Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Nop53 e uma protena nucleolar, conservada evolutivamente e essencial na levedura Saccharomyces cerevisiae para a biogênese da subunidade maior do ribossomo, 60S. O principal fenotipo causado pela repressão da expressão de Nop53 e o acumulo do intermedi ario de processamento de pre-Rrna, 7S, que tambem e substrato do complexo exossomo na formação do rRNA maduro 5:8S. Nop53 interage diretamente com a subunidade do exossomo Rrp6 e com a subunidade Mtr4 do co-ativador do exossomo TRAMP. O objetivo principal deste trabalho foi o de analisar como a interação entre Nop53 e o exossomo pode modular a atividade deste ultimo. Para isso, foram utilizados metodos bioqumicos, geneticos e de biologia molecular. Os resultados mostrados aqui demonstram que a depleção de Nop53 faz com que mais protenas ribossomais, principalmente da subunidade maior, sejam co-imunoprecipitadas com o core do exossomo, sugerindo que Nop53 possa ter um papel na liberação do exossomo da subunidade pre-60S depois da formação do rRNA maduro 5:8S. Esta hipotese foi conrmada atraves da separação de complexos por centrifugação em gradiente de glicerol, que mostrou a presenca de subunidades do exossomo em complexos maiores na ausência de Nop53, provavelmente correspondendo a partculas pre-ribossomais. Co-imunoprecipitação de RNA com o exossomo na ausência de Nop53 tambem conrmou uma maior associação deste complexo com o pre-rRNA 7S. Como tambem mostrado aqui, alem de interagir com Rrp6, Nop53 interage com subunidades do core do exossomo e a superexpressão de uma destas subunidades, Rrp43, complementa parcialmente a ausência de Nop53 na celula. Estes resultados levaram a conclusão de que Nop53 pode recrutar o exossomo para a partcula ribossomal pre-60S para a maturação do pre-rRNA 7S a 5:8S, e atue tambem na liberação do exossomo, possivelmente atraves de sua interação com a helicase Mtr4. / Abstract Nop53 is a nucleolar, conserved and essential protein in the yeast Saccharomyces cerevisiae, involved in the biogenesis of the large ribosomal subunit 60S. The main phenotype of the depletion of Nop53 in yeast cells is the accumulation of the prerRNA processing intermediate 7S, which is also the substrate of the exosome complex for the formation of the mature rRNA 5:8S. Nop53 directly interacts with the exosome subunit Rrp6, and with the subunit Mtr4 of the TRAMP complex, an exosome co-activator. The main objective of this work was the analysis of the interaction between Nop53 and the exosome and the identication of the mechanism through which Nop53 regulates the exosome activity. The results shown here demonstrate that the depletion of Nop53 leads to a more stable association of the exosome with the pre-60S ribosome particle, as determined by co-immunoprecipitation of proteins with one of the exosome core subunits, and by fractionation of complexes through glycerol gradients. These results suggested that Nop53 could play a role in the release of the exosome after the formation of the mature rRNA 5:8S. This hypothesis was conrmed through the co-immunoprecipitation of pre-rRNA 7S with the exosome in the absence of Nop53. In addition to the interaction with the exosome subunit Rrp6, as shown here, Nop53 also interacts with core subunits of the complex. Interestingly, overexpression of one of these subunits, Rrp43, partially complements the depletion of Nop53. These results led to the conclusion that Nop53 may recruit the exosome to the pre-60S particle for the maturation of the pre-rRNA 7S to the mature 5:8S, but Nop53 may also be involved in the release of the exosome, possibly through its interaction with the helicase Mtr4.

