By determining the Calpha and Cbeta chemical shift of P2 and its relaxation properties, together with secondary structure prediction, P2 was found to have an N-terminal 4-helices structural domain and a C-terminal unstructured coil. / P2 was found to interact with P1, forming heterodimer and with P2, forming homodimer. It was found that dimerization is carried out by their N-terminal, forming NTD-P1/NTD-P2 heterodimer and NTD-P2 homodimer. / Ribosome is the organelle responsible for protein synthesis and it was suggested that P-proteins located at the lateral stalk are involved in this process. Until now, structure of any P-protein is still not known although crystal structure of ribosome was solved. In order to know more about the biological role of P-proteins, structural characterization was carried out on human ribosomal protein P2. / The C-terminal tail which is conserved among P0, P1 and P2 of various species was found to interact with ribosome inactivating protein (TCS). This helps delivering TCS to its RNA substrate and carrying out its N-glycosidase activity. It was also found that TCS and EF2 are close in space suggesting that binding of TCS to P-proteins may hinder the association of EF2 to P-protein, thus inhibiting protein translation. / The solution structure of NTD-P2 homodimer was solved. It has 8 helices, 4 from each monomer. The surface is hydrophilic and the core is hydrophobic with a hydrophobic dimeric interface. By circular dicroism measurement, structural alignment and secondary structure prediction, we hypothesize that the dimerization mode of NTD-P1/NTD-P2 heterocomplex should be similar to NTD-P2 homodimer. Therefore, homology modeling was used to model the structure of NTD-P1/NTD-P2 using NTD-P2 as template. Interestingly, there is a small exposed hydrophobic patch on NTD-P1 which is lack in NTD-P2. This exposed hydrophobic patch may be the potential P0 binding site, forming P0(P1/P2)2 complex. Moreover, this exposed hydrophobic pocket is smaller than that of prokaryotic counterpart, thus providing insight in ribosome assembly and regulation in protein translation. / Lee, Ka Ming. / Advisers: K. B. Wong; P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0096. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 121-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Adiponectin, a hormone that homo-oligomerizes into trimer, hexamer, or higher molecular weight (HMW) species, is involved in maintaining insulin sensitivity in muscle and liver. Interestingly, its functions appear to be oligomer-specific. Recent data suggest that HMW levels are decreased in obesity and insulin resistance, although, the cause for this decrease is not known. Impaired assembly to the octadecamer represents one possible reason for decreased HMW adiponectin in insulin resistance and type 2 diabetes, but mechanisms by which HMW adiponectin assembles are unknown. This dissertation discusses the progress that we have made regarding formation of HMW adiponectin in vitro.I found that disulfide bonds are important in the assembly process to octadecameric adiponectin, but are not required for stability of the octadecamer itself. We showed that hydrogen peroxide accelerated oligomerization to the octadecamer through formation of disulfide bonds, while alkylation of the cysteines led to inhibition of both oligomerization and disulfide bond formation. Using comparative native/denaturing polyacrylamide gel electrophoresis (PAGE), dynamic light scattering, and tandem mass spectrometry, we demonstrated that octadecamer is stable in the absence of disulfide bonds by using multiple biochemical and biophysical assays. In addition, oxidized adiponectin oligomerizes to octadecamer far slower than reduced adiponectin. To further evaluate the role of disulfide bonds in the formation to octadecamer, we analyzed the role of reduction potential on adiponectin oligomerization. We observed that under immediate oxidizing conditions, hexamers and trimers form. Oxidized hexamer can form HMW adiponectin through disulfide bond rearrangement using beta-mercaptoethanol (βME) or increasing the total concentration of glutathione under oxidizing conditions. To further understand the role of disulfide bonds, we showed that zinc increased the oligomerization to octadecamer. This effect was associated with decreased initial disulfide bonding during the assembly to the octadecamer. In summary, these data suggest the rate of disulfide bond formation and the ability to undergo disulfide bond isomerization are important in the oligomerization process of HMW adiponectin.

