Protein:protein interactions are central to the regulation of the intrinsic programmed cell death (apoptosis) pathway. Opposing members of the Bcl-2 family of proteins, which have distinct sequence features, interact with each other on the outer mitochondrial membrane to regulate apoptosis. Pro-survival proteins such as Bcl-2, Bcl-x[L], Bcl-w, Mcl-1 and A1 protect cells from apoptosis and contain up to four regions of homology to Bcl-2 (Bcl-2 homology domains 1 - 4, BH1-4). Pro-apoptotic BH3-only proteins such as Bim, Puma, Noxa, Bad, Bmf, and Bid promote apoptosis by interacting with and inactivating pro-survival proteins, and contain just the BH3-domain. The pro-apoptotic proteins Bax and Bak are essential for apoptosis and contain three regions of homology to Bcl-2 (the BH1-, BH2- and BH3-domains). In this study, two different sets of interactions involving pro-survival proteins were investigated. Initially, the pro-apoptotic protein Bnip3 was examined to determine if it was a mitochondrial anchor for the pro-survival protein Bcl-w. Secondly, to characterise the interactions between a pro-survival protein and different BH3-domains, structures were solved of the pro-survival protein A1 in complex with four different BH3-domains. In the structure of Bcl-w, the hydrophobic C-terminus is bound to its own BH3-domain binding groove. This location of the C-terminus is consistent with the observation that Bcl-w is only loosely associated with the outer mitochondrial membrane in healthy cells. Upon interaction of Bcl-w with a BH3-domain, Bcl-w becomes tightly associated with the mitochondrial membrane, presumably due to displacement of the C-terminal residues by the BH3-only protein. In healthy cells it has been suggested that Bcl-w is associated with the membrane due to an interaction with an unidentified membrane protein, which preliminary experiments suggested may be Bnip3. Protein interaction experiments performed in vitro and in vivo did not reveal an interaction between Bnip3 and Bcl-w. It was originally thought that each pro-apoptotic BH3-only protein could interact with all pro-survival proteins. However, it has recently become clear that there is selectivity within the pathway suggesting functional groupings. Bim and Puma behave as originally predicted and can interact with all pro-survival proteins and are potent killers. In contrast, Noxa and Bad interact with distinct subsets of pro-survival proteins. Noxa only binds Mcl-1 and A1, while Bad binds Bcl-2, Bcl-x[L] and Bcl-w. As a result, either Noxa or Bad acting alone is a weak killer, but together they are potent. Other BH3-only proteins bind tightly to some pro-survival proteins and weakly to others. The diversity that exists between BH3-domain sequences precludes sequence-based identification of the determinants of specificity. In this study, crystal structures of A1:Puma BH3-domain, A1:Bmf BH3-domain, A1:Bak BH3-domain and A1:Bid BH3-domain complexes have been solved. Differences identified between these structures explain some of the variation in affinities observed in pro-survival protein:BH3-domain complexes. These observations, in combination with published data, suggest that BH3-domains bind weakly when the optimal interactions with conserved residues cannot be formed. Additionally, differences were observed in the A1:Bak BH3-domain structure that may be functionally important for the regulation of Bak.

Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references (p. 98-107).

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 117-122).

Understanding normal and cancer cell biology requires the development and application of systems biology approaches capable of probing the functional human proteome, and the protein-protein interactions (PPIs) within it. Such technologies will facilitate our understanding of how molecular events drive phenotypic outcomes, and how these processes are perturbed in disease conditions. In this thesis, I first describe the development of a mammalian, Gateway compatible, lentivirus-based protein-fragment complementation assay (magical-PCA), for the in vivo high-throughput identification of PPIs in mammalian cells. This technology provides a vast improvement over current PCA methodologies by allowing for pooled, proteome-scale mapping of PPIs in any mammalian cell line of interest, using any bait protein of interest. A proof-of-concept pooled genome-scale screen using the magical-PCA approach was performed using the human mitochondrial protein TOMM22 as a bait, providing evidence that this technology is amenable to proteome-wide screens. Moreover, the TOMM22 screens offered novel insight into links between TOMM22 and proteins involved in mitochondrial organization, apoptosis, and cell cycle dynamics. Second, I performed a pooled genome-scale magical-PCA screen with the oncoprotein BMI1, a component of the E3 ubiquitin ligase complex involved in histone H2A mono-ubiquitination and gene silencing, to identify novel BMI1 protein interactors. Consequently, I have uncovered a novel physical and functional association between BMI1 and components of the mammalian splicing machinery. I further discovered that BMI1 knockdown influenced the alternative splicing of a number of cellular pre-mRNAs in colon cancer cell lines, suggesting that the association between BMI1 and cellular splicing factors impinges on pre-mRNA processing. Importantly, BMI1 expression was shown to influence the alternative splicing of the SS18 oncoprotein towards an exon 8-excluded isoform, which was shown in this study to promote cell proliferation when assessed in an anchorage-independent growth assay. Together, these studies highlight the development of a new methodology for the detection and proteome-scale screening of mammalian PPIs. A proof-of-concept screen with human TOMM22 highlighted the utility of the approach, as I was able to detect both strong and weak or transient PPIs. Application of my screening methodology to BMI1 provided crucial insight into the function of this oncoprotein, and BMI1-driven tumorigenesis.

