SUPPLEMENTARY
METHODS
Compilation of dendrogram
Protein-protein interactions listed in the Human Protein Reference Database (HPRD) were converted into matrix format containing entries of 1 if two proteins interacted directly or 0 if they did not interact. For example, if protein X interacts with protein Y and protein Z interacts with protein X, then
The HPRD contains over 36,617 protein-protein interactions listed within it [12] creating a large matrix with > 36,617 x 36,617 entries. The matrix was reduced so that it contained only our 15 enzymes expressed in the decidua and their 62 protein partners (i.e. 77 x 77 entries). The resulting matrix, termed ‘c’ is represented mathematically as c = {cij}.
If protein i interacts with one protein, protein j interacts with two different proteins and i and j interact directly with each other, there are a number of protein paths of different lengths connecting i and j (Box 1). The smallest path length connecting i and j (lij) is 1. The number of path lengths from i to j of length l is the product of all paths connecting i and j over the path length l. Mathematically this is represented as follows:
where
A penalizing factor g, allowed the effective connectivity, Sij(g), between i and j to be examined:
where g is greater than 0 but less than 1
For example, a large g of 0.01 penalises longer path lengths while a small g of 0.0000001 penalises shorter path lengths. Conventional protein network pathway analyses examine the shortest protein-protein path. In distinction to this, by using a g of 0.0000001 our approach allowed visualization of a long protein-protein path within the network.
The effective distance between i and j, Dij(g), is defined as giving
The above definitions were applied to the matrix containing the 77 enzymes and others involved in placental ARA metabolism using a custom written C ++ code. The output of the programme was a matrix containing 77 x 77 entries, each representing an effective distance between two interacting proteins. Each distance entry in this matrix underwent hierarchical clustering as defined by S.C. Johnson [13]. Briefly, this process was carried out in three steps:
Step 1: Each distance was assigned to an individual cluster. The distances between the clusters were equivalent to the distances between the items they contained.
Step 2: The most similar pair of clusters in terms of distance were merged into a single cluster reducing the number of total clusters by 1.
Step 3: Distances were re-calculated between the new cluster and each of the old clusters. We performed average hierarchical clustering whereby the distance between one cluster and another cluster was considered to be equal to the average distance from any member of one cluster to any member of the other cluster.
Steps 1 to 3 were repeated until all items were clustered in one single cluster of size 77.
Table I: Identification of 15 proteins expressed in deciduaThe cited literature identifies proteins from evidence using either immunohistochemistry or immunoblotting. Target proteins are highlighted in bold.
Decidua expression
Gene / Protein / Human Protein Atlas / Literature / HPRD
