In this thesis we propose some new approaches to the study of complex networks, and apply them to multiple domains, focusing in particular on protein-protein interaction networks. We begin by examining the roles of individual proteins; specifically, the influential idea of 'date' and 'party' hubs. It was proposed that party hubs are local coordinators whereas date hubs are global connectors. We show that the observations underlying this proposal appear to have been largely illusory, and that topological properties of hubs do not in general correlate with interactor co-expression, thus undermining the primary basis for the categorisation. However, we find significant correlations between interaction centrality and the functional similarity of the interacting proteins, indicating that it might be useful to conceive of roles for protein-protein interactions, as opposed to individual proteins. The observation that examining just one or a few network properties can be misleading motivates us to attempt to develop a more holistic methodology for network investigation. A wide variety of diagnostics of network structure exist, but studies typically employ only small, largely arbitrarily selected subsets of these. Here we simultaneously investigate many networks using many diagnostics in a data-driven fashion, and demonstrate how this approach serves to organise both networks and diagnostics, as well as to relate network structure to functionally relevant characteristics in a variety of settings. These include finding fast estimators for the solution of hard graph problems, discovering evolutionarily significant aspects of metabolic networks, detecting structural constraints on particular network types, and constructing summary statistics for efficient model-fitting to networks. We use the last mentioned to suggest that duplication-divergence is a feasible mechanism for protein-protein interaction evolution, and that interactions may rewire faster in yeast than in larger genomes like human and fruit fly. Our results help to illuminate protein-protein interaction networks in multiple ways, as well as providing some insight into structure-function relationships in other types of networks. We believe the methodology outlined here can serve as a general-purpose, data-driven approach to aid in the understanding of networked systems.

The G-protein coupled receptors (GPCRs) are seven-transmembrane receptors that transduce signals from the cell surface to intracellular effectors. There are more than 1000 GPCRs in metazoans, while no GPCR has been definitively identified in plants. The most promising plant GPCR candidate, Arabidopsis G-protein coupled receptor 1 (GCR1), physically couples to the G-protein < subunit GPA1 and is involved in cell cycle regulation, blue light and phytohormone responses, but its signalling network remains largely unknown. This project aimed to achieve a better understanding of GCR1 signalling by identifying its interactors using a novel yeast two hybrid system – the Ras Recruitment System (RRS). Screening of an Arabidopsis cDNA library using a bait comprising intracellular loop 1 (i1) and 2 (i2) of GCR1 resulted in the isolation of 20 potential interactors. Extensive reconfirmation screening demonstrated that three of these interactors: Thioredoxin h3 (TRX3), Thioredoxin h4 (TRX4) and a DHHC type zinc finger family protein (zf-DHHC1) interact specifically with both i1 and i2 of GCR1. This was supported by the reverse RRS (rRRS) and 6xHis-pull-down assays. It is speculated that TRX3 and TRX4, which can reduce disulfide bridges of target proteins and act as powerful antioxidants, may regulate GCR1-mediated signalling events in response to oxidative stress. Alternatively, they may modulate GCR1 targeting or signalling through their chaperone activities. zf-DHHC1 has a predicted membrane topography that is shared by most DHHC domain-containing palmitoyl acyl transferases. It may modify GCR1 activity through palmitoylation of the two cysteines located at the cytoplasmic end of the first transmembrane domain. Together, these findings contribute to the growing understanding of the GCR1 signalling network, and provide valuable starting points for further investigation.

Le terme de sulfoprotéomique est utilisé pour désigner l’étude de la sulfatation des protéines. Bien que la sulfatation soit depuis peu considérée comme une MPT d’une importance majeure, il y a toujours peu de travaux scientifiques qui y sont consacrés en comparaison avec ce qui se fait sur la phosphorylation notamment. Ce retard s’explique notamment par la difficulté à analyser les espèces protéiques sulfatées dans les conditions classiques utilisées en protéomique, notamment par spectrométrie de masse. Ces travaux de thèse visent justement à développer des méthodes d’analyses par spectrométrie de masse dédiées à l’étude de la sulfatation des protéines, afin d’augmenter le champ des connaissances de cette MPT. Pour cela, nous avons largement utilisé le mode d’ionisation négatif, très peu, voire jamais utilisé en protéomique, avec deux techniques de fragmentation pour réaliser des spectres MS/MS, à savoir les fragmentations CID et HCD. Les résultats obtenus nous ont permis de mettre en évidence une méthode d’analyse permettant la formation d’ions spécifiques de la sulfatation et de la phosphorylation (qui sont isobariques), permettant ainsi une identification certaine de chacune des deux MPTs. Nous avons également entrepris d’étudier le rôle de la sulfatation d’un récepteur cellulaire, CXCR4, dans son interaction avec son ligand naturel, la chimiokine SDF-1/CXCL12. Cette étude a été menée par électrophorèse capillaire, et pourra constituer une base de travail solide pour des futures analyses mettant en œuvre le couplage entre l’électrophorèse capillaire et la spectrométrie de masse pour une meilleure caractérisation des complexes formés entre les partenaires protéiques. / Sulfoproteomics term designs protein sulfation studies. It appears during the 2000’s, when the interest for others Post-Translational Modifications (PTMs) than phosphorylation and glycosylation was growing up. Even though sulfation is thought to be an important PTM, a weak number of publications has emerged about it, notably if we compare with the huge quantity of phosphorylation papers. This difference is mainly due to the difficulty to correctly analyze sulfated proteins and peptides in the classical ways of proteomics, as in mass spectrometry for example. The goal of this thesis is to develop mass spectrometry methods dedicated to the characterization of sulfated species, in order to improve the knowledge of this PTM. To do that, we have mainly used negative ion mode, which is almost never used, with two fragmentations techniques for the MS/MS spectra, which are CID and HCD. Results obtained allow us to pinpoint an analytical method allowing the differentiation between sulfation and phosphorylation (they are isobaric), based on the presence of specific ion for each PTM in MS/MS. In another part of the project, we have investigated the role of sulfation in the interaction between a cellular receptor, CXCR4, and its in vivo ligand, the chemokine SDF-1/CXCL12. We used capillary electrophoresis for this work, and it could be a good basis for future analyses using capillary electrophoresis coupled with mass spectrometry, in order to have a better characterization of the observed complexes.

氫化酶作為一種催化劑，能催化氫分子成為質子及電子的相互轉換。 [鎳鐵]- 氫化酶散播最廣的一種氫化酶，從古菌到細菌都能找到 [鎳鐵]- 氫化酶。完整成熟的 [鎳鐵]-氫化酶需要插入鐵、氰化物、一氧化碳以及鎳到它的催化核心。這複雜的過程需要其它由若干 hyp 基因編譯的輔助蛋白酶的幫助，其中蛋白HypA 與 HypB 負責將鎳運送到[鎳鐵] -氫化酶的催化核心。敲除了 hypA 或hypB 基因的細菌株缺失[鎳鐵] -氫化酶的活性，如在生長介質裡添補鎳可恢復部份[鎳鐵] -氫化酶的活性。當HypB 與鳥嘌呤核苷酸結合時會變成蛋白二聚體。對比HypB 脫輔基蛋白及與HypB 與鳥嘌呤三核苷酸類似物的蛋白複合物的晶體結構可發現，HypB 透過一個保守賴氨酸殘基( Archaeoglobus fulgidus HypB 的殘基 148 )組成分子間鹽橋以構成蛋白二聚體。Escherichia coli 的體內實驗顯示，此保守賴氨酸殘基對活性氫化酶的製造起必要的作用，反映由此殘基所構成的鹽橋對HypB 功能的重要性。此外，本研究展示了A. fulgidusHypA 及 HypB 蛋白之間的相互作用。通過在A. fulgidus HypB 上進行系統性的突變，發現HypB 利用其GTP 酶域上的一段氨基端區域與HypA 相互作用。