Maintaining genomic integrity is important for any organism. DNA mismatch repair (MMR) serves to correct errors that occur during DNA replication and recombination, such as unpaired bases or mismatched bases. Mutl is a key player and serves to coordinate protein-protein interactions. Recently it has been shown that human Mutl functions as an endonuclease and that this activity is imperative for functioning MMR. In this work, the X-ray crystal structure of the C-terminal endonuclease domain of Bacillus subtilis Mutl (BsMutL-CTD) is presented. Diffraction quality crystals of BsMutL-CTD were grown using vapor diffusion. The crystal structure of BsMutL-CTD was solved using multiwavelength anomalous diffraction. The structure reveals a putative metal binding site which clusters closely in space with endonuclease motif. Using the structure and sequence homology, several mutations were made and an investigation into the endonuclease activity of BsMutL was performed. BsMutL was confirmed to be a manganese-dependent endonuclease and key residues which contribute to endonuclease function were identified. / Thesis / Master of Science (MSc)

Adherens junctions are a main cell-cell adhesion structure found in epithelial cells. The stability of adherens junctions is attributed to various protein-signaling cascades and importantly the interaction between the transmembrane protein E-cadherin and cytoplasmic p120 catenin. This interaction is critical for cell adhesion and prevention of uncontrolled growth in normal cells. The interaction interface between these two binding partners was previously determined to comprise p120's Armadillo repeat domain (p120Arm) and Ecadherin's cytoplasmic juxtamembrane domain (Ecadc). Based on this information, peptide aptamers were derived from p120Arm and their interaction with Ecadc was tested in vitro. We reasoned that those could be expressed in vivo to stabilize adherens junctions at the cell-cell junction. In this study, we established protein-protein interaction assays to demonstrate p120Arm's ability to bind Ecadc and then used these assays to determine if p120Arm-derived peptides may competitively bind Ecadc. We demonstrated the interaction between p120Arm and Ecadc using assays that were not previously used such as: co-precipitation, analytical gel filtration and the bacterial-2-hybrid assay. However, the p120Arm-derived peptides did not bind to Ecadc or compete its interaction with p120Arm. This may be due to the nature of the assays that may not reflect competitive binding or the aptamers may not adopt the native conformation preventing binding to Ecadc. / Thesis / Master of Science (MSc)

The last few decades have witnessed an upsurge in the availability of large-scale data on genomes and genome-scale information. The development of methods to understand the trends and patterns from large scale data promised potentially to unravel the mechanisms responsible for the enormous diversity observed in biological systems. Of the many mechanisms adopted, protein-protein interactions represent one of the commonly adopted mechanisms to achieve functional diversity using a limited genetic repertoire. Protein-proteins interactions bring about several fundamental cellular processes and also modulate regulation at the cellular level. Different types of protein-protein interactions have evolved to carry out myriad functions in a cell. Mainly, interactions can be permanent or transient in nature, depending on the duration of interaction. In terms of affinity ,they are classified as obligate or non-obligate interactions. Structural studies on the various kinds of complexes have enabled the identification of the distinctive features characterizing the different types of complexes. Further, identifying the mechanisms involved in the evolution of protein-protein interactions are important in understanding the forces involved in their maintenance. Such studies also provide clues for the development of methods to predict protein-protein interactions from the large repertoire of sequence and structural data available. In spite of significant understanding of various aspects of protein-protein interactions, several questions still remain unanswered. The work embodied in this thesis studies two main aspects of protein-protein interactions for various types of complexes: structural and evolutionary features. The first part of the thesis(comprising of chapters 2,3,4 and 5) involves structural studies on the following features of protein-protein interactions: structural change, flexibility, symmetry, and residue conservation. The second part of the thesis(comprising of chapters 6,7,8 and 9) involves study of evolutionary aspects of protein-protein interactions, based on both large-scale data as well as case studies. Chapter1 provides a background and literature survey of the area of protein-protein interactions. The different classification schemes commonly used for describing the various protein-protein interactions are outlined. The key small-scale and large-scale experimental methods used for the identification of protein-protein interactions are described along with the details of the databases storing such experimentally derived information. Further, a comprehensive account of structural and evolutionary studies performed so far using the available data is provided. The computational(prediction)methods developed to address various aspects of protein-protein interactions are also outlined. In addition, the