as well as the ready dissociation of subunit interactions, either between trans-membrane subunits or between trans-membrane and cytoplasmic subunits [Barrera et al. 2008]. Further, membrane protein structure is difficult to study by commonly used high-resolution methods including X-ray crystallography and NMR spectroscopy.
A major avenue by which one can understand membrane proteins and their complexes is by mapping the membrane-protein “subinteractome”—the subset of interactions involving membrane proteins. Conventional Y2H system is confined to the nucleus of the cell thereby excluding the study of membrane proteins. New biochemical techniques have been developed to facilitate the characterization of interactions among membrane proteins. Among these is the split-ubiquitin membrane yeast two-hybrid (MYTH) system [Miller et al. 2005, Kittanakom et al. 2009, Stagljar et al. 1998, Petschnigg et al. 2012]. This system is based on ubiquitin, an evolutionarily conserved 76-amino acid protein that serves as a tag for proteins targeted for degradation by the 26S proteasome. The presence of ubiquitin is recognized by ubiquitin-specific proteases (UBPs) located in the nucleus and cytoplasm of all eukaryotic cells. Ubiquitin can be split and expressed as two halves: the amino-terminal (N) and the carboxyl terminal (C). These two halves have a high affinity for each other in the cell and can reconstitute to form pseudo-ubiquitin that is recognizable by UBPs.
In MYTH, the bait proteins are fused to the C-terminal of a split-ubiquitin, and the prey proteins are fused to the N-terminal. The two halves reconstitute into a pseudo-ubiquitin protein if there is affinity between the bait and prey proteins. This pseudo-ubiquitin is recognized by UBPs, which cleaves after the C-terminus of ubiquitin to release the transcription factor, which then enters the nucleus to activate reporter genes.
Two of the earliest studies using the MYTH screens reported a fair number of interactions among membrane proteins from yeast: 343 interactions among 179 proteins by Lalonde et al. [2010], and 808 interactions among 536 proteins by Miller et al. [2005]. PCA has also been adopted to identify and/or verify membrane-protein interactions. For example, Babu et al. [2012] used PCA to validate and integrate 1,726 yeast membrane-protein interactions obtained from multiple studies, and these encompassed 501 putative membrane protein complexes.
The mammalian version of membrane yeast two-hybrid, MaMTH, is also based on the split-ubiquitin assay and is derived from the MYTH assay. Stagljar and colleagues [Petschnigg et al. 2014, Yao et al. 2017] used MaMTH to probe interactions involving the epidermal growth factor receptor/receptor tyrosine-protein kinase (RTK) ErbB-1 (EGFR/ERBB1), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and other RTKs that localize to the plasma membrane in human cells. When applied to human lung cancer cells, the assay identified 124 interactors for wild-type and mutant EGFR [Petschnigg et al. 2014].
2.2 Data Sources for PPIs
Several public and proprietary databases now catalog protein interactions from both lower-order model and higher-order organisms (summarized in Table 2.2). These databases contain PPI data in an acceptable format required for data deposition, such as IMEx (http://www.imexconsortium.org/submit-your-data) [Orchard et al. 2012]. The Biomolecular Interaction Network Database (BIND) [Bader et al. 2003], now called Biomolecular Object Network Database (BOND), includes experimentally determined protein-protein, protein-small molecule, and protein-nucleic acid interactions. BioGrid [Stark et al. 2011] catalogs physical and genetic interactions inferred from multiple high-throughput experiments. The Database of Interacting Proteins (DIP) [Xenarios et al. 2002] contains experimentally determined protein interactions with a “core” subset of interactions that have passed quality assessment (for example, based on literature verification). The Centre for Cancer Systems Biology (CCSB) Interactome Database at Harvard [Rolland et al. 2014, Yu et al. 2008, Yu et al. 2011] contains yeast, plant, virus, and human interactions. STRING [Von Mering et al. 2003, Szklarczyk et al. 2011] catalogs physical and functional interactions inferred from experimental and computational techniques. MIPS Comprehensive Yeast Genome Database (CYGD) [Güldener et al. 2005] and MIPS Mammalian Protein-Protein Interaction Database (MPPI) [Pagel et al. 2005] catalog protein interactions and also expert-curated protein complexes from yeast and mammals. The Human Protein Reference Database (HPRD) [Peri et al. 2004, Prasad et al. 2009] mainly contains experimentally identified human interactions. IRefIndex [Razick et al. 2008, Turner et al. 2010] and Integrated Interaction Database (IID) [Brown and Jurisica 2005] integrate experimental and computationally predicted interactions for human and several other species.
Table 2.2 Public and proprietary databases for protein-protein, protein-small molecule, and protein-DNA interactions
| PPI Database | Source | Reference |
| BioGrid | http://thebiogrid.org | [Stark et al. 2011] |
| BIND | http://bind.ca | [Bader et al. 2003] |
| CCSB | http://interactome.dfci.harvard.edu/ | [Rolland et al. 2014, Yu et al. 2008, Yu et al. 2011] |
| CYGD | http://mips.helmholtz-muenchen.de/genre/proj/yeast/ | [Güldener et al. 2005] |
| DIP | http://dip.doe-mbi.ucla.edu | [Xenarios et al. 2002, Salwinski et al. 2004] |
| EMBL-EBI IntAct | http://www.ebi.ac.uk/intact/ | [Hermjakob et al. 2004, Kerrien et al. 2012] |
| HAPPI | http://discern.uits.iu.edu:8340/HAPPI/ | [Chen et al. 2009] |
| HPRD | http://www.hprd.org/ | [Peri et al. 2004, Prasad et al. 2009] |
| OPHID/IID | http://ophid.utoronto.ca/ | [Brown and Jurisica 2005] |
| InnateDB | http://www.innatedb.com/ | [Lynn et al. 2008] |
| iRefIndex | http://irefindex.org/wiki/index.php?title=iRefIndex | [Razick et al. 2008, Turner et al. 2010] |
| MINT/HomoMINT | http://mint.bio.uniroma2.it/mint/ | [Zanzoni et al. 2002, Chatr-Aryamontri et al. 2007, Persico et al. 2005] |
| MIPS | http://mips.helmholtz-muenchen.de/proj/ppi/ | [Mewes et al. 2008] |
| MPPI | http://mips.helmholtz-muenchen.de/proj/ppi/ | [Pagel et al. 2005] |
| STRING | http://string-db.org/ | [Von Mering et al. 2003, Szklarczyk et al. 2011] |
2.3 Topological Properties of PPI Networks
A simple yet effective way to represent interaction data is in the form of an undirected network called a protein-protein interaction network or simply PPI network, given as G = 〈V, E〉, where V is the set of proteins and E is the set of physical interactions between the proteins. Such a network presents a global or “systems” view of the entire set of proteins and their interactions, and provides a topological (mathematical) framework to interrogate the interactions. In the definitions throughout this book, we also use V (G) and E(G) to refer to the set of proteins and interactions of a (sub)network of G. For a protein v ∈ V, the set N(v) or Nv includes all immediate neighbors of v, and deg(v) = |N(v)| is the degree of v. These neighbors together with their interactions, Ev = E(v) = {(v, u) : u ∈ N(v)} ∪ {(u, w) : u ∈ N(v),