跟據這個結果，我們進而在E. coli HypB 上發現了兩個保守的非極性殘基與HypA 相互作用。當以丙氨酸取代在HypB 上的這兩個非極性殘基時，HypB 無法激活E. coli 中的氫化酶，導置降低的氫化酶活性，這表明了HypA 和HypB 的相互作用對[鎳鐵] -氫化酶成熟過程的必要性。 / Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons. [NiFe]-hydrogenase is the most widely distributed hydrogenases, which is found in organisms ranging from archaea to bacteria. Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. The maturation process of hydrogenase is a complicated procedure, which requires many accessory proteins encoded by hyp genes. HypA and HypB participate in the nickel delivery step to the catalytic core of hydrogenase, which is supported by the fact that strain deficient in hypA or hypB gene lack hydrogenase activity which can be recovered partially by elevating nickel content in the medium. HypB is capable to form dimer in solution upon guanine nucleotide binding. By comparing the crystal structures of HypB in dimer and monomer form, an important lysine residue (residue 148 in A. fulgidus HypB) which is required to form an intermolecular salt bridge during GTP-dependent dimerization, has been identified. Substitution of this lysine resiue with alanie would break HypB dimer in vitro. In vivo complementation study in E. coli showed that the corresponding lysine residue in E. coli HypB is required for active hydrogenase production indicating the importance of this intermolecular salt bridge to the biological function of HypB. Besides, interaction between A. fulgidus HypA and HypB are demonstrated in this work. By making systematic mutation to A. fulgidus HypB, the N‐terminal region of the GTPase‐domain has been identified to be important for its interaction with HypA. Further mutagenesis study has been done on E. coli HypB and two conserved non‐polar residues responsible for interaction with HypA have been identified. Alanine substitution of these conserved non‐polar residues result in HypB mutants which failed to rescue hydrogenase activity in vivo in E. coli showing that HypA/HypB interaction is required for hydrogenase maturation. / Detailed summary in vernacular field only. / Chan, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 88-95). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction: Hydrogenase biosynthesis requires insertion of nickel facilitated by protein HypA and HypB --- p.1 / Chapter 1.1 --- What is hydrogenase? --- p.1 / Chapter 1.2 --- [NiFe] hydrogenase contains a complex catalytic core composed of metal atoms and diatomic ligands --- p.2 / Chapter 1.3 --- The [NiFe] catalytic core --- p.4 / Chapter 1.4 --- Building the catalytic [NiFe] core --- p.4 / Chapter 1.5 --- Nickel insertion into the hydrogenase precursor involves the proteins HypB, HypA and SlyD --- p.7 / Chapter 1.5.1 --- Protein HypB --- p.7 / Chapter 1.5.2 --- Protein HypA --- p.11 / Chapter 1.5.3 --- Protein SlyD --- p.12 / Chapter 1.6 --- Objectives - How HypB dimerization and HypA/HypB interaction are involved in hydrogenase maturation process? --- p.13 / Chapter Chapter 2 --- A conserved Lys residue is required for GTP-dependent dimerization and hydrogenase maturation --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Recombinant Plasmid Construction --- p.22 / Chapter 2.2.2 --- HypB mutant construction by site-directed Mutagenesis --- p.22 / Chapter 2.2.3 --- Protein Expression and purification --- p.23 / Chapter 2.2.4 --- HypB protein purification --- p.23 / Chapter 2.2.5 --- Analytical gel filtration chromatography coupled with Light Scattering (SEC/LS) --- p.24 / Chapter 2.2.6 --- Nucleotide binding affinity determination --- p.25 / Chapter 2.2.7 --- GTPase activity determination --- p.26 / Chapter 2.2.8 --- Sample preparation for hydrogenase activity assay --- p.26 / Chapter 2.2.9 --- Hydrogenase activity determination --- p.27 