PLA2G1B / Secretory phospholipase A2 group IB pancreas / û / [14, 15] / ü
PLA2G2A / Secretory phospholipase A2 group IIA / û / [14, 15] / ü
PLA2G2D / Secretory phospholipase A2 group IID / û / û
PLA2G2E / Secretory phospholipase A2 group IIE / - / û
PLA2G2F / Secretory phospholipase IIF / _ / û
PLA2G3 / Secretory phospholipase A2 group III / û / û
PLA2G5 / Secretory phospholipase A2 group V / û / [14, 15] / ü
PLA2G6 / Cytosolic phospholipase A2 group VI calcium independent / û / û
PLA2G7 / Lipoprotein associated acetylhydrolase / û / ü
PLA2G10 / Secretory phospholipase A2 group X / û / [14, 15] / ü
PLA2G12A / Secretory phospholipase A2 group XIIA / û / û
PLA2G12B / Secretory phospholipase A2 group XIIB / û / û
PLA2G15 / Lysophospholipase 3 / ü / ü
PLA2G4A / Cytosolic phospholipase A2 calcium dependent alpha / û / [3, 15] / ü
PLA2G4C / Cytosolic phospholipase A2 gamma calcium dependent / û / û
PLA2G4D / Cytosolic phospholipase A2 delta / û / û
PLA2G4E / Cytosolic phospholipase A2 group IVE / û / û
ALOX15 / Arachidonate 15-lipoxygenase / û / û
ALOX15B / Arachidonate 15-lipoxygenase type B / _ / û
ALOX12 / Arachidonate 12-lipoxygenase / ü / ü
ALOX12B / Arachidonate 12-lipoxygenase 12 R types / û / ü
ALOX5 / Arachidonate 5-lipoxygenase / ü / [4] / ü
ALOXAP5 / Arachidonate 5-lipoxygenase activating protein / û / ü
LTC4S / Leukotriene-C4 synthase / _ / ü
LTA4H / Leukotriene A4 hydrolase / ü / ü
PTGS1 / Prostaglandin G/H synthase, cyclooxygenase 1 / û / [16] / ü
PTGS2 / Prostaglandin G/H synthase, cyclooxygenase 2 / ü / [16] / ü
PTGIS / Prostacyclin synthase / û / û
TBXAS1 / Thromboxane A synthase / û / û
PTGES / Prostaglandin E synthase / û / [17] / ü
PTGES2 / Membrane associated prostaglandin E synthase 2 / ü / [18] / ü
PTGES3 / Cytosolic prostaglandin E synthase 3 (23kDa) / û / [18] / ü
CBR1 / Carbonyl reductase / û / ü
HPGDS / Hematopoietic prostaglandin D synthase / û / û
PTGDS / Prostaglandin D2 synthase / û / ü
AKR1C3 / Aldo- keto reductase family 1 member C3 / û / [19] / ü
CYP2B6 / Cytochrome p450 family 2 subfamily B polypeptide 6 / ü / û
CYP4A / Cytochrome p450 family 4 subfamily A / _ / û
EPHX1 / Microsomal epoxide hydrolase / û / û
EPHX2 / Soluble epoxide hydrolase / û / ü
CYP4A11 / Cytochrome p450 family 4 subfamily A polypeptide 11 / _ / û
CYP2U1 / Cytochrome p450 family 2 subfamily U polypeptide 1 / û / û
CYP2E1 / Cytochrome p450 family 2 subfamily E polypeptide 1 / û / ü
CYP2C19 / Cytochrome p450 family 2 subfamily C polypeptide 19 / û / ü
CYP4F8 / Cytochrome p450 family 4 subfamily F polypeptide 8 / Not there / û
CYP2J2 / Cytochrome p450 family 2 subfamily J polypeptide 2 / _ / û
CYP4F2 / Cytochrome p450 family 4 subfamily F polypeptide 2 / û / û
Key to symbols:
(ü) Positive identification of target proteins in the Human Protein Atlas (HPA) using at least one antibody with three different human placental decidua tissue sections and protein-protein interactions listed in the Human Protein Reference Database (HPRD).
(û) Negative staining in the HPA, data not available, or reliable.
(_) Not yet characterised by the HPA.
2
Table II: Expression and function of network proteinsTarget and previously uncharacterised proteins are highlighted in bold.