Antimicrobial peptides are active against bacteria, fungi and viruses as part of the innate immune system in animals and insects. Such peptides are currently produced by extracting them from the host organism or by solid phase peptide synthesis; both techniques are expensive and produce low yields. Recombinant DNA technology opens a window to produce these peptides inexpensively and in large quantities utilizing E. coli expression systems. Two antimicrobial peptides, indolicidin and PGQ, were the focus of this work. They are short amphipathic alpha helical antimicrobial peptides that target a broad range of microorganisms. Genes encoding multimers of indolicidin, PGQ and a hybrid of indolicidin:PGQ were placed into protein expression vectors pET32a+ and pET43.1a+, for peptide production in E. coli. A combination of multimerization and the use of a fusion protein were utilized to mask the toxicity of these peptides in E. coli. The multimerized peptide fusion construct was purified using Ni/NTA affinity chromatography. Methionine residues flanking each monomeric unit were utilized to enable cleavage of the multimerized protein and liberating a biologically active peptide. A Trx:indolicidin trimer fusion was produced in the greatest yield of all constructs investigated. Upon cyanogen bromide cleavage, a band corresponding to the theoretical molecular weight of an indolicidin monomer was observed with SDS-PAGE. Antimicrobial activity of monomeric recombinant indolicidin was tested resulting in zones of clearing. Overall the results indicate that multimerizing antimicrobial peptide genes can potentially produce a larger quantity of peptide per bacterial cell. These studies suggest that multimerization of antimicrobial peptide genes represents a means to control in vivo toxicity of the recombinant peptides and increase production relative to single gene fusions.

TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) est un récepteur exprimé par les cellules de l’immunité innée qui amplifie l’inflammation initiée par les TLRs (Toll-like Receptors) et est impliquée dans plusieurs pathologies inflammatoires aigües et chroniques. À ce jour, peu de choses sont connues quant aux mécanismes moléculaires d’activation de TREM-1. Ainsi, nous avons développé des outils pour pouvoir stimuler TREM-1 de façon monovalente et multivalente. Ces travaux montrent que TREM-1 est activé par multimérisation et qu’il est régulé différemment sur les neutrophiles et monocytes. En effet, sur les monocytes activés au LPS, l’activation de TREM-1 s’effectue en deux étapes tandis qu’une seule est nécessaire dans les neutrophiles. Grâce à des approches de protéomiques, nous avons confirmé la dimérisation de l’ectodomaine de TREM-1 en solution. De plus, la multimérisation semble aussi être médiée par le ligand naturel de TREM-1, qui est, entre autres, libéré par les neutrophiles activés au LPS. Le travail présenté ici est une première étape vers la compréhension des mécanismes moléculaires conduisant à l'interaction de TREM-1 et son ligand, ouvrant ainsi de nouvelles perspectives thérapeutiques / Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells which amplifies inflammatory signals initially triggered by TLRs (Toll-like receptors) and TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases. Currently, the molecular mechanisms leading to the activation of TREM-1 remain unknown. We have developed specific tools to stimulate TREM-1 in a monovalent and divalent way. Here we show that TREM-1 is activated by multimerization and is differentially regulated on neutrophils and monocytes. Indeed, TREM-1 activation on primary human monocytes by LPS required a two-step process while one is required on neutrophils. Using proteomic approaches, we have confirmed that TREM-1 ectodomain dimerizes in solution. Furthermore, the multimerization seems to be mediated by the natural ligand of TREM-1, which is released by LPS activated neutrophils. Collectively, our findings uncover molecular mechanisms leading to TREM-1 and its ligand interaction, painting the way of new therapeutics

No description available.

With an expanding life-science sector and growing production of recombinant proteins, the need for efficient downstream processing is increasing. Certain proteins are sensitive to the harsh conditions often used in protein purification, such as low pH, which can result in aggregation and denaturation. ZCa is a domain derived from Protein A that can be used for calcium-dependent purification of antibodies without the need for acidic pH. Based on this domain, the CaRA library has been constructed, which targets other therapeutic proteins than human antibodies. Four of the proteins isolated from the CaRA library, namely CaRA_scFv_1, CaRA_scFv_2, CaRA_G-CSF_1 and CaRA_G-CSF_3, are presented here for the purification of single chain variable fragment and granulate colony stimulating factor. The four proteins were produced as monomers, trimers and hexamers in an attempt to increase the binding capacity and attached to a matrix for purification using site-specific coupling. The successful binders CaRA_scFv_1 and CaRA_scFv_2 showed high affinity for their target protein scFv and were able to selectively capture an increased number of molecules through multimerization. Calcium-dependent binding was demonstrated by elution at neutral pH using the calcium chelator citrate, thus concluding that these multimerized CaRA variants can be used to considerably increase the efficiency in scFv purification while providing excellent purity and significantly reducing the risk of aggregation.