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- AfHypB undergoes GTP-dependent dimerization --- p.29 / Chapter 2.3.2 --- Analysis of Structural difference between the apo form and GTP S-bound form suggests a mechanism of GTP-dependent dimerization for HypB --- p.30 / Chapter 2.3.3 --- Lys-148 is essential for GTP-dependent dimerization --- p.31 / Chapter 2.3.4 --- Disruption of dimerization by K148 mutation did not affect nucleotide binding and GTP hydrolysis activity significantly --- p.32 / Chapter 2.3.5 --- The conserved lysine residue is required for hydrogenase maturation in E. coli --- p.33 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- A conserved intermolecular salt‐bridge is required for GTP-dependent dimerization of HypB and hydrogenase maturation --- p.45 / Chapter 2.4.2 --- The extra metal binding site at the dimeric interface of HypB may provide a mechanism of why GTP-dependent dimerization is essential to Ni insertion --- p.46 / Chapter Chapter 3 --- N-terminal region of GTPase‐domain of HypB is required for interaction with HypA --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Methods and materials --- p.53 / Chapter 3.2.1 --- Recombinant Plasmid Construction --- p.53 / Chapter 3.2.2 --- HypB variant construction by site‐directed Mutagenesis --- p.53 / Chapter 3.2.3 --- Protein Expression --- p.54 / Chapter 3.2.4 --- Tag‐free AfHypA and AfHypB purification --- p.54 / Chapter 3.2.5 --- Analytical size exclusion chromatography coupled with Light Scattering --- p.54 / Chapter 3.2.6 --- GST pull‐down of GST‐AfHypA and AfHypB --- p.55 / Chapter 3.2.7 --- Tandem affinity pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.55 / Chapter 3.2.8 --- GST pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.56 / Chapter 3.2.9 --- Hydrogenase activity determination --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- HypA and HypB from A. fulgidus form 1:1 heterodimer in solution --- p.58 / Chapter 3.3.2 --- The N‐terminal regions upstream of the first helix of A. fulgidus HypB is required for HypA-HypB interaction --- p.59 / Chapter 3.3.3 --- Two conserved hydrophobic residues on HypB from E. coli are required to interact with HypA --- p.60 / Chapter 3.3.4 --- HypA-HypB interaction is required for hydrogenase maturation in E. coli --- p.62 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- The N‐terminal region of the GTPase domain is required for interaction with HypA and hydrogenase maturation in E. coli --- p.73 / Chapter 3.4.2 --- Location of interaction site on HypB reveals possible role for HypA/HypB interaction --- p.74 / Chapter 3.4.3 --- Mode of specific interaction with HypA: Interaction via a disordered region implies a coupled folding and binding process --- p.75 / Chapter Chapter 4 --- Conclusion and Future Perspectives --- p.80 / Chapter A1.1 --- Summary of findings in this work --- p.80 / Chapter A1.2 --- Implications in hydrogenase maturation --- p.81 / Chapter A1.3 --- Questions unresolved --- p.82 / Chapter 4.3.1 --- Factors that activate GTPase activity of HypB are still elusive --- p.82 / Chapter 4.3.2 --- How nickel delivery is regulated by HypA/HypB complex is still unclear --- p.83 / References --- p.88 / Chapter Appendix 1 --- Preliminary results of HypA/HypB protein complex structural study --- p.96 / Chapter A1.1 --- Structural study may provide invaluable insights to the role of HypA‐HypB interaction --- p.96 / Chapter A1.2 --- X‐ray crystallography as an approach to determine HypA/HypB complex structure --- p.96 / Chapter A1.3 --- Initial crystal hits were obtained with purified AfHypA/HypB complex --- p.97 / Chapter Appendix 2 --- Publications associated to the thesis --- p.100 / Chapter Appendix 3 --- Constructs and Primers used --- p.101

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.