importance of protein-protein interactions in the context of diseases and the development of methods used to inhibit these interactions are discussed. Finally, the efforts towards design of protein-protein interactions based on the understanding of the principles governing their formation are outlined. Chapter 2 and chapter 3 describe different aspects of transient protein-protein interactions, which form an important subset of interactions, and are mainly involved in the regulation of Cellular processes. In chapter 2, the structural changes occurring upon complex formation are described. In chapter 3, roles of interface residues in the unbound form are described. In chapter 2, the nature, extent and location of structural changes upon binding is analyzed using a non-redundant and curated dataset of 77 structures of protein-protein complexes available in both bound and unbound forms. Structural change has been captured using two metrics: protein blocks and root mean square deviation of Cα positions. The relevance of the structural changes observed in protein-protein complexes is established by comparison with a control dataset of proteins not bound to any small or macromolecule. Results indicate that the observed changes are much larger than those observed due to random fluctuations. Given this background, the following observations were made on the nature, extent and location of structural changes in protein-protein complexes.(i) The nature of structural changes occurring at the interface is largely conformational with few rigid-body movements.(ii)The interfaces in the dataset are segregated into three types based on the extent of structural changes at the interface. A significant fraction of the interfaces are ‘pre-made’(almost in variant interface) or‘ induced-fit’(interface with large structural changes), while the rest are interfaces with moderate structural changes(‘others’). Analysis of structural changes using protein blocks reveals that pre-made interfaces are not completely invariant and are characterized by conformational changes of small magnitude. Pre-made interfaces are also observed to bind preferentially to‘ induced fit ’or‘ other’ interfaces. These observations implicate that non-obligate interactions possess in-built regulatory mechanisms in terms of conformational features to control the timely association-dissociation of transiently interacting proteins. (iii) Interestingly, significant structural changes away from the interface were observed in almost one-half of the complexes in the dataset. The analysis of these changes forms a major focus of this chapter. Crystallographic temperature factors, crystal packing, and normal mode analysis of these regions were studied to analyze the structural changes in these regions. Normal mode analysis along with literature survey indicates that most of the structural changes observed in non-interacting surface regions may be functionally relevant, with many of them corresponding to allosteric transitions. The majority of these changes occur in signaling proteins. The chapter summarizes that these observations suggest a much higher prevalence of allostery caused due to protein binding than appreciated before. The data set generated in this chapter can serve as a starting point to uncover potentially new allosteric modulators in signaling systems. In chapter 3, the question‘ Do residues at the interface of transient protein-protein interactions have any role in the unbound form?’ has been investigated. A high resolution, non-redundant and functionally diverse dataset of 67 proteins with known structures available both in the form of protein-protein complex and unbound forms has been used. Significantly low B-factors at the core of the interface in the unbound form are observed in these structures, indicating high rigidity. Many of these residues also show B-factors comparable to those of buried residues in a protein, which formed the basis for classifying interface residues as ‘rigid’ and‘ non-rigid’. These two types of residues have differential contribution towards the energetics of complex formation. It is also observed that rigid interfacial residues are conserved better in evolution than non-rigid interfacial residues. Their stronger selection is highlighted by substantial conservation of microenvironment (rigidness), sequence(amino acid identity/similarity) and structure(specific side-chain orientation) in homologous proteins. These observations coupled with the absence of any specific type of amino acid to occur preferentially at a rigid site indicates that rigidness is a property of the topological location of the residue at the interface and not the type of the amino acid present at that site. Analysis of the energetic parameters of these residues indicates that the y contribute significantly to the molecular recognition process by reducing the entropic cost on complexation by virtue of their pre-ordered conformation. This chapter also explores the contribution of interface residues towards the stability of the self-protein vis-à-vis that of the complex. It was seen that most interface residues contribute towards stabilizing the bound form. Interestingly, some of the interfacial residues predominantly stabilize the self-protein(the protein in which they are situated) and have a negligible contribution towards stabilization of the bound form. Thus, though these residues are located in the protein-protein interface their main role seems to be in the