Alx4 is a paired-type homeodomain transcription factor that plays a key role in development, strongly expressed in the first branchial arch and craniofacial region. Pax3 also belongs to this family, and it displays a similar pattern of expression to that of Alx4. When Pax3 or Alx4 activity is lost individually, defects arise in an overlapping set of embryonic structures. In addition to their expression patterns, this suggests that these two factors may interact to play a role in normal murine development. We demonstrate an overlapping pattern of expression of Pax3 and Alx4 in the developing embryo and that Pax3 and Alx4 physically interact in vivo and in vitro. Pax3 and Alx4 can activate transcription from a P3 homeodomain consensus site, and preliminary analysis of mice null for both Pax3 and Alx4 show a novel mutant phenotype. We have therefore demonstrated a physical and genetic interaction between Pax3 and Alx4.

The currently known network of human protein-protein interactions (PPIs) is providing new insights into diseases and helping to identify potential therapies. However, according to several estimates, the known interaction network may represent only 10% of the entire interactome - indicating that more comprehensive knowledge of the interactome could have a major impact on understanding and treating diseases. The primary aim of this thesis was to develop computational methods to provide increased coverage of the interactome. A secondary aim was to gain a better understanding of the link between networks and phenotype, by analyzing essential mouse genes. Two algorithms were developed to predict PPIs and provide increased coverage of the interactome: FpClass and mixed co-expression. FpClass differs from previous PPI prediction methods in two key ways: it integrates both positive and negative evidence for protein interactions, and it identifies synergies between predictive features. Through these approaches FpClass provides interaction networks with significantly improved reliability and interactome coverage. Compared to previous predicted human PPI networks, FpClass provides a network with over 10 times more interactions, about 2 times more proteins and a lower false discovery rate. This network includes 595 disease related proteins from OMIM and Cancer Gene Census which have no previously known interactions. The second method, mixed co-expression, aims to predict transient PPIs, which have proven difficult to detect by computational and experimental methods. Mixed co-expression makes predictions using gene co-expression and performs significantly better (p < 0.05) than the previous method for predicting PPIs from co-expression. It is especially effective for identifying interactions of transferases and signal transduction proteins. For the second aim of the thesis, we investigated the relationship between gene essentiality and diverse gene/protein features based on gene expression, PPI and gene co-expression networks, gene/protein sequence, Gene Ontology, and orthology. We identified non-redundant features closely associated with essentiality, including centrality in PPI and gene co-expression networks. We found that no single predictive feature was effective for all essential genes; most features, including centrality, were less effective for genes associated with postnatal lethality and infertility. These results suggest that understanding phenotype will require integrating measures of network topology with information about the biology of the network’s nodes and edges.

Alx4 is a paired-type homeodomain transcription factor that plays a key role in development, strongly expressed in the first branchial arch and craniofacial region. Pax3 also belongs to this family, and it displays a similar pattern of expression to that of Alx4. When Pax3 or Alx4 activity is lost individually, defects arise in an overlapping set of embryonic structures. In addition to their expression patterns, this suggests that these two factors may interact to play a role in normal murine development. We demonstrate an overlapping pattern of expression of Pax3 and Alx4 in the developing embryo and that Pax3 and Alx4 physically interact in vivo and in vitro. Pax3 and Alx4 can activate transcription from a P3 homeodomain consensus site, and preliminary analysis of mice null for both Pax3 and Alx4 show a novel mutant phenotype. We have therefore demonstrated a physical and genetic interaction between Pax3 and Alx4.

The currently known network of human protein-protein interactions (PPIs) is providing new insights into diseases and helping to identify potential therapies. However, according to several estimates, the known interaction network may represent only 10% of the entire interactome - indicating that more comprehensive knowledge of the interactome could have a major impact on understanding and treating diseases. The primary aim of this thesis was to develop computational methods to provide increased coverage of the interactome. A secondary aim was to gain a better understanding of the link between networks and phenotype, by analyzing essential mouse genes. Two algorithms were developed to predict PPIs and provide increased coverage of the interactome: FpClass and mixed co-expression. FpClass differs from previous PPI prediction methods in two key ways: it integrates both positive and negative evidence for protein interactions, and it identifies synergies between predictive features. Through these approaches FpClass provides interaction networks with significantly improved reliability and interactome coverage. Compared to previous predicted human PPI networks, FpClass provides a network with over 10 times more interactions, about 2 times more proteins and a lower false discovery rate. This network includes 595 disease related proteins from OMIM and Cancer Gene Census which have no previously known interactions. The second method, mixed co-expression, aims to predict transient PPIs, which have proven difficult to detect by computational and experimental methods. Mixed co-expression makes predictions using gene co-expression and performs significantly better (p < 0.05) than the previous method for predicting PPIs from co-expression. It is especially effective for identifying interactions of transferases and signal transduction proteins. For the second aim of the thesis, we investigated the relationship between gene essentiality and diverse gene/protein features based on gene expression, PPI and gene co-expression networks, gene/protein sequence, Gene Ontology, and orthology. We identified non-redundant features closely associated with essentiality, including centrality in PPI and gene co-expression networks. We found that no single predictive feature was effective for all essential genes; most features, including centrality, were less effective for genes associated with postnatal lethality and infertility. These results suggest that understanding phenotype will require integrating measures of network topology with information about the biology of the network’s nodes and edges.