Gene / Protein / Decidua expression / Biological process (Molecular function)
PTGER2 / Prostaglandin E receptor 2, 53kDa / _ / Adenylate cyclase-activating G-protein coupled receptor signalling pathway; cellular response to prostaglandin E stimulus; regulation of cell proliferation; response to lipopolysaccharide
PTGES
PTGER1 / Prostaglandin E receptor 1, 42kDa / _ / Response to lipopolysaccharide
EXOSC10 / Exosome component 10 / ü / Nonsense-mediated mRNA decay; rRNA processing
PTGES2
PTGFR / Prostaglandin F receptor / _ / Negative regulation of apoptotic processes; parturition; response to lipopolysaccharide
PLA2R1 / Phospholipase A2 receptor 1, 180kDa / ü / Endocytosis
MYO10 / MyosinX / ü / Transport
PLA2G10
DCN / Decorin / û / Aging; carbohydrate metabolic process; chondroitin sulphate biosynthetic and catabolic processes; dermatan sulphate biosynthetic process; extracellular matrix organisation; kidney development; organ morphgenesis; peptide cross-linking via chondroitin-4 sulfate glycosaminoglycan; placenta development, response to lipopolysaccharide; response to mechanical stimulus; skeletal muscle tissue development; wound healing
VCAN / Versican / û / Carbohydrate metabolic process; cell adhesion; cell recognition; chondroitin sulphate catabolic process; dermatan sulphate biosynthetic process; extracellular matrix organization; glial cell migration; heart development; multicellular organismal development
PLA2G2A
PLAA / Phospholipase A2-activating protein / ü / Inflammatory response; phospholipid metabolic process; signal transduction
PLA2G1B
BGN / Biglycan / ü / Blood vessel remodelling; carbohydrate metabolic process; chondroitin sulphate catabolic process; dermatan sulphate biosynthetic process; extracellular matrix organization; peptide cross-linking via chondtroitin 4-sulfate glycosaminoglycan
ARPC3 / Actin related protein 2/3 complex, subunit 3, 21 kDa / ü / Arp 2/3 complex-mediated actin nucleation; Fc gamma receptor signalling pathway involved in phagocytosis; cellular component movement; innate immune response
LTA4H
TMEM62 / Transmembrane protein 62 / ü / UNKNOWN
NUCB1 / Nucleobindin 1 / ü / (DNA binding, calcium ion binding)
PTGS1
BAT5 / Abhydrolase domain-containing protein 16A / û / (Hydrolase activity)
PTGS2
CAV1 / Caveolin 1, caveolae protein, 22kDa / ü / Host-virus interaction
MAPKAPK2 / Mitogen activated protein kinase- activated protein kinase 2 / ü / 3’UTR mediated mRNA stabilization; G2 DNA damage checkpoint; MyD88-dependent toll-like receptor signalling pathway; activation of MAPK activity; arachidonic acid metabolic process; cellular response to vascular endothelial growth factor stimulus; Inflammatory response; innate immune response; leukotriene metabolic process; inner ear development; mRNA metabolic process; macropinocytosis; neutrophil TRK receptor signalling pathway; peptidyl serine phosphorylation; regulation of interleukin 6 production; regulation of tumour necrosis factor production; response to cytokine stimulus; response to lipopolysaccharide; stress activated MAPK cascade; toll like receptor 10, 2, 3, 4, 5, 9 signalling pathways; toll like receptor TLR1:TLR2 signalling pathway; toll like receptor TLR6:TLR2 signalling pathway; vascular endothelial growth factor receptor signalling pathway
COTL1 / Cactosin-like 1 (Dicyostelium) / û / Defence response to fungus
TGFBRAP1 / Transforming growth factor, beta receptor associated protein / ü / Intracellular protein transport; regulation of transcription, DNA-dependent; transforming growth factor beta receptor signalling pathway; vesicle-mediated transport
ALOX5
ALOX5AP / Arachidonate 5 lipoxygenase activating protein / û / Arachidonic acid metabolic process; cellular response to calcium ion; leukotriene biosynthetic process; lipoxin metabolic process; lipoxygenase pathway; positive regulation of acute inflammatory response; positive regulation of catalytic activity; protein homotrimerization
RNF7 / Ring finger protein 7 / ü / Intrinsic apoptotic signalling pathway in response to oxidative stress; negative regulation of apoptotic process; protein neddylation; protein ubiquitination; response to redox state
IMMT / Inner membrane protein, mitochondrial / ü / Mitochondrial calcium ion homeostasis; response to cold
LYPLA3
UBE2W / Ubiquitin-conjugating enxyme E2W (putative) / ü / DNA repair; cellular response to misfolded protein; misfolded or incompletely synthesized protein catabolic process; proteasomal ubiquitin dependent protein catabolic process; protein K11 linked ubiquitination; protein mono ubiquitination
