P42345” with the
interaction identifier “742389” (first row) was adopted, we use:
## author
## 9096 -
## 143905 Thedieck K (2007)
## 143974 Takahara T (2006)
## 143979 Urano J (2007)
## 387372 -
## 387373 -
## 586846 remy-2001-1
## pmids
## 9096 pubmed:15467718
## 143905 pubmed:18030348
## 143974 pubmed:16870609
## 143979 pubmed:17360675
## 387372 pubmed:10702316|pubmed:11438723|pubmed:12030785|pubmed:16870609
## 387373 pubmed:10702316|pubmed:11438723|pubmed:12030785|pubmed:16870609
## 586846 pubmed:11438723
## sourcedb
## 9096 MI:0462(bind)
## 143905 MI:0463(biogrid)
## 143974 MI:0463(biogrid)
## 143979 MI:0463(biogrid)
## 387372 MI:0468(hprd)
## 387373 MI:0468(hprd)
## 586846 MI:0469(intact)In this manner you are able to search for each entry provided by the iRefIndex table.
Export results
Now, we want to work with the predicted PPI network and do further
analyses either directly in R or using an advanced network analysis tool
like Cytoscape (Cline et al., 2007). To do so, we can use the method
getPPI, which gives us the edge list of the PPI
network:
## source.id target.id score r.species
## 1 B2AT71 B2AE79 1.989472 559292,9606
## 2 B2AE79 B2AWL8 1.969948 559292,9606
## 3 B2AQV0 B2B5M3 1.000000 559292
## 4 B2AQV0 B2AXK6 1.000000 559292
## 5 B2AM44 B2AXK6 1.000000 559292
## 6 B2AM44 B2AUW3 1.000000 559292
## 7 B2AQV0 B2AUW3 1.000000 559292
## 8 B2AE79 B2AQV0 1.000000 559292
## 9 B2AXK6 B2AUW3 1.000000 559292
## 10 B2AXK6 B2B5M3 1.000000 559292
## 11 B2AXK6 B2AE79 0.820000 9606
## 12 B2AT71 B2AQV0 0.820000 559292
## 13 B2AWL8 B2AQV0 0.780000 559292If you want this PPI network with each single (not the combined one)
predicted interaction, use the additional argument
raw=TRUE.
We also want the edge list of the hybrid network which includes the
PPIs of the reference species. For this purpose we use the method
getHybridNetwork:
## source.id target.id t.species.id score type
## 1 B2AT71 P42345 9606 0.710000 homology
## 2 B2AT71 P35169 559292 0.640000 homology
## 3 B2AT71 P32600 559292 0.650000 homology
## 5 B2AE79 P42345 9606 1.000000 homology
## 6 B2AE79 P35169 559292 1.000000 homology
## 7 B2AE79 P32600 559292 1.000000 homology
## 13 B2AWL8 P42345 9606 0.510000 homology
## 14 B2AWL8 P35169 559292 0.560000 homology
## 15 B2AWL8 P32600 559292 0.610000 homology
## 17 B2AXK6 O75385 9606 0.640000 homology
## 18 B2AQV0 Q06628 559292 1.000000 homology
## 20 B2AM44 Q12527 559292 1.000000 homology
## 26 B2AXK6 P53104 559292 1.000000 homology
## 45 B2B5M3 P39968 559292 1.000000 homology
## 48 B2AUW3 Q06410 559292 1.000000 homology
## 16 P42345 P42345 9606 1.000000 interaction
## 24 P35169 P32600 559292 1.000000 interaction
## 31 P32600 P32600 559292 1.000000 interaction
## 4 P35169 P35169 559292 1.000000 interaction
## 171 O75385 P42345 9606 1.000000 interaction
## 181 Q06628 P39968 559292 1.000000 interaction
## 19 Q06628 P53104 559292 1.000000 interaction
## 201 Q12527 P53104 559292 1.000000 interaction
## 21 Q12527 Q06410 559292 1.000000 interaction
## 22 Q06628 Q06410 559292 1.000000 interaction
## 23 P35169 Q06628 559292 1.000000 interaction
## 261 P53104 Q06410 559292 1.000000 interaction
## 27 P53104 P39968 559292 1.000000 interaction
## 110 B2AT71 B2AE79 5145 1.989472 interaction
