The emergence of genomics as a discrete field of biology has changed humanity’s understanding of our relationship with bacteria. Sequencing the genome of each newly-discovered bacterial species can reveal novel gene sequences, though the genome may contain genes coding for hundreds or thousands of proteins of unknown function (PUFs). In some cases, these coding sequences appear to be conserved across nearly all bacteria. Exploring the functional roles of these cases ideally requires an integrative, cross-species approach involving not only gene sequences but knowledge of interactions among their products. Protein interactions, studied at genome scale, extend genomics into the field of interactomics. I have employed novel computational methods to provide context for bacterial PUFs and to leverage the rich genomic, proteomic, and interactomic data available for hundreds of bacterial species. The methods employed in this study began with sets of protein complexes. I initially hypothesized that, if protein interactions reveal protein functions and interactions are frequently conserved through protein complexes, then conserved protein functions should be revealed through the extent of conservation of protein complexes and their components. The subsequent analyses revealed how partial protein complex conservation may, unexpectedly, be the rule rather than the exception. Next, I expanded the analysis by combining sets of thousands of experimental protein-protein interactions. Progressing beyond the scope of protein complexes into interactions across full proteomes revealed novel evolutionary consistencies across bacteria but also exposed deficiencies among interactomics-based approaches. I have concluded this study with an expansion beyond bacterial protein interactions and into those involving bacteriophage-encoded proteins. This work concerns emergent evolutionary properties among bacterial proteins. It is primarily intended to serve as a resource for microbiologists but is relevant to any research into evolutionary biology. As microbiomes and their occupants become increasingly critical to human health, similar approaches may become increasingly necessary.

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Aspergillus flavusis a filamentous fungusthat causes an ear and kernel rot in maize (Zea mays L.). It produces a toxic secondary metabolite, aflatoxin, on the colonized maize kernels. Aflatoxin is a carcinogen to humans and animals. The toxin is also an immunosuppressant and causes aspergillosis in immune compromised individuals. Therefore, the presence of aflatoxin in food is strictly regulated by governmental agencies. Contaminated food leads to severe loss in profit and in marketable yield. There has been extensive research to investigate resistance of maize toA. flavus. Certain lines of maize exhibit increased resistance to A. flavus and aflatoxin accumulation compared to others and correlated with that are proteins and metabolites that differ in abundance in those lines. Among them are members of the cupin superfamily of proteins and products of special nitrogen metabolism (derived from glutamate). The goal here was to identify networks underlying disease resistance indifferent maize genotypes through the identification of protein-protein interactions and the analysis of transcript abundance profiles realting to cupins and glutamate. The outcome will be an understanding of host resistance to A. flavussufficient to develop methods to prevent pre-harvest contamination by aflatoxin. A protein abundant in resistant maize was identified as a cupin and named ZmCUP1. The cDNA isolation, expression in E. coliand characterization of the protein encoded by the mRNA, Zmcup1, lead to the discovery that the ZmCUP1 protein had anti fungal properties and oxalate decarboxylase activity (EC 4.1.1.2). Another part of the project aimed at understanding the involvement of a transgene that encoded bacterial NADPH-glutamate dehydrogenase (GDHA; EC 4.2.3.1) that reduced aflatoxin accumulation by half. A maize partial predicted protein to protein interactome was built and used to identify potential interactions between proteins expressed differentially in lines of maize resistant to A. flavus. These interactions were characterized in-silico and one specific interaction, between Zmcup1 and a maize zinc finger protein was characterized in vitro.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions.

Proteins are the functional units of cells. However, a major portion of the proteome does not have a known functional annotation. This dissertation explores protein -protein interactions, involving these uncharacterized or unknown function proteins. Initially, protein – protein interactions were tested and analyzed for paralogous proteins in Escherichia coli. To expand this concept further and to get an overview, protein – protein interactions were analyzed using ‘comparative interactomics’ for four pathogenic bacterial species including Escherichia coli, Yersinia pestis, Vibrio cholerae and Staphylococcus aureus. This approach was used to study unknown function protein pairs as well as to focus on uncharacterized hub proteins. The dissertation aims at using protein – protein interactions along with other research data about proteins as a possible approach to narrow down on functions of proteins.