stabilization of the self-protein both in the unbound and bound forms. These residues are classified as Self-protein Stabilizing Residues(SSR -6.93%) and the rest as Neutrally Stabilizing Residues(NR -42.60%) and Complex Stabilizing Residues(CSR -50.46%). In addition, it was noted that the proportion of rigid residues is more in SSR(73.33%) than in NR(58.13%) and CSR(48.90%)sites. Apart from the favorable energetic contribution by rigid residues to the free energy of the unbound form than non-rigid residues, their predominance in SSRs suggests that rigid residues play an important role in the stabilization of the unbound form of the protein.The analyses performed in this chapter suggest that not all the protein-protein interfacial residues have the major role of stabilizing the complex; some of these residues seem to have more significant role in the unbound form than the bound form. Chapter4 provides a discussion on the prevalence and relevance of a symmetry in homodimeric proteins. One of the main features characterizing homodimers is the symmetric arrangement of subunits in the three-dimensional structures.Typically, asymmetric arrangements of subunits are associated with disease states; however, they are also observed in normal homodimers performing specialized functions. Two measures to quantify structural asymmetry in homodimers (global asymmetry and interface asymmetry)have been used on an on-redundant dataset of 223 biologically relevant homodimers. The survey for globally asymmetric homodimers in the dataset indicates that they are very rare(n=11).The chapter discusses cases where a globally asymmetric arrangement of homodimeric proteins has been utilized by the nature to perform certain specialized functions, such as linking of a dimeric system with a monomeric system(half-of-sites reactivity) and the transmission of signals emanating from asymmetric DNA repeats. Analysis of the 3-D structures of homologues reveals that there is no clear conservation of asymmetry. Specifically, the function of the homologous protein appears to dictate the pattern of structural organization. This chapter also describes the structural and evolutionary analyses of the 11 globally asymmetric complexes, which suggest possible mechanisms adopted by nature for preventing infinite array formation. The postulated mechanisms are:(i) In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used.(ii) In case of homodimers associating via partially topologically equivalent surfaces, steric hindrance serves as the preventive mechanism of infinite array. Since most of the biologically relevant homodimers exhibit gross structural symmetry, this chapter explores further the extent of interface asymmetry in symmetric homodimers. It was observed that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Further, binding of small ligands at the interface does not cause any significant variation in interface asymmetry.The chapter provides new insights regarding accommodation of structural asymmetry in homodimers. Chapter 5 describes the ability of residue conservation of interface residues vis-à-vis surface residues near interface residues to identify fitting errors caused due to mis-orientation in cryo-electron microscopy maps. Cryo-electron microscopy is the most popular technique for solving structures of large assemblies in physiological conditions. However, the structures are usually solved at low resolution and atomic resolution is desired to get insights at the molecular-level. Although several methods have been developed for the fitting of atomic structures or models in to low-resolution cryo-electron microscopic maps, inaccurate fitting is observed in several cases. Using a non-redundant and high-resolution dataset of 125 permanent interactions and 95 transient interactions, it was observed that interface residues are significantly conserved better than residues near to the interface. The chapter describes the ability of this differential conservation to identify probable mis-fittings in cryo-EM maps for three case-studies: ribosomal complex from Escherichia coli, transferring-transferrin receptor complex from Homosapiens, and glutamate synthase complex from Azospirillum brasilense. For these cases, the use of conservation information resulted in the identification of a few residues in the vicinity of the interface with significantly higher conservation, implying their probable occurrence in the interface. These findings were verified against the high-resolution structures for two of these complexes (ribosomal assembly and transferring-transferrin receptor complex).These analyses suggest that residue conservation information can be useful in the fitting process to arrive at the fitted structure with an improved accuracy. Further, the discriminative power of the simplistic measure of residue conservation coupled with residue surface accessibility in identifying interacting residues on protein structures is also analyzed in this chapter. Testing on a set of signaling and scaffolding molecules indicates that this simplistic measure can identify interface residues in protein structures, indicating that conservation contains a distinct, although weak, signal for functional regions. Chapters 6 to 9 discuss studies involving evolutionary aspects of protein-protein interactions. Chapter 6 describes the usage of phylogenetic tree construction using maximum likelihood method to understand the origin of the signal captured by the mirror tree approach. Mirror tree is one of the most popular approaches for identifying interacting proteins based on co-evolution. This method uses the similarity in phylogenetic trees as an indicator of protein-protein interaction. The origin of the evolutionary signal detected by the mirror tree method is a subject of some controversy. Two broad hypotheses have been postulated in the literature to explain the origin of the signal(i)site-specific co-evolution alone and(ii)correlation induced by external factors with only minor, if any, contribution from site-specific co-evolution. In the typical mirror tree protocol, inferences from phylogenetic tree are optional and only genetic distances are analysed. Even if the tree is constructed, usually the Neighbor-Joining approach is used. However ,with Neighbor-Joining method the inferred tree topology and genetic distances are directly linked. With maximum likelihood the tree topology is not derived directly from the genetic distances and therefore the contributions of the signals arising from tree topology and genetic distances can be studied separately. Tree topologies can be considered to serve as indicators of compensatory substitutions(implicated in site-specific co-evolution)as well as shared evolutionary history. Genetic distances correspond to evolutionary rates(implicated in correlation induced by external factors).Using this method, phylogenetic trees for a range of datasets of interacting and non-interacting proteins corresponding to yeast(S.cerevisiae) have been derived. The analysis performed in this chapter reveals no strong correlation between phylogenetic tree topologies, and significant correlation between genetic distance matrices for interacting proteins. The chapter discusses the implications of these findings and attempts to understand the origin of the signal captured by mirror tree protocol using the following points.(i) The near lack of correlation in tree topologies is not surprising since compensatory substitutions accounts for only a minority of the sites in a protein.(ii) The influence of shared evolutionary history has also been tested in the chapter by comparison of tree topologies of interacting proteins and non-interacting with 18S rRNA tree. Tree topologies of both interacting and non-interacting proteins do not mirror the 18S rRNA tree, ruling out shared evolutionary history as the signal of correlated evolution.(iii) By contrast, the significant correlation observed between branch lengths(genetic distances) of interacting proteins in all the variant datasets demonstrates correlation between evolutionary rates, independent of evolutionary divergence. In summary, the chapter concludes by providing support for the theory of correlation induced by external factors with only minor contribution from site-specific co-evolution. Chapter 7 explores the homology based transfer of interactions by quantifying the extent of retention/variation of interaction partnerships amongst a set of homologous proteins related at SCOP family level(which indicates clear evolutionary relationship).A large dataset of domain-domain interacting pairs(n=20,254)culled from SCOP1.73 was used for this analysis. Study involving this dataset shows that in around~80% of the cases, interacting partners are completely retained(evaluated as proteins belonging to the same SCOP family).If‘common’ partnership is evaluated at the level of SCOP folds, which are known to be structurally similaral though not necessarily evolutionarily related, the percentage of homologous domains with complete retention of partnership increases only by~5%. This indicates that only the presence of a common structural scaffold is not a sufficient feature for interaction. Further, the chapter also describes the retention/variation in partnerships analyzed as a function of sequence divergence between the homologous proteins. It is observed that there is a higher tendency to vary interacting partners as the evolutionary divergence between the homologues increases. In spite of this, interaction partnerships appear to be retained for homologous domains irrespective of their sequence divergence if the function mandates the presence of the interaction. However, all these observations could be influenced by the incomplete nature of information on the interactions available in the structural space. This problem has been addressed in this chapter by studying variation in interaction partnerships for Saccharomyces cerevisiae proteins. Yeast was chosen since it is extensively studied and interactions are available for~87% of proteins yielding a comprehensive list of interactions. To study this aspect, the SCOP dataset of interacting proteins(which represents a generic dataset) was compared with interactions of homologous proteins from yeast. The dataset of interacting proteins for yeast collated from all sources and documented in BIOGRID v50 was used. In this analysis, the proportion of homologous domains showing complete retention of interacting partners was only ~12%. This observation is the reverse of the trend observed for the dataset of homologous SCOP domains. Further analysis of homologous pairs of yeast-SCOP domains, containing only those pairs whose interacting protein families are found both in yeast and SCOP dataset, was performed to ascertain the extent of contribution of organism-specific proteins to the variation in interaction partnership for homologous domains. The proportion of homologous domains showing complete retention of interaction partners increases to~50% for these cases. These observations indicate that organism-specific proteins contribute significantly to the variation of interaction partnerships in homologous proteins. The next two chapters(8 and 9) discuss two contrasting scenarios of interaction partnerships. Chapter 8 describes the study of two protein families showing variation in interaction partnerships/interface structure and analyzes the drift in protein-protein interaction surfaces in each of the cases. The analysis in this chapter is facilitated by the large number of sequences available for the case studies. The first case study involves members of the glutamine amido transferase (GAT) superfamily of enzymes. Three remote homologues in this superfamily could also be related by sequence: intracellular protease(DJ-1/PfpIfamily),C-terminal domain of the small subunit of carbomoyl phosphate synthetase (ClassI glutamine amidotransferase-like family), and C-terminal domain of catalase (Catalase ,C-terminal domain family).In two cases, it is seen that domain recruitment influences the interacting surface(catalase, carbamoyl phosphate synthetase). The tethered domains, which are involved in interaction with the GAT domain, are from different SCOP folds, indicating that partnerships are not retained at extreme divergence. However, members of the DJ-1/PfpIfamily form homodimers with differing quaternary structures i.e. different orientations of the dimers. Four members have been studied in detail in this chapter (intracellular protease–two distinct interfaces–forming hexamer, stress-induced protein -dimer, DJ-1protein -dimer, sigma cross-reacting protein -dimer). Since the members are sequentially less divergent(as they are within the same family), it is possible to trace the drift in interfaces among these members based on the multiple sequence alignments of members with the differing quaternary structures and the sequences bridging them. The second case study involves analysis of the family of legume lectins, which corresponds to another set of proteins exhibiting differing quaternary structures for remarkably well conserved tertiary structures and sequences. Analysis of variations in protein-protein interaction surfaces when they show only slight differences between homologous members indicates that the drift is gradual, as seen when tracing the dynamics of DJ-1 family members and legume lectin family members. There exist sequences containing many different intermediate combinations of the interacting residues involved in both the sets of proteins. Comparisons of homologues where an entire interface seems to be lost show a different trend(intracellular protease and DJ-1).The most prominent interacting residues show an abrupt shift between the two different subfamilies. However, inspection of the other interacting residues reveals that there is a gradual change occurring generally, although a drastic change in the important(although quantitatively smaller) residues would have led to loss of interface. In summary, analysis of the evolutionary dynamics of the consensus interface residues of different quaternary structure types of DJ-1/PfpI family of enzymes and legume lectins shows that nature employs only the most important mutations to Prevent a specific interface and form a new interface and the rest of the positions drift and accumulate changes in the course of evolution. Chapter 9 describes the opposite scenario i.e. conservation of an interface even at high sequence divergence, using the RNA polymerase assembly as a case study. The multi-molecular assembly consists of four core subunits–alpha (I and II), beta, betaprime, and omega. These four subunits are common to RNA polymerase complexes of eubacteria, eukaryota and archaea. The sigma subunit aids in initiation of transcription in eubacteria (cor eenzyme +sigma = holoenzyme). Remarkably, prokaryotic and eukaryotic structures exhibit high degree of structural similarity, although their sequence similarity is low(19-28% sequence identity).However, this is expected as the obligatory interaction between the various subunits is essential to successfully carry out transcription. This chapter investigates the structural accommodation of diverse sequences at the interface of RNA polymerase machinery of eubacteria, using sequence analysis and homology modelling. Analysis of domain composition and order of domains for the core subunits of the RNA polymerase assembly in>85 eubacterial species indicates complete conservation. However, conservation analysis of the various core subunits indicates that the interface residues are more divergent for alpha and omega subunits. Although beta and beta prime are generally well-conserved, the residues involved in interaction with the divergent subunits(i.e.alpha, omega) are not conserved. Insertions/deletions are also observed near the interacting surfaces even in the cases of most conserved subunits(beta and betaprime). The chapter describes the homology modeling of three divergent RNApolymerase complexes from Helicobacter pylori, Mycoplasma pulmonis and Onion yellows phytoplasma, highlighting that insertions/deletions can be accommodated near the interface as they generally occur at the periphery. The development of a generalized matrix capturing preferences of interface environment is documented, along with results comparing the similarity of the modeled interfaces to that of the template interface. It is observed that the modeled interfaces are physico-chemically similar to that of the template interfaces in Thermus thermophilus, indicating that nature accommodates substantial substitutions and insertions/deletions at and near the interface in order to retain the structure of the obligate complex, which is in dispensable for the process of transcription. The main conclusions of the entire thesis work are summarized in chapter10, which also places the work in the context of the field of protein-protein interactions. The new insights obtained for transient interactions and homodimers from structural studies are highlighted. The application of evolutionary conservation to improve fitting of atomic structures in cryo-electron microscopic maps is discussed. The understanding gained from study of different evolutionary aspects of protein-protein interactions, ranging from correlated evolution to evolutionary dynamics of variations in interactions is also highlighted. Appendix 1 of this thesis describes the homology modeling of the hexameric form of AAA ATPase domain of spastin along with associated structural analysis.

L’identification de molécules organiques capables de moduler des interactions protéine-protéine (PPIs) est longtemps restée un domaine peu exploité par la recherche pharmaceutique privée comme académique. Cependant, le développement de méthodologies innovantes pour l’étude des PPIs et la validation récente de ce type d’inhibiteurs dans des essais précliniques, démontrent que les PPIs constituent une nouvelle source de cibles importantes. Les composés capables de moduler ces interactions représentent une nouvelle classe d’outils prometteurs, tant en recherche fondamentale qu’en thérapeutique. Elles peuvent aider à différencier les multiples fonctions portées par une même protéine, à replacer la protéine dans une cascade de réactions, ainsi qu’à disséquer et reconstituer des réseaux de signalisations protéiques. Ces molécules permettront également de faire émerger de nouvelles familles d’agents pharmacologiques actifs dans diverses pathologies.Mon travail de thèse s'est projeté dans l'avenir de la recherche biomédicale, en ciblant les interactions protéine-protéine. J’ai pu durant mon doctorat mettre en œuvre plusieurs méthodologies pour étudier et caractériser des interactions protéiques afin de développer des inhibiteurs de ces interactions. J’ai ainsi pu travailler sur l’optimisation d’un composé inhibiteur de l’interaction de la protéine virale Nef VIH-1 avec les domaines SH3 des Src kinases, le composé DLC27. J’ai également pu mettre en évidence la pertinence biologique de ce composé, qui cible un mode d’interaction unique, ou très rare, au niveau cellulaire en étudiant l’interaction avec les domaines SH3 de deux protéines, ALIX (ALG2-Interacting Protein X) et la sous-unité p85 de la PI3K (phosphatidylinositol 3-kinase).J’ai également pu caractériser la surface et le mode d’interaction de protéines virales impliquées dans le complexe de réplication du virus du SRAS (Syndrome Respiratoire Aigu Sévère). Cette étude tend à montrer que la protéine virale nsp10 agit comme une plateforme de reconnaissance pour ses partenaires, les protéines virales nsp14 et nsp16. Ces interactions permettent l’activation ou l’augmentation des activités respectives de nsp16 et nsp14 et jouent un rôle au niveau de la réplication virale. Suite à l’identification d’un ‘point chaud’ d’interaction, le résidu Tyr96 à la surface de nsp10, nous avons mis en évidence la première famille de molécules inhibitrices du complexe nsp10-nsp14 en couplant des méthodes informatiques (in silico) à des criblages expérimentaux. Ces molécules pourraient être utilisées comme antiviraux ou servir d’outils pour la recherche, en permettant par exemple de mieux comprendre et d’élucider les mécanismes moléculaires impliqués dans la réplication du virus du SRAS et des coronavirus en général. / Protein-protein interactions (PPIs) participate in and regulate almost all essential cellular functions. As a consequence, they are frequently involved in various pathologies (going from cancer development to viral replication and host cell infection) but their study remains a challenge.Thus understanding those interactions as well as finding small drug candidates able to modulate them, a field of research not currently fully developed, appear as the future of the healthcare industry.In this context, I chose to learn different techniques to study PPIs that are usually employed in academic (IMR laboratory, CNRS, France) or corporate environments (Genentech, USA). Moreover, I also worked on the development of small organic inhibitors of PPIs coupling in silico methodologies (chemo-informatics, Drug Design) to biological and structural validations.During my PhD, I could manage and work on different projects involving the study of PPIs involved in cancer signaling pathways as well as the development of potent antiviral drugs targeting the HIV and SARS viruses.My organizational, personal and scientific skills as well as the practical experience I developed on various techniques (from cell biology to biophysics, structural biochemistry and Drug Design), make me feel confident on the management of PPIs drug discovery projects.I am thus able to efficiently work on, and manage, the study of protein-protein interactions in various pathologies as well as the development of potent PPIs inhibitors, that will be a major breakthrough for Biotech/Pharma companies in the coming years.

Le nombre considérable d'interactions protéine-protéine (PPIs) existant au sein d'un organisme, ainsi que leur implication cruciale dans la vie cellulaire et dans de nombreuses pathologies, font des PPIs un immense réservoir de cibles potentielles pour la recherche de médicaments. Les PPIs sont aujourd'hui sur le devant de la scène grâce au développement de méthodologies innovantes et la validation récente de molécules chimiques modulant ces interactions dans des essais précliniques.L'étude des modulateurs d'interactions protéine-protéine (PPIM), a des implications tant dans la recherche fondamentale que thérapeutique. Les PPIMs peuvent aider à la compréhension des réseaux d'interactions. Elles permettront également de faire émerger de nouvelles familles d'agents thérapeutiques actifs dans diverses pathologies.Mon travail de thèse a principalement porté sur deux aspects de l'étude de l'inhibition des PPIs. D'une part, l'étude de l'implication des divers paramètres physicochimiques gouvernant une PPI dans sa capacité à être modulée (étude dite de la « druggabilité »), m'a amené à participer à la création d'une base de données structurale des interactions protéine-protéine : 2P2IDB (http://2p2idb.cnrs-mrs.fr/). D'autre part, j'ai contribué à l analyse de l'espace chimique des molécules présentes dans la base de données 2P2IDB. Nous avons défini la « Rule Of 4 » comme ligne de conduite pour caractériser ces molécules. Nous avons de plus utilisé le SVM afin de créer un protocole innovant (2P2IHUNTER) qui nous a permis de filtrer de grandes collections de composés afin de créer des chimiothèques dédiées aux PPIs. / The number of protein-protein interactions (PPIs) existing in an organism, and their crucial implication in cellular life and in many pathologies, demonstrates the importance of PPIs as a large reservoir of potential targets for medicinal research. Neglected for a long time by both pharmaceutical companies and academic laboratories because they were historically classified as difficult targets, PPIs are now getting into the groove due to the development of innovative methodologies and the growing number of small molecule compounds modulating these interactions.The study of PPI modulators has implications in both fundamental and therapeutics research. On the one hand, PPI modulators can be used in basic research to decipher the role of PPIs in biological networks. On the other hand, they represent a valuable source of new families of therapeutic agents in pathologic processes.In the first part of my PhD, I contributed to the development of a structural database dedicated to protein-protein interactions: 2P2IDB (http://2p2idb.cnrs-mrs.fr/). The interface descriptors of protein-protein interfaces which are typical of complexes present in 2P2IDB have been used to develop a qualitative scoring function to assess the ‘druggability' of PPI targets.In the second part of my PhD, I contributed to the analysis of the chemical space of PPI inhibitors present in the 2P2I database using chemoinformatics tools. We defined the ‘Rule-of-4' as a guideline to characterize these compounds. We have used support vector machine approaches to elaborate a protocol: 2P2IHUNTER, which allows filtering large collection of compounds to design chemical libraries dedicated to PPI targets.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.

The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

published_or_final_version / Computer Science / Master / Master of Philosophy