L’Integrase du VIH est une ADN recombinase catalysant deux réactions qui permettent l'intégration de l'ADN viral dans l'ADN hôte. L’intégrase du VIH comprend 3 domaines : N-terminal impliqué dans la réaction de « 3' processing » et le transfert de brin, le domaine catalytique contenant le site actif et le domaine C-terminal liant l'ADN non-spécifiquement (CTD). Des recherches récentes mettent en évidence l'importance du CTD dans la liaison avec d'autres protéines virales comme la transcriptase inverse. Le but de la thèse était de comprendre les rôles et l'importance du domaine C-terminal de l’intégrase dans deux contextes : l'intégration dans la chromatine et la coévolution, avec l'objectif de comprendre le rôle de la multimerisation dans la fonction de l’intégrase. Globalement, les résultats de mon projet indiquent que l'IN-CTD joue un rôle important, en contribuant à la formation de multimères d'ordre supérieur importants pour la fonction de l’IN. / HIV Integrase is a DNA recombinase that catalyzes two endonucleolytic reactions that allow the viral DNA integration into host DNA for replication and subsequent viral protein production. HIV Integrase consists of 3 structural and functional domains: The N-terminal zinc domain involved in 3’ processing and strand transfer, the catalytic core domain which contains the active site, and the C-terminal domain that binds DNA non- specifically. Recent research highlights the importance of the CTD in binding with other viral proteins such as Reverse Transcriptase. The aim of the thesis was to understand the roles and importance of the C-terminal domain of HIV-1 Integrase in two contexts: chromatin integration, and co-evolution, with the overall purpose of understanding the role of multimerization in IN function. Overall, results from my project indicate that the IN-CTD plays an important role, by contributing to the formation of higher order multimers that are important for IN functionality.

No description available.

Les virus Nipah (NiV) et Hendra (HeV) sont des paramyxovirus zoonotiques appartenant au genre Henipavirus. Les paramyxovirus possèdent un génome ARN simple brin de polarité négative encapsidé par la nucléoprotéine (N) au sein d’une nucléocapside hélicoïdale. Cette dernière sert de substrat pour la transcription et la réplication, réalisées par la polymérase virale qui consiste en un complexe entre la protéine L et la phosphoprotéine (P). A l’aide d’approches biophysiques, j’ai établit une cartographie de l’interaction entre la région C-terminale désordonnée de N (NTAIL) et la région C-terminale de P (PXD) chez NiV, HeV et MeV. L’observation à l’échelle atomique par RMN a confirmé l’intervention d’un élément de reconnaissance moléculaire (MoRE) qui subit un repliement α-hélical au contact de PXD. J’ai également montré la capacité des domaines NTAIL et PXD des henipavirus à former des complexes hétérologues soulignant leur proximité structurale. L’interaction NTAIL-PXD, cruciale pour le recrutement de la polymérase virale constitue une cible idéale pour des approches antivirales. Ainsi, un test de criblage à haut débit par HTRF a été mis en place dans le but d’identifier des inhibiteurs. Enfin, une approche structurale a révélé une organisation trimérique de la protéine P de NiV et HeV en solution. La résolution de la structure cristalline de la région de tétramérisation de P du virus de la rougeole montre la présence d’une région désordonnée à proximité du site putatif de recrutement de L. Collectivement, ces résultats représentent une étape clé vers l’élucidation du l’impact fonctionnel de l’oligomérisation de la protéine P sur le cycle réplicatif des paramyxovirus. / Nipah (NiV) and Hendra (HeV) viruses are zoonotic paramyxoviruses that belong to the Henipavirus genus. Paramyxoviruses possess a single-stranded negative-sense RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. This latter is the substrate for both transcription and replication that are carried out by the polymerase, consisting of a complex between the large protein (L) and the phosphoprotein (P). Using various biophysical approaches, I was able to map the interaction between the C-terminal disordered region of N (NTAIL) and the C-terminal region of P (PXD) in NiV, HeV and MeV. Atomic resolution description of the HeV NTAIL-PXD interaction by NMR confirms the involvement of a molecular recognition element (MoRE) of α−helical nature in binding to PXD. I also showed that Henipavirus NTAIL-PXD form heterologous complexes, involving a structural similarity. As this interaction is crucial for the recruitment of the viral polymerase, it is a promising target for antiviral approaches. This prompted me to set up a protein-protein interaction (PPI) assay based on the HTRF technology to identify inhibitors. Finally, I provided the first experimental evidence of a trimeric organization of P proteins in NiV and HeV. We also solved the crystal structure of two different forms of MeV P tetramerization domain who unveiled the presence of a disordered region located near the putative L-binding site and reveal significant structural variations in coiled-coils organization. Collectively, these results represent a key step towards the elucidation of the functional impact of P protein oligomerization on the replicative cycle of paramyxoviruses.

Dans la cellule, les ARN pré messagers contenant des introns sont normalement retenus au noyau par leur interaction avec des facteurs d’épissage. Cependant, les ARN partiellement et non épissés des rétrovirus doivent entrer dans le cytoplasme pour servir de matrice pour la synthèse de certaines protéines telles que Env, Gag et Gag-Pol ainsi que d’ARN génomique qui sera empaqueté dans les nouveaux virions. Un mécanisme post-transcriptionnel utilisé par les lentivirus pour éviter la séquestration nucléaire de ces ARNm dépend d’une protéine virale appelée Rev. Pour assurer sa fonction d’exportation, Rev doit transiter entre le noyau et le cytoplasme et doit aussi pouvoir former des multimères. Par conséquent, Rev est dotée de domaines fonctionnels lui procurant ces habiletés. On retrouve le domaine riche en arginines qui contient le domaine de liaison à l’ARN et le signal de localisation nucléaire (NLS), un second domaine, riche en leucines, porte le signal d’exportation nucléaire (NES) et finalement le domaine de multimérisation. Bien que les protéines Rev du virus de l’immunodéficience humaine de type 1 (VIH-1) et bovine (VIB) aient été caractérisées, aucune étude n’a été réalisée pour la protéine Rev du virus de la maladie de Jembrana (JDV) et très peu sur le virus de l’immunodéficience féline (VIF). Comme les domaines fonctionnels et la voie d’importation des protéines Rev déjà caractérisées sont différents, nous supposons que chaque protéine Rev possède une organisation qui lui est propre et que les mécanismes de transport nucléo-cytoplasmique diffèrent entre les virus. Ce projet a pour objectif de caractériser ces domaines pour la protéine Rev du JDV et ceux du VIF ainsi que les mécanismes permettant leur transport nucléaire. L’utilisation de mutants de la protéine Rev de ces virus couplés à la protéine de fluorescente verte (EGFP) exprimés dans des cellules appropriées et observés par microscopie a permis d’identifier des séquences NLS et NES différentes de celles déjà caractérisées. Le NLS de la protéine Rev du JDV a été identifié et est composé des résidus arginines de la séquence 76-RRPARRPPIRR-87 avec un NoLS composé des mêmes résidus en plus des arginines R74, R103 et R104. Son NES est composé des résidus hydrophobes de la séquence 116-MAELEERFEDLAL-128 et est du type de l’inhibiteur de la protéine kinase (PKI pour « protéine kinase inhibitor »). Pour la protéine Rev du VIF, son NLS est composé des résidus basiques de la séquence 84-KKKRQRRRRKKKAFKK-99. Le NoLS est composé des mêmes acides aminés en plus du résidu K82. De plus, les essais d’importation nucléaires et d’interaction semblent indiquer que les voies d’importation utilisées diffèrent entre les virus et que plusieurs voies peuvent être utilisées. Ces travaux pourront éventuellement servir de base pour identifier de nouvelles cibles thérapeutiques contre les lentivirus. / In the cell, pre-messenger RNAs containing introns are normally retained in the nucleus by their interaction with splicing factors. However, the partially and unspliced ​​RNAs of retroviruses must enter the cytoplasm to serve as a template for the synthesis of certain proteins such as Env, Gag and Gag-Pol as well as genomic RNA to be packaged in the new virions. A post-transcriptional mechanism used by lentiviruses to prevent nuclear sequestration of these mRNAs depends on a trans-activator, the viral protein Rev. To ensure its export function, Rev must be able to shuttle between the nucleus and the cytoplasm and to form multimers. As a result, Rev has functional domains that provide these abilities: the arginine-rich domain, which contains the RNA binding domain and the nuclear localization signal (NLS), a second domain, rich in leucine, corresponding to the nuclear export signal (NES) and finally the multimerization domain. Although the Rev proteins of the human and bovine immunodeficiency virus (HIV-1 and BIV respectively) have been characterized, no studies have been performed for the Jembrana disease virus (JDV) Rev protein and very little on the feline immunodeficiency virus (FIV). Since the functional domains and import pathway of the already characterized Rev proteins are different, we assume that each Rev protein has its own organization and that the nucleo-cytoplasmic transport mechanisms differ between viruses. The goal of this project is to characterize these domains for the JDV and FIV Rev proteins as well as to elucidate mechanisms for their nuclear transport. The use of Rev mutants fused to the EGFP expressed in appropriate cells and observed by microscopy has identified NLS and NES sequences that differ from those already characterized. JDV Rev NLS is composed of arginine residues in the 76-RRPARRPPIRR-87 sequence with a NoLS composed of the same residues with the addition of arginine R74, R103 and R104. JDV Rev NES is composed of hydrophobic residues in the 116-MAELEERFEDLAL-128 sequence and is of the protein kinase inhibitor type (PKI). For the FIV Rev protein, its NLS is composed of basic residues in the 84-KKKRQRRRRKKKAFKK-99 sequence. FIV Rev NoLS is composed of the same residues with the addition of the lysine at position 82. In addition, the nuclear import and interaction tests suggest that the import routes used by Rev differ between the different viruses studied and that more than one import pathway may be used. This work could serve as a basis for identifying new therapeutic targets against lentiviruses.