ACIN1 / Apoptotic chromatin condensation inducer 1 / ü / Apoptotic chromosome condensation; erythrocyte differentiation; negative regulation of mRNA splicing, via spliceosome; positive regulation of apoptotic process; positive regulation of monocyte differentiation
MAGEA11 / Melanoma antigen family A, 11 / û / (tumour antigen)
AKR1C3
ZHX1 / Zinc finger and homeoboxes 1 / _ / Regulation of transcription
C1orf103 / Ligand dependent nuclear receptor interacting factor 1 / û / Regulation of transcription, DNA-dependent; transcription, DNA-dependent
CRMP1 / Collapsin response mediator protein 1 / û / Axon guidance, nucleobase-containing compound metabolic process; pyrimidine nucleobase catabolic process
VIM / Vimentin / û / Bergmann glial cell differentiation, astrocyte development; cellular component disassembly involved in execution phase of apoptosis; Intermediate filament organization; lens fibre cell development; modulation by virus of host morphology of physiology; muscle filament sliding; negative regulation of neuron projection development
CASP3 / Caspase 3, apoptosis related cysteine peptidase / ü / Apoptosis
HTATIP / Histone acetyltransferase 5 / ü / Growth regulation, host-virus interaction, transcription, transcription regulation
ACTB / Beta actin / ü / “de novo” posttranslational protein folding; Fc gamma receptor signalling pathway involved in phagocytosis; adherens junction organization; axon guidance; blood coagulation; cell junction assembly; innate immune response
LMNA / Lamin A/C / ü / Activation of signalling protein activity involved in unfolded protein response; cellular component disassembly involved in execution phase of apoptosis; cellular protein metabolic process; cellular response to hypoxia; establishment or maintenance of microtubule cytoskeleton polarity; mitotic nuclear envelope disassembly; muscle organ development; positive regulation of apoptotic process; regulation of cell migration; sterol regulatory element binding protein import into nucleus; ventricular cardiac muscle cell development
TRIB1 / Tribbles homolog 1 (Drosophila) / _ / JNK cascade; negative regulation of lipopolysaccharide mediated signalling pathway; negative regulation of sequence specific DNA binding transcription factor activity; negative regulator of smooth muscle cell proliferation and migration; positive regulation of proteosomal ubiquitin dependent protein catabolic process; regulation of MPA kinase; response to lipopolysaccharide;
KRT85 / Keratin 85 / _ / Epidermis development
KRT5 / Keratin 5 / û / Epidermis development; hemidesmosome assembly
ITGB4 / Integrin, beta 4 / û / Cell adhesion
ALOX12
COPG2 / Coatomer protein complex, subunit gamma 2 / _ / ER-golgi transport; protein transport; transport
COPG (10) / Coatomer protein coplex, subunit gamma 1? / û / ER-golgi transport; protein transport; transport
DNAJB1 / DnaJ (Hsp40) homolog, subfamily B, member 1 / ü / Stress response
TERT / Telomerase reverse transcriptase / _ / DNA strand elongation; RNA dependent DNA replication; negative regulation of extrinsic apoptotic signalling pathway in absence of ligand; replicative senescence; telomere formation and maintenance via telomerase
AIP / Aryl hydrocarbon receptor interacting protein / û / Negative regulation of cyclic nucleotide phosphodiesterase activity; protein maturation by protein folding; protein targeting to mitochondrion; regulation of protein kinase A signalling cascade; Xenobiotic metabolic process
PTGES3
ARNT / Aryl hydrocarbon receptor nuclear translocator / ü / Cell differentiation; embryonic placenta development; mRNA transcription from RNA polymerase II promoter; positive regulation of endothelial cell differentiation, erythrocyte differentiation; glycolysis, hormone biosynthetic process, protein sumoylation; transcription from RNA polymerase II promoter, DNA dependent transcription; vascular endothelial growth factor receptor signalling pathway; vascular endothelial growth factor production; regulation of transcription from RNA polymerase II promoter in response to hypoxia; regulation of transcription from RNA polymerase II promoter in response to oxidative stress
AHR / Aryl hydrocarbon receptor / ü / Cell cycle, transcription, transcription regulation
STIP1 / Stress induced phosphoprotein 1 / ü / Response to stress
NR3C2 / Nuclear receptor subfamily 3, group C, member 2 / ü / Transcription, transcription regulation
TP53 / Tumour protein p53 / û / Apoptosis, cell cycle, host-virus interactions, necrosis, transcription, transcription regulation.