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On estime que 10%-20% de la population mondiale est infectée chaque année par des virus influenza A (IAV) saisonniers, causant 250 à 500 000 morts. De plus ces virus présentent des risques de pandémie, et sont à ce titre un problème de santé publique majeur. Le cycle viral est dépendant de la capacité du virus à manipuler le protéome cellulaire. Par ailleurs, le système ubiquitine-protéasome (SUP) cellulaire est impliqué dans de nombreux processus de régulation cellulaires par l'induction de la dégradation de protéines, ou par la modification de leur activation ou de leur localisation sub-cellulaire. Le SUP est une cible privilégiée des virus lors de l'infection. Des études récentes indiquent qu'un réseau d'interactions entre les protéines virales des IAV et les protéines du SUP pourrait contribuer à la réplication virale et l’échappement du virus face au système immunitaire. Cependant ces interactions restent encore mal connues. Nous avons construit une banque contenant 570 facteurs du SUP, ce qui représente environ 60% des facteurs SUP humains connus. Puis nous avons mis au point une méthodologie permettant de réaliser un crible comparatif des interactions entre cette banque SUP et cinq PB2 provenant de souches de virus influenza A de virulence différentes chez l’homme : deux souches saisonnières circulant actuellement dans la population humaine (H1N1pdm09 et H3N2), deux souches hautement pathogènes chez l’homme (H7N9 et H1N1-1918) et une souche de laboratoire (H1N1-WSN). Cette première phase de cartographie a permis de sélectionner 42 facteurs du SUP interagissant avec au moins une des protéines PB2 étudiées. Par ailleurs, l’analyse des similarités de profils d’interaction PB2/UPS des souches étudiées a permis de mettre en évidence une corrélation avec le temps de circulation de chaque souche dans la population humaine. Nous avons ensuite caractérisé le rôle fonctionnel des partenaires de PB2 dans le cycle viral par des expériences de déplétion transitoire de l’expression des facteurs cellulaires par siARN, et validé 36 des 42 facteurs testés. La très grande quantité de facteurs identifiés impliqués dans le cycle viral démontre la qualité de la méthodologie développée pour l’identification de ces interacteurs. Parmi ces facteurs, nous avons étudié plus en détail le rôle de trois deubiquitinases (DUBs) dans l’infection. Nous avons montré que les DUBs sont impliquées dans les phases précoces et tardives du cycle viral. De plus, avec des collègues de Hong Kong nous avons mis en évidence que la DUB OTUB1 est impliquée dans la réponse cellulaire à l’infection produisant des cytokines, et probablement dans l’assemblage des nouveaux virions. Nous avons identifié que la DUB OTUD6A est également impliquée dans les phases tardives du cycle viral. A l’inverse PAN2 qui fait partie des complexes de poly-d’adénylation est impliqué dans les phases précoces. Nous poursuivons nos études afin d’élucider le rôle de ces DUBs dans l’infection par IAV. / An estimated 10%-20% of the world's population is affected each year by seasonal epidemic influenza, causing about 250,000 to 500,000 fatal cases. The pandemic risk reinforces the trait of influenza A virus (IAV) infection as a public health issue. The virus life cycle critically relies on its ability to manipulate the host proteome. Besides, the ubiquitin-proteasome system (UPS) is involved in many regulatory processes in mammalian cells by inducing protein degradation, mediating protein activation or shaping their sub-cellular localisation. Therefore, UPS is a prime target hijacked by viruses. Recent evidence indicates that an intricate regulatory network involving viral proteins and the cellular UPS is likely to contribute to viral replication and immune evasion of influenza A viruses. However, usurpation of the host UPS by IAV is far from being comprehensively deciphered. To gain better understanding, we assessed the interplay between the human UPS and the PB2 subunit of the influenza A virus polymerase through a global proteomic profiling approach. For that purpose, an UPS-dedicated library of 590 human cDNAs, comprising 63% of the whole human UPS, was constituted and characterised. In an initial screen, UPS factors were challenged using a high-throughput split luciferase assay for interaction with the PB2 protein from 5 influenza A strains of different pathogenicity in human. A total of 80 UPS factors emerged as potential PB2 partners, of which 42 were validated as high-confidence PB2 partners for at least one of the strains. Further comparison of interaction profiles of the 5 PB2 with the UPS by hierarchical clustering revealed an interaction dendrogram fitting with the circulation time in the human population.Functional importance of interactors was tested by siRNA-mediated knock down experiments using luciferase tagged recombinant IAV viruses. Depletion of 36 out of the 42 tested UPS factors showed an effect on the infection with all or a subset of IAV strains, underlying the strong functional output of the developed methodology. Among these factors three deubiquitinases (DUBs) were further studied to decipher their involvement in IAV viral cycle. We have shown that they are involved in early and late stage of the infection and began to draw their function in viral cycle. We demonstrated with our colleagues in Hong-Kong that OTUB1 is involved in the host cytokine response and most probably in virus assembly. OTUD6A was also shown to be implicated in late stages of the infection but we still don't know its exact role. Contrariwise, the inactive DUB PAN2, which is part of poly-deadenylation complexes, is implicated in early phase of IAV infection, but surprisingly apparently not through viral mRNA regulation. More work is on-going to precise by which mechanisms these DUBs are implicated in IAV infection.

Proteins in Escherichia coli were compared in terms of essentiality, centrality, and conservation. The hypotheses of this study are: for proteins in Escherichia coli, (1) there is a positive, measureable correlation between protein conservation and essentiality, (2) there is a positive relationship between conservation and degree centrality, and (3) essentiality and centrality also have a positive correlation. The third hypothesis was supported by a moderate correlation, the first with a weak correlation, and the second hypotheis was not supported. When proteins that did not map to orthologous groups and proteins that had no interactions were removed, the relationship between essentality and conservation increased to a strong relationship. This was due to the effect of proteins that did not map to orthologus groups and suggests that protein orthology represented by clusters of orthologus groups does not accurately dipict protein conservation among the species studied.

Bien que le Virus Respiratoire Syncytial, responsable de la bronchiolite du nourrisson, soit aujourd’hui un problème de santé publique majeur, il n’existe encore aucun vaccin ou antiviral curatif contre ce pathogène. Le manque de données sur les étapes clés du cycle viral et sur les interactions virus-cellule freine le développement de nouvelles molécules antivirales.Nous avons étudié l’interactome de deux protéines virales : la polymérase L et le facteur de transcription M2-1. Dans ce but, nous avons mis au point un crible s’appuyant à la fois sur des critères d’interactomique et sur des critères fonctionnels.La première étape consistait à identifier des partenaires potentiels de M2-1 et L par des co-immunoprécipitations couplées à une approche de protéomique quantitative. Pour plus de pertinence, ce crible a été réalisé sur cellules infectées, grâce des virus recombinants produits par génétique inverse. Ceci nous a permis d’identifier 45 et 137 partenaires potentiels de L et M2-1 respectivement. Une étude systématique de l’impact de l’inhibition de 15 partenaires potentiels de M2-1 sur la multiplication virale a mis en avant trois candidats : ILF2, PABPN1 et PABPC1.Nous nous sommes par la suite concentrés sur PABPC1. L’inhibition de l’expression de PABPC1 altère la multiplication virale, mais nous n’avons pas pu mettre en évidence un effet spécifique sur la transcription ou la traduction virale. Son interaction avec M2-1 a été confirmée, et le domaine MLLE de PABPC1 a été identifié comme le site de liaison à M2-1. L’interaction entre M2-1 et PABPC1 a été observée à la fois dans le cytoplasme et dans les IBAGs, des sous-structures concentrant les ARNm viraux au sein des corps d’inclusion viraux. Nous avons formulé l’hypothèse que M2-1, liée à PABPC1, accompagne les ARNm viraux après leur sortie des corps d’inclusion. Ceci suggère un rôle de M2-1 dans le devenir des ARNm viraux en aval de leur transcription. / Although the Respiratory Syncytial Virus, responsible of bronchiolitis in infants, represents a major public health problem, there are currently no vaccine or curative antiviral directed against it. The lack of information on key steps of its viral cycle and on virus-cell interactions hinders the development of new antiviral molecules.We chose to study the interactome of two viral proteins: the polymerase L and the transcription factor M2-1. To do so, we developed a screen based on interactomic and functional criteria.The first step consisted in identifying potential binding partners of M2-1 and L by co-immunoprecipitations coupled to quantitative proteomics. For better relevance, this screen was realised on infected cells, thanks to recombinant viruses produced by reverse genetics. 45 and 137 potential binding partners of M2-1 and L respectively were thus identified. A systematic study of the inhibition of 15 potential partners of M2-1 and its impact on viral multiplication enabled the selection of three candidates: ILF2, PABPN1 and PABPC1.We chose to concentrate on PABPC1. The inhibition of PABPC1’s expression reduces viral multiplication, but no specific effect on viral transcription or translation was brought to light. Its interaction with M2-1 was confirmed, and the MLLE domain of PABPC1 was identified as the M2-1 binding site. The interaction between M2-1 and PABPC1 was observed both in the cytoplasm and in IBAGs, substructures of viral inclusion bodies where viral mRNA accumulate. We formulated the hypothesis that M2-1, with PABPC1, stays with viral mRNA after leaving inclusion bodies and during their translation. This suggests a role for M2-1 in the fate of viral mRNA downstream of transcription.