## 25 B2AE79 B2AWL8 5145 1.969948 interaction
## 32 B2AQV0 B2B5M3 5145 1.000000 interaction
## 41 B2AQV0 B2AXK6 5145 1.000000 interaction
## 51 B2AM44 B2AXK6 5145 1.000000 interaction
## 61 B2AM44 B2AUW3 5145 1.000000 interaction
## 71 B2AQV0 B2AUW3 5145 1.000000 interaction
## 8 B2AE79 B2AQV0 5145 1.000000 interaction
## 9 B2AXK6 B2AUW3 5145 1.000000 interaction
## 10 B2AXK6 B2B5M3 5145 1.000000 interaction
## 11 B2AXK6 B2AE79 5145 0.820000 interaction
## 12 B2AT71 B2AQV0 5145 0.820000 interaction
## 131 B2AWL8 B2AQV0 5145 0.780000 interactionIf you want to work with an igraph object instead, use the
additional argument igraph=TRUE in both methods. To export
the edge lists, use default R functions like
write.table.
References
Camacho, C. et al. (2009). BLAST+: architecture and applications. BMC Bioinformatics, 10(1), 421.
Cline, M. S. et al. (2007). Integration of biological networks and gene expression data using Cytoscape. Nature Protocols, 2(10), 2366-2382.
Csardi, G. and Nepusz, T. (2006). The igraph software package for complex network research. InterJournal Complex Systems, 1695(5), 1-9
Razick, S. et al. (2008). iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics, 9(1), 405.
Kanehisa, M. et al. (2014). Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Research, 42(D1), D199-D205.
Session info
## R version 4.4.2 (2024-10-31)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.1 LTS
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## BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
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##
## time zone: Etc/UTC
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] Path2PPI_1.37.0 igraph_2.1.1 BiocStyle_2.35.0
##
## loaded via a namespace (and not attached):
## [1] digest_0.6.37 R6_2.5.1 fastmap_1.2.0
## [4] xfun_0.49 magrittr_2.0.3 maketools_1.3.1
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## [10] pkgconfig_2.0.3 htmltools_0.5.8.1 rmarkdown_2.29
## [13] buildtools_1.0.0 lifecycle_1.0.4 cli_3.6.3
## [16] sass_0.4.9 jquerylib_0.1.4 compiler_4.4.2
## [19] sys_3.4.3 tools_4.4.2 evaluate_1.0.1
## [22] bslib_0.8.0 yaml_2.3.10 BiocManager_1.30.25
## [25] jsonlite_1.8.9 rlang_1.1.4Appendix
Biological evidence of the predicted PPI network
The example of autophagy induction was choosen, since it is well known and described for many eukaryotic organisms, including yeast, human and even P. anserina. Representatively and briefly described for yeast, the induction step works as follows:
Generally, nutrient availability activates the TOR-kinase, which phosphorylates ATG13. Hyper-phosphorylated ATG13 cannot interact with ATG1 and ATG17 to build the ATG1 complex (including ATG11 and VAC8) , which is important for autophagosome nucleation. Hence, autophagy is inhibited. In contrast, under nutrient-depleted conditions, TOR is deactivated, ATG13 is dephosphorylated and available for complex formation with ATG1 and ATG17. The next step of the autophagy process - autophagosome nucleation - can begin.
As reference proteins we used those provided by the KEGG database (Kanehisa et al., 2014) for yeast (seven) and human (five). We compared the predicted PPI with that provided by the KEGG database for P. anserina.