NR3C1 (10) / Nuclear receptor subfamily 3, group C, member 1 / û / Transcription, transcription regulation
HSPA1A / Heat shock 70kDa protein 1A / ü / Stress response
HSP90AA1 / Heat shock protein 90kDa alpha (cytosolic), class A, member 1 / û / Cardiac muscle cell apoptotic process; neuron migration; positive regulation of cardiac muscle contraction; cell size, nitric oxide biosynthetic process; protein import into nucleus; protein folding, response to oestrogen stimulus, heat, salt stress; skeletal muscle contraction
EIF2AK2 / Eukaryotic translation initiation factor 2-alpha kinase 2 / ü / Antiviral defence; host-virus interaction; immunity; innate immunity; transcription; transcription regulation.
CSF3R / Colony stimulating factor 3 receptor (granulocyte) / ü / Cell adhesion
JAK1 / Janus kinase 1 / ü / Enzyme linked receptor protein signalling pathway; interferon gamma mediated signalling pathway; interleukin 2 mediated signalling pathway; intracellular signal transduction; regulation of interferon gamma mediated signalling pathway; regulation of type I interferon mediated signalling pathway; response to antibiotic; type I interferon mediated signalling pathway
ERBB3 / V-erb-b2 avian erythroblastic leukemia viral oncogene homolg 3 / ü / Fc epsilon receptor signalling pathway; Schwann cell differentiation; cranial nerve development; endocardial cushion development; epidermal growth factor receptor signalling pathway; extrinsic apoptotic signalling pathway in absence of ligand; fibroblast growth factor receptor signalling pathway; heart development; innate immune response; negative regulation of cell adhesion, neuron apoptotic process, serotonin, signal transduction; neuron apoptotic process; neurotrophin TRK receptor signalling pathway; phosphatidylinositol 3-kinase cascade; positive regulation of Phosphatidylinositol 3-kinase cascade; regulation of cell proliferation; would healing
MAPK14 / Mitogen activating protein kinase 14 / ü / Apoptosis, stress response, transcription, transcription regulation
GRB2 / Growth factor receptor bound protein 2 / ü / Fc epsilon receptor signalling pathway; Fc gamma receptor signalling pathway involved in phagocytosis; Ras protein signal transduction; T cell costimulation; aging; anatomical structure formation involved in morphogenesis; axon guidance; branching involved in labyrinthine layer morphogenesis; cell-cell signalling; cellular response to ionizing radiation; epidermal growth factor receptor signalling pathway; fibroblast growth factor signalling pathway; innate immune response; insulin receptor signalling pathway; leukocyte migration; modulation by virus of hosy morphology of physiology; negative regulation of epidermal growth factor receptor signalling pathway; neurotrophon TRK receptor signalling pathway; phosphatidylinositol mediated signalling; platelet activation; positive regulation of actin filament polymerization; positive regulation of reactive oxygen species metabolic processes; protein heteroologomerization; receptor internalization; regulation of MAPK cascade; signal transduction in response to DNA damage
ESR1 / Estrogen receptor 1 / û / Transcription; transcription regulation
CEBPB (10) / CCAAT/enhancer binding protein (C/EBP), beta / û / Differentiation; transcription; transcription regulation
RPS6KA5 / Ribosomal protein S6 kinase, 90kDam polypeptide 5 / ü / Inflammatory response; stress response
MKNK1 / MAP kinase interacting serine/threonine kinase 1 / _ / Translation regulation
MAPKAPK5 / Mitogen activated protein kinase-activated protein kinase 5 / ü / Ras protein signal transduction; negative regulation of TOR signalling cascade; protein autophosphorylation; regulation of translation; stress induced premature senescence
PLA2G5
CASP4 / Caspase 4, apoptosis-related cysteine peptidase / ü / Apoptosis
CASP1 / Caspase 1, apoptosis-related cysteine peptidase / û / Apoptosis
PLA2G4A
Key to symbols: