京都大学 / 0048 / 新制・課程博士 / 博士(情報学) / 甲第19109号 / 情博第555号 / 新制||情||98(附属図書館) / 32060 / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 阿久津 達也, 教授 山本 章博, 教授 鹿島 久嗣 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

Mass spectrometry (MS) is emerging as an important tool for studying protein and protein complexes. When applying this tool, it is important to understand and investigate whether some of the intramolecular and intermolecular interactions of proteins in solution and are maintained in the gas phase. To investigate if some of these interactions are maintained in the gas phase, we develop and use a method in which the electron-transfer dissociation (ETD) spectra of native proteins are compared with spectra from ETD followed by low amplitude collisional induced dissociation (CID). From these experiments, we find that some intramolecular interactions from solution are maintained in the gas phase for ubiquitin and beta-2-microglobulin (β2m). However, using these approaches, cytochrome c’s structure in the gas phase appears to be quite different than its structure in solution. We also investigated if ETD spectra of intact protein complexes reflect contact site information in these complexes

No description available.

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.

Os trabalhos realizados e apresentados nesta tese de doutorado compreendem diversos estudos computacionais e experimentais aplicados ao planejamento de candidados a novos fármacos para o tratamento do câncer, de uma metodologia inovadora para investigar a formação de complexos proteicos e de uma base de compostos naturais reunindo parte da biodiversidade brasileira com a finalidade de incentivar e auxiliar a descoberta e o desenvolvimento de novos fármacos no país. No primeiro capítulo, são descritos estudos que permitiram a identificação e o desenvolvimento de novas moléculas com atividade anticâncer, através da integração de ensaios bioquímicos e métodos de modelagem molecular na área de química medicinal. Dessa forma, estudos de modelagem molecular e ensaios bioquímicos utilizando uma base de compostos disponibilizada pela colaboração com o Laboratório de Síntese de Produtos Naturais e Fármacos (LSPNF) da UNICAMP, permitiram identificar uma série de moléculas da classe ciclopenta-β-indóis como inibidores da polimerização de microtúbulos com considerável atividade anti-câncer. Estes compostos apresentaram-se capazes de modular a polimerização de microtúbulos em ensaios in vitro frente ao alvo molecular e a células cancerígenas, com valores de IC50 na faixa de 20 a 30 μM. Além disso, estudos experimentais permitiram identificar o sítio da colchicina na tubulina como a região de interação desta classe e ensaios de migração celular comprovaram sua atividade antitumoral. A partir dos resultados obtidos, estudos mais aprofundados de docagem e dinâmica molecular permitiram elucidar as interações moleculares envolvidas no processo de ligação à proteína tubulina, e a utilização destes modelos moleculares no planejamento, síntese e avaliação de uma nova série de compostos. Com base nos dados obtidos por estudos computacionais, modificações foram propostas e novos inibidores da polimerização de tubulina foram planejados, sintetizados e avaliados, resultando na identificação de um inibidor de elevada atividade e perfil farmacodinâmico superior dentre as moléculas planejadas, com IC50 de 5 μM. Concomitantemente, ensaios de citotoxicidade in vitro demostraram uma interessante seletividade destes compostos por células cancerígenas em comparação a células saudáveis. Os estudos desenvolvidos com inibidores de tubulina aqui apresentados permitiram identificar moduladores da polimerização de microtúbulos com excelente perfil anti-câncer, que servirão como modelo para o desenvolvimento de novos tratamentos eficazes contra o câncer. No segundo capítulo é apresentado um novo método para predizer modificações conformacionais e a formação de complexos multiméricos em sistemas proteicos. Este método foi elaborado durante os estudos desenvolvidos ao longo de um programa de intercâmbio no laboratório The Center for Theoretical and Biological Physics (CTBP, Rice University, Estados Unidos), sob orientação do professor Dr. José Nelson Onuchic. Durante este projeto, estudos de modelagem computacional foram realizados utilizando métodos computacionais modernos desenvolvidos no próprio CTBP, tal como o método de Análise de Acoplamento Direto (DCA, do inglês Direct-Coupling Analysis) e um método de simulação conhecido como Modelagem Baseada em Estrutura (SBM, do inglês Structure-Based Modeling). Nos estudos aqui apresentados, os métodos DCA e SBM desenvolvidos no CTBP foram combinados, modificados e ampliados no desenvolvimento de uma nova metodologia que permite identificar mudanças conformacionais e elucidar mecanismos de enovelamento e oligomerização em proteínas. Os resultados obtidos através da predição de diversos complexos proteicos multiméricos com uma alta precisão mostram que este sistema é extremamente eficaz e confiável para identificar regiões de interface de contato entre proteínas a a estrutura quaternária de complexos macromoleculares. Esta nova metodologia permite a elucidação e caracterização de sistemas proteicos incapazes de serem determinados atualmente por métodos puramente experimentais. No terceiro capítulo desta tese de doutorado, é descrito a construção de uma base virtual de dados em uma iniciativa pioneira que tem como principal objetivo reunir e disponibilizar o máximo possível de toda a informação já obtida através do estudo da biodiversidade brasileira. Esta base, intitulada NuBBE DataBase, reúne diversas informações como estrutura molecular 2D e 3D e informações de atividades biológicas de diversas moléculas já isoladas pelo Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), localizado na Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP). A NuBBEDB será de grande utilidade para a comunidade científica, fornecendo a centros de pesquisa e indústrias farmacêuticas informações para estudos de modelagem molecular, metabolômica, derreplicação e principalmente para o planejamento e a identificação de novos compostos bioativos. / The work developed during a doctorate program and shown here as a PhD thesis reports the accomplishment of a series of computational and experimental studies focused on the development of new anticancer agents, an innovative methodology for the investigation of protein complexes formation and of a new database for natural products based on the Brazilian biodiversity, in an effort to assist and encourage the discovery and development of new pharmaceutical drugs inside country. The first chapter describes studies that resulted in the identification and development of new molecules with anticancer activity through the integration of biochemical experiments and molecular modeling methods in the area of medicinal chemistry. Thus, molecular modeling studies and biochemical assays using a library of compounds provided by collaboration with the Laboratório de Síntese de Produtos Naturais e Fármacos (LSPNF) from the University of Campinas (Unicamp) have identified a number of molecules of the cyclopenta-b-indole class as inhibitors for microtubule polymerisation, with substantial anti-cancer activity. These compounds showed to be able to modulate microtubule polymerisation on in vitro assays against the molecular target and cancer cells with IC50 values in the range of 20 to 30 μM. Moreover, experimental studies have identified the colchicine site of tubulin as in the region of interaction of this class and cell migration assays have proven their antitumour activity. Based on these results, further studies using molecular docking and molecular dynamics allowed to elucidate the molecular interactions involved in the binding process to tubulin protein, and these molecular models were used to guide the design, synthesis and evaluation of a novel series of compounds. From the data obtained by computational studies, modifications were proposed to design, synthesise and evaluate new tubulin polymerisation inhibitors, resulting in identification of a high-activity inhibitor and superior pharmacodynamic profile and IC50 of 5 μM. Alongside, in vitro cytotoxicity assays demonstrated an interesting selectivity of these compounds for cancer cells when compared to healthy cells. The studies presented here with tubulin inhibitors allowed to identify modulators of microtubule polymerisation with excellent anti-cancer profile, that will provide a valuable scaffold for the development of new effective treatments against cancer. The second chapter presents a new method for predicting changes on the conformational and the formation of multimeric protein complexes. This method was developed during the studies carried out over an exchange program in the Center for Theoretical and Biological Physics (CTBT, Rice University, USA), under the supervision of professor Dr. José Nelson Onuchic. During this project, computer modeling studies were carried using modern methods developed in the CTBT itself, such as Direct-Coupling Analysis (DCA) and a simulation method known as Modeling Based Structure (SBM). In the studies presented here, the DCA and SBM methods developed in CTBP were combined, modified and expanded to develop a new methodology able to identify the conformational changes and to elucidate mechanisms folding and oligomerization of proteins. The results obtained through prediction of various multimeric protein complexes with high accuracy show that this system is extremely effective and reliable to identify interface contacts between proteins and to predict the quaternary structure of macromolecular complexes. This new method allows the characterization and elucidation of protein systems that are currently unable to be solely determined by experimental methods. The third chapter of this doctoral thesis describes the construction of a virtual database in a pioneering initiative that aims to gather and make available all the information already obtained through the study of Brazilian biodiversity. This database, entitled NuBBE DataBase, brings together various information such as 2D and 3D molecular structure and biological activity of several molecules already isolated by the Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais(NuBBE), located at the Universidade Estadual Paulista Julio de Mesquita Filho (UNESP). The NuBBEDB will be useful to the scientific community, providing research and pharmaceutical centers information for molecular modeling studies, metabolomics, derreplication and principally for the planning and identification of new bioactive compounds.

Saccharomyces yeasts are ideal candidates for genomic and evolutionary studies in eukaryotes due to their small genome, short generation time and availability of genomic data. Species freely hybridize producing viable but largely sterile cells. A hybridization event can be a swift mechanism for evolutionary innovation that if successful, may produce individuals fitter than either parents. It is largely unclear which mechanisms contribute to such hybrid vigour. This thesis investigated three mechanisms by which a natural hybrid may utilise one or both subgenomes to its advantage: recombination, the formation of chimeric protein complexes and the inheritance of mitochondrial DNA. Three strains of Saccharomyces pastorianus, a natural hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, used in the lager fermentation process were sequenced using a NGS SOLiD platform. An analysis of recombination between each subgenome revealed the presence of 30 breakpoints, 28 of which are found within coding regions. Two breakpoints, present within the genes XRN1 and HSP82 have been reused in all three strains of S. pastorianus. This thesis investigated the formation of chimeric protein complexes in S. pastorianus by determining the configuration of protein complex-forming gene pairs to see whether they were mainly uni-specific, with all members belonging to the same parent, or chimeric, comprising one member from each parental species. A total of 21 pairwise protein complexes were found to be obligatorily chimeric in three strains of S. pastorianus. We used PCR-mediated gene deletion to recreate chimeric protein complexes in laboratory hybrids of S. cerevisiae and S. uvarum. The allelic configuration of one protein-complex forming gene pair, MLP2 and SPC110, impacted the growth of hybrid strains in a temperature-dependent manner. Finally, we looked at the mitochondrial inheritance in hybrids. Yeast hybrids can initially inherit mitochondrial DNA (mtDNA) from both parents, but rapidly become homoplasmic. To investigate the mechanisms influencing mtDNA inheritance, strains of Saccharomyces cerevisiae and Saccharomyces uvarum were crossed under different environmental conditions. The majority of hybrids inherited S. cerevisiae mtDNA when crossed in glycerol, a carbon source that can only be respired by yeast, in a range of temperatures. Those crossed in glucose, a fermentable source, did not show a preference for the inheritance of mtDNA at 30°, but at 10°C preferentially inherited S. uvarum mtDNA. In subsequent growth assays, hybrids with S. cerevisiae mtDNA grew better than those with S. uvarum mtDNA at 30°C and 20°C. However, at 10°C, the reverse was true: hybrids with S. uvarum mtDNA grew better that those with S. cerevisiae mtDNA, although only in glycerol. Overall this works sheds light on the molecular mechanisms contributing to fitness and evolutionary vigour in yeast hybrids.

Clathrin-mediated endocytosis is a process by which cells can internalise diverse molecules such as nutrients, antigens and signalling-surface receptors. The creation of clathrin-coated vesicles demands interplay between the plasma membrane lipids, cargo molecules and the proteins that build up the coat. This thesis deals with the identification and characterisation of sorting nexin 9 (SNX9) as a novel component of the endocytic machinery. SNX9 belongs to a large family of proteins based on the presence of a PX domain. In addition, SNX9 harbours an SH3 domain followed by a region with predicted low-complexity and a C-terminal BAR homology domain. Binding studies demonstrated that SNX9 interacted with the endocytic core components clathrin and AP-2 and dynamin-2, a GTPase known to be crucial for vesicle scission. The C-terminal region bound to phosphatidylinositols and targeted SNX9 to artificial liposomes and cellular membranes. Consistent with a role in endocytosis, a large portion of SNX9 co-localised with AP-2 and dynamin-2 but not with markers for early endosomes, Golgi. Over-expression of truncated variants of SNX9 in K562 and HeLa cells interfered with the uptake of transferrin. SNX9 recycles between a membrane-bound and a cytosolic pool. In cytosol, SNX9 formed a resting complex together with dynamin-2 and the metabolic enzyme aldolase. Activation for membrane binding involved ATP hydrolysis and correlated with phosphorylation of SNX9 and the release of aldolase. Aldolase bound to a tryptophan-containing acidic region near the clathrin and AP-2 motifs and blocked lipid binding of purified SNX9 derivatives. SNX9 was required for membrane targeting of dynamin2 in vitro and knockdown of SNX9 in HeLa cells by RNAi resulted in impaired membrane localisation. Together these results argue strongly for a role of SNX9 in recruiting and linking of dynamin-2 to sites of vesicle creation.

The major goal of this study was to develop an experimental proteomics approach for deciphering protein complexes and protein interaction networks in the budding and fission yeasts. Key steps of the employed analytical routine, including the purification of complexes and mass spectrometric identification of their subunits, were investigated in detail. Archiving, storage and handling of polyacylamide gels, visualization of protein bands and their effect on the efficiency of in-gel digestion and mass spectrometric identification of proteins were quantitatively evaluated. It was further demonstrated that a combination of several mass spectrometric techniques based on MALDI and ES ionization provided complementary data and enabled comprehensive characterization of protein digests. The optimized analytical procedures were employed in deciphering protein complexes and protein interaction networks in the budding and fission yeasts. A combination of Tandem Affinity Purification (TAP) and mass spectrometric identification of gel separated protein subunits is generic and robust strategy that provided accurate and reproducible data. The evaluation of TAP success rate, reproducibility and typical protein background presented in this work is based on TAP tagging and immunoprecepitation of 75 genes in S. cerevisiae and 22 in S. pombe. The molecular composition of characterized protein complexes was compared with protein-protein interactions uncovered by other established methods, such as yeast two hybrid screens or proteome-wide purification of protein complexes. We found that repetitive purification of protein complexes using different subunits as baits is crucially important for confident charting of proteomic environments. Accurate dissection of individual protein complexes and identification of their proteomic hyperlinks enabled to consider proteomic environments in the phylogenetic perspective and paved the way to reliable projection of proteomics data obtained in lower eukaryotic model organisms to higher eukaryotes, including humans.

Predicting the structure of complexes formed by two interacting proteins is an important problem in computation structural biology. Proteins perform many of their functions by binding to other proteins. The structure of protein-protein complexes provides atomic details about protein function and biochemical pathways, and can help in designing drugs that inhibit binding. Docking computationally models the structure of protein-protein complexes, given three-dimensional structures of the individual chains. Protein docking methods have two phases. In the first phase, a comprehensive, coarse search is performed for optimally docked models. In the second refinement and reranking phase, the models from the first phase are refined and reranked, with the expectation of extracting a small set of accurate models from the pool of thousands of models obtained from the first phase. In this thesis, new algorithms are developed for the refinement and reranking phase of docking. New scoring functions, or potentials, that rank models are developed. These potentials are learnt using large-scale machine learning methods based on mathematical programming. The procedure for learning these potentials involves examining hundreds of thousands of correct and incorrect models. In this thesis, hierarchical constraints were introduced into the learning algorithm. First, an atomic potential was developed using this learning procedure. A refinement procedure involving side-chain remodeling and conjugate gradient-based minimization was introduced. The refinement procedure combined with the atomic potential was shown to improve docking accuracy significantly. Second, a hydrogen bond potential, was developed. Molecular dynamics-based sampling combined with the hydrogen bond potential improved docking predictions. Third, mathematical programming compared favorably to SVMs and neural networks in terms of accuracy, training and test time for the task of designing potentials to rank docking models. The methods described in this thesis are implemented in the docking package DOCK/PIERR. DOCK/PIERR was shown to be among the best automated docking methods in community wide assessments. Finally, DOCK/PIERR was extended to predict membrane protein complexes. A membrane-based score was added to the reranking phase, and shown to improve the accuracy of docking. This docking algorithm for membrane proteins was used to study the dimers of amyloid precursor protein, implicated in Alzheimer's disease.R. DOCK/PIERR was shown to be among the best automated docking methods in community wide assessments. Finally, DOCK/PIERR was extended to predict membrane protein complexes. A membrane-based score was added to the reranking phase, and shown to improve the accuracy of docking. This docking algorithm for membrane proteins was used to study the dimers of amyloid precursor protein, implicated in Alzheimer’s disease. / text

Despite a great deal of research, the functional significance of tightly bound DNA-protein complexes is not yet clear, therefore these complexes are perfect object for pioneering research. Very little is known about plant TBP-DNA complexes. In this work we investigated barley TBP-DNA complexes from different organs (first leaves, roots and coleoptiles) at different developmental stages. We characterized individual components of tightly bound DNA-proteins complexes: polypeptides (TBP) and DNA. We isolated and characterized TBP proteins from barley first leaves, roots and coleoptiles of different age and differentiation stage. Also we isolated and characterized the DNA fragments from barley first leaves and water ripe and milky ripe grain TBP-DNA complexes. We demonstrated that in different developmental stages of coleoptiles, first leaves and roots TBP-DNA complexes were identified as a group of 15-160 kDa proteins, most of TBPs are acidic. Some of barley TBPs (10, 25, 38, 40 and 55 kDa) exhibit phosphatase, maybe Ser/Thr activity. We have identified also that some of TBPs tyrosines were phosphorylated, this modification depends on organ and developmental stage. Identified barley TBPs were involved in fundamental genetic processes, as well as in chromatin rearrangement and regulation processes. Nuclear matrix proteins, enzymes, transcription factors, serpins, immunophilins, and transposon polypeptides were identified among TBPs. We demonstrated that expression of TBPs depends... [to full text] / Žinoma, kad pastovi nehistoninių polipetidų frakcija yra išgryninama kartu su eukariotine DNR ir sudaro labai tvirtus (galbūt kovalentinius) kompleksus tarp branduolio baltymų ir DNR. Nustatyta, kad Erlicho ascito tvirtuose DNR-baltymų kompleksuose yra baltymas C1D, baltymai, pasižymintys fosfataziniu ir kinaziniu aktyvumais, kai kurie proteazių slopikliai ir kiti, dar neištirti baltymai. Nepaisant intensyvių tyrinėjimų, eukariotinių ląstelių tvirti DNR-baltymų kompleksai vis dar lieka menkai aprašyti ir yra objektas tolimesniems tyrimams. Augalų TBP-DNR kompleksai kol kas buvo tyrinėti labai mažai. Šiame darbe charakterizuojami miežių Hordeum vulgare tvirti DNR-baltymų kompleksai. Mes tyrėme TBP-DNR kompleksus iš miežių skirtingų ūglių organų ir skirtingų vystymosi stadijų ląstelių: lapų, šaknų, koleoptilės. Norint ištirti tokių nukleoproteidų funkcijas, svarbu charakterizuoti individualius komplekso komponentus: polipeptidus ir DNR. Taigi, išskyrėme tvirtai su DNR sąveikaujančius baltymus iš miežių skirtingos diferenciacijos bei skirtingo amžiaus ląstelių: pirminių lapelių, šaknų, koleoptilės ir juos charakterizavome. Taip pat išskyrėme ir charakterizavome DNR fragmentus iš miežių pirminių lapelių bei vandeninės brandos ir pieninės brandos grūdų TBP-DNR kompleksų. Parodėme, kad miežių TBP baltymai yra 15-160 kDa, dauguma baltymų yra rūgštiniai. Kai kurie iš miežių TBP baltymų (10, 25, 38, 40 ir 55 kDa) pasižymi fosfataziniu, galbūt, Ser/Thr aktyvumu. Nustatėme, kad tam... [toliau žr. visą tekstą]