We found that two of the five proteins of human, ULK1 and TOR, and all of the seven proteins of yeast have been taken into account from the algorithm. This is due to the closer evolutionary distance of the both fungi and the corresponding proteins. The alogrithm predicts all of the proteins and interactions provided by the KEGG database for the induction step of autophagy in P. anserina. Additionally, it also includes two additional proteins, which are not characterized yet. This is propably due to the fact that these two proteins are putative serine/threonine kinases with similar motifs and sequences like the TOR kinase.
Summarizing, this example shows that Path2PPI is able to transfer interaction data amongst organisms. The resulting PPI networks can serve as starting points for further analyses and PPI studies.
The prediction algorithm
Aim: We aim to predict an interaction network in a target organism based on the interaction networks of well established model organisms. The networks are based on a predefined set of proteins which may belong to a certain pathway. We consider the degree of homologies and the number of reference species.
Requirements: The major initial requirements for the algorithm are the PPIs for each reference species, the BLAST results of the target species (P. anserina) against each reference species, and an E-value range defined by the upper bound hu and the lower bound hl. The range is required to map the BLAST E-value interval [hl, hu] to a homology score in the interval [1, 0] where 0 is the worst and 1 the best value. We will exemplarily describe the algorithm based on the two reference species, human and yeast.
Computing preliminary reference species-specific PPIs
Figure 1. Steps of
the prediction algorithm a) It searches for each interaction
(blue edges) in each reference species (blue) the corresponding homologs
(dotted edges) in the target species (red). Each valid homologous
protein from the first set, Ha, will now be
connected with each homologous protein from the other set, Hb. The deduced
interactions are scored, using the function described below. b) The
linear function to map the BLAST E-values to the interval [0, 1]. c) Combining the redundantly
predicted interactions to one final PPI network, PPIp,
of the target species.
At the beginning, we have the (undirected) PPI graphs of human PPIh = (Vh, Eh) and yeast PPIy = (Vy, Ey) (figure 1a, the blue graph corresponds to one reference species), a set of unassigned nodes Vp which represents all P. anserina proteins (figure 1a, red nodes) and an empty preliminary PPI graph of P. anserina PPIpre = (Vp, Ep) = ∅. The homology comparing approach leads to a directed homology graph
H = (VH, EH) where
VH ⊆ (Vh ∪ Vy ∪ Vp) and
EH ⊆ {(a, b) ∣ a ∈ Vp, b ∈ (Vy ∪ Vh)}.
Each edge in H is weighted by the E-values (EV) of the BLAST search.
Before starting the algorithm we have to set three major parameters. First, the parameter ethresh, which defines a threshold for the E-values. Homologous relations which exceed that theshold are immeditely declined and will not be taken into account anymore. The second and third parameters are the upper and the lower bound for an E-value range [hl, hu], where hl < hu ≤ ethresh which will be mapped to the interval [1, 0] by the scoring function s where 1 is the best score and 0 the worst (figure 1b). Since generally, homology-based network inference has to consider that more homologous proteins exist, we aim to distinguish and weight these relations by different scores provided by the scoring function. If only one homologous relationship exists, the scoring function will lead to the best score of 1, as well. This exception is due to the unambigous and desired situation if only one protein in the target species is homologous to the corresponding protein in the reference species.
Each interaction of each reference species is handled one by one and contributes to the prediction and scoring with an equal weight of 1, i.e. we do not distinguish between the underlying experimental methods. Furthermore, it is assumed that interactions and the reference species are stochastically independent, i.e. the occurance of one interaction does not influence the probality of the occurence of another interaction. We start with the first reference species, here, PPIh:
For each eh = {ah, bh} ∈ Eh (figure 1a, vertices ah and bh):
Get Ha ⊆ H with each ea = {(aea, bea) ∣ aea, bea ∈ VH, bea = ah} and Hb ⊆ H with each eb = {(aeb, beb) ∣ aeb, beb ∈ V, beb = bh} (figure 1a, dotted edges pointing to set Ha or set Hb)
Predefinition: Given the graph H and V(H) gives the set of vertices in H and E(H) the set of edges in H then ∣(V(H)∣ gives the number of vertices in H and ∣(E(H)∣ the number of edges in H.
If V(Ha) = ∅ or V(Hb) = ∅, decline eh, otherwise compute the homology score sh → [0, 1] for each edge ea ∈ Ha and each edge eb ∈ Hb based on the single E-values and the cardinalities of Ha and Hb:
If ∣(E(Ha)∣ = 1 then sh(ea) = 1 else sh(ea) = ∣mlog10(EV(ea)) + b∣,
if ∣(E(Hb)∣ = 1 then sh(eb) = 1 else sh(eb) = ∣mlog10(EV(eb)) + b∣
with $m=\frac{1}{log_{10}(h_l)-log_{10}(h_u)}$ and b = −(mlog10(hu)) (figure 1b).
Decline edges which are now scored with 0, i.e. set Ha = Ha \ {(ea ∈ Ha), sh(ea) = 0} and
Hb = Hb \ {(eb ∈ Hb), sh(eb) = 0}.
Add each remaining vertex {v ∈ V({Ha ∪ Hb}) ∧ v ∈ Vp} from target species to PPIpre and connect each node deduced from set Ha with each node deduced from set Hb (figure 1a, red edges). Compute a score si for each of these new predicted edges (interactions) using the arithmetic mean:
$s_i=\frac{s_h(e_a)+s_h(e_b)}{2}$.
Decline edges where si ≤ sthresh with sthresh as a predefined threshold.
Repeat step 1-4 with PPIy and each other reference species.
Combining PPIs deduced from each reference species
Now, the algorithm has to combine all predicted and probably redundant interactions in PPIpre to one combined PPI of P. anserina, PPIp = (Vp, Ep). In particular, it has to consider interactions which were suggested by both reference species PPI networks (figure 1c):
For each ei = {aei, bei} ∈ E(PPIpre):
Get Ia, b ⊆ PPIpre with each ej = {aej, bej ∣ aej, bej ∈ E(PPIpre), ((aej = aei) ∧ (bej = bei)) ∨ ((aej = bei) ∧ (bej = aei))}, i.e. further interactions with the same interacting partners of ei (each edge X-Y in figure 1c).
Divide Ia, b into subsets Sh ∈ Ia, b and Sy ∈ Ia, b, depending on the reference species where the corresponding interactions have been found (black bordered areas in figure 1c).
Combine each score si(sh ∈ Sh) to one intra species score s(Sh) and each score si(sy ∈ Sy) to one intra species score s(Sy). Since a higher number of interactions increases the probability that Ia, b exists in the target species, and the predicted interaction should be rated at least with the highest score, we sum up each score of each interaction in Sh or Sy, respectively, using a recursive function where sk is the kth score in si(sy ∈ Sy). Additionally, si(sy ∈ Sy) has been sorted in a decreasing order:
s(sk) = sk if k = 1,
f(sk) = f(sk − 1) + (1 − f(sk − 1)) * sk otherwise.
Hence, the intra species score is at least as high as the biggest sub
score. For example, if we have three interactions deduced from one
reference species, predicting all the same interaction in the target
species with the scores 0.7, 0.9 and 0.6.
Then, the final species score is:
s(0.6, 0.7, 0.9) = 0.9 + (1 − 0.9) * 0.7 + (1 − (0.9 + (1 − 0.9) * 0.7)) * 0.6 = 0.988.
Combine the two intra species scores to one common inter-species or final-score ia, b (figure 1c lower area):
$i_{a,b}=\frac{s(S_h)+s(S_y)}{m}+m-1$, where m is the number of reference species for which the interaction Ia, b ⊆ PPIpre was found (i.e. m = 1, if only in yeast or human and m = 2, if in both).Finally, add the new edge (interaction) Ia, b to PPIp with the score ia, b.