Pulchellina é uma proteína inativadora de ribossomo (RIP) isolada de sementes de Abrus pulchellus fragmento que codifica a cadeia A da pulchellina (PAC) foi clonado e inserido no vetor pGEX-5X para expressar a cadeia A recombinante (rPAC) como uma proteína de fusão em Escherichia coli. A análise da seqüência de aminoácidos mostrou que a rPAC apresenta uma alta identidade seqüencial (&#62 86%) com a cadeia A da abrina-c. A habilidade que a rPAC possui para depurinar rRNA em ribossomos de levedura também foi demonstrada em testes in vitro. Objetivando verificar a atividade tóxica do produto heterólogo, nós promovemos a associação in vitro da rPAC com a cadeia B recombinante da pulchellina (rPBC). Ambas as cadeias foram incubadas na presença de um sistema de redução/oxidação, originando um heterodímero ativo (rPAB). O rPAB apresentou uma massa molecular aparente de aproximadamente 60 kDa, similar a pulchellina nativa. As atividades tóxicas do rPAB e da pulchellina nativa foram comparadas através da injeção intraperitonial em camundongos, usando diferentes diluições de cada proteína. O rPAB foi capaz de matar 50% dos animais testados com doses de 45&#956g.kg-1. Nossos resultados mostraram que o heterodímero recombinante apresenta tanto toxicidade quanto um padrão conformacional similar a pulchellina nativa. Estudos usando cultura de tecidos também foram realizados com o objetivo de investigar a presença da pulchellina em calos obtidos a partir de sementes de A. pulchellus. Segmentos de cotilédones de sementes imaturas foram inoculados em meio MS suplementado com diferentes concentrações de auxina, citocinina e sacarose para promover a indução dos calos. A expressão da pulchellina nos calos foi monitorada através de RT-PCR e testes de atividade biológica. Os calos obtidos após 35 dias foram congelados, macerados e submetidos a extração de RNA total e proteínas. Um fragmento específico de DNA que codifica a cadeia A da pulchellina foi amplificado a partir do RNA total sugerindo a síntese da proteína nos calos. Isto foi confirmado no extrato bruto de calos, que mostrou atividade hemaglutinante contra sangue de coelho e uma alta toxicidade quando injetado via intraperitoneal em camundongos.O extrato bruto também foi submetido à cromatografia de afinidade em coluna de Sepharose-4B. A fração retida na coluna apresentou duas bandas protéicas quando analisadas em gel de poliacrinamida, sob condições desnaturantes, apresentando um padrão similar ao obtido com a pulchellina de semente. / Pulchellin is a type 2 ribosome-inactivating protein (RIP) isolated from seeds of the Abrus pulchellus tenuiflorus plant. The DNA fiagment encoding Pulchellin A-chain (PAC) was cloned and inserted in pGEX-5X to express the recombinant pulchellin Achain (rPAC) as a fusion protein in Escherichin coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (&#62 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. Intending to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reducedloxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of about 60 D a similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection in mice using different dilutions. The rPAB was able to kill 50% of the tested mice with doses of 45&#956g.kg-1. Our results indicated that the recombinant heterodimer presented toxic activity and a conformational pattern similar to pulchellin. Studies using tissue cultures were also performed to investigate the presence of the pulchellin in callus established from seed explants of A. pulchellus. Cotyledon segments of immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin, citokinin and sucrose in order to determine the best callus induction. The pulchellin expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained aRer 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins. A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the synthesis of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to the results obtained with seeds pulchellin.

As the first structural example of this class of proteins, crystals of Pro-RIP and MOD were grown and diffracted to 2.4 and 2.5 A respectively. The structures of the two proteins are solved and found to be highly similar, with main chain RMSD of 0.519. Each protein has two domains. The N-terminal domain consists of five alpha-helices and five-stranded mixed beta-sheet. The conserved active site residues Y94, Y130, E207, R210 and W241, similar to those of other RIPs, are located at the cleft between the N-terminal and C-terminal domains. In Pro-RIP, the 25-amino acid internal inactivation region is found on the surface of the N-terminal domain and consists of a flexible loop followed by a long alpha-helix. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure of maize RIP reveals that the active site is too small to accommodate two glutamate residues and suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. / Pull-down assay indicated that the internal inactivation region diminished the interaction of Pro-RIP with purified ribosomes and ribosomal proteins P0, P1 and P2. Surface plasmon resonance assays showed that Pro-RIP has a slower association rate and faster dissociation rate on intact ribosomes when compared to MOD, resulting 80-fold decrease in binding affinity. These evidences strongly suggested that the reduced ribosome-inactivating activity and cytotoxicity of Pro-RIP is the result of its diminished interaction with the ribosomes. The ribosome binding site of MOD is found to be different from TCS and saporin, which are located between the anti-parallel beta-sheet in the C-terminal domain. In MOD, the positive-charged residues K158, K159, K160 and K161 that were found to be important for ribosome binding are located in the N-terminal domain, underneath the internal inactivation region. / Ribosome-inactivating proteins (RIPs) are rRNA N-glycosidases, which hydrolyze the N-glycosidic bond of A-4324 in 28S rRNA of eukaryotic ribosomes. Based on the number of subunits, RIPs are grouped into three classes. Type I RIPs (e.g. trichosanthin and saporin) are monomeric polypeptide with molecular weights of 25-32 kDa. Type II RIPs (e.g. ricin and cinnamomin) are heterodimeric proteins whose subunits are linked by a disulphide bridge, with molecular weights of 60-65 kDa. Chain A of type II RIPs is the catalytic subunit, while chain B is the lectin subunit, which facilitates the cellular entry of the protein by interacting with carbohydrates on the cell surface. Maize ribosome-inactivating protein is classified as a type III RIP, or an atypical RNA N-glycosidase. It is synthesized and stored in the kernel as a 34 kDa inactive precursor (Pro-RIP). During germination, the precursor undergoes proteolysis to generate a two-chain active RIP (MOD). Previous study has found that the 25-amino acid residues at the acidic internal inactivation region, which are removed during activation of Pro-RIP, is the major control element to suppress its in vitro protein synthesis inhibition activity. / Since the internal inactivation region of Pro-RIP controls the ribosome-inactivating activity and cytotoxicity, it provides an opportunity to engineer an on/off switch forits activity by HIV-1 protease through engineering HIV-1 protease recognition sites into the internal inactivation region of Pro-RIP. A variant that contains two HIV-1 protease recognition sites incorporated to the 25-amino acid internal inactivation region was found to be activated by HIV-1 protease in vitro. This variant entered cells more efficiently than Pro-RIP and was as cytotoxic as MOD. This switch may be applied to other RIPs such as ricin A chain and other protease recognition sequences may be used for increasing the specificity of an RIP toward viral infected cells. / The internal inactivation region of Pro-RIP greatly decreases its cytotoxicity, but not cellular uptake through alpha-2 macroglobulin receptor. On the contrary, the acidic residues within the region hinder fluid-phase endocytosis. Moreover, it is found that the internal inactivation region does not affect sub-cellular localization of the protein - MOD and Pro-RIP locate in the same cellular compartment (nucleus in JAR or cytoplasm in J774A.1 and C8166). / Mak, Nga Sze Amanda. / "July 2007." / Adviser: Shaw Pang Chui. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 216-236). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ribosome-inactivating proteins (RIPs) are cytotoxins that remove a specific adenine from the sarcin-ricin loop (SRL) of large ribosomal RNA and in turn inhibit protein synthesis. Apart from N-glycosidase activity, some RIPs are found to possess antiviral activity and the suppression on human immunodeficiency virus (HIV) has been extensively studied. / Maize RIP stands out from other members for having an internal inactivation region and requires proteolytic removal to regain full activity. We have exploited the innate regulatory mechanism of maize RIP and increased its specificity towards HIV by adding the HIV protease recognition site to the inhibitory segment. Our results demonstrated the wild-type maize RIP is inhibitory on simian immunodeficiency virus (SIV) replication in rhesus macaque and showed the HIV sensitive variant undergoes specific proteolytic activation upon viral infection and exerts enhanced in vitro antiviral effects. Therapeutic applications of RIPs are often restricted by short in vivo half-life and strong allergic responses and we attempted to improve the therapeutic potential of maize RIP by coupling with polyethylene glycol (PEG). Two mutants were generated for PEGylation and the resultant MOD-PEG₂₀ₖ variant was shown to be less sensitive to antibody recognition and has a prolonged plasma half-life, suggesting it may have enhanced therapeutic values. Besides, the applicability of protease-activation system in RIPs without inactivation loop was tested using ricin A chain (RTA) as the test case and HIV recognition sites were introduced either within or at C-terminus of the protein. The C-terminal RTA variants were specifically processed and had the anti-HIV activity increased in HIV-infected cells. / The present work illustrates the potential development of maize RIP as an anti-HIV agent and shows PEGylation can serve to enhance the protein for in vivo applications. Besides, the engineering of RTA with HIV recognition site suggests the potential of the protease-activation strategy to other RIPs for activity control. / 核糖體失活蛋白（RIPs）是一種細胞毒素蛋白，能特異地水解核糖體sarcin-ricin環（SRL），通過脫嘌呤抑制核糖體的蛋白合成功能。除此功能以外，很多RIP還具有抗病毒的活性，如抗人免疫缺陷病毒（HIV）的活性。 / 玉米RIP與其他的RIP不同，它含有一段內部失活結構域，需通過蛋白水解作用移除該結構域才能成為活性體。我們利用玉米RIP的這一特性，通過對內部結構域的改造，獲得了兩個可被HIV蛋白酶特異識別的突變體。體外實驗証明HIV可以特異地識別突變體上的蛋白酶切割位點，從而將其啟動產生抗病毒活性。另外，我們以蓖麻毒素A鏈（RTA）為例，分別於蛋白質的內部和碳端插入HIV識別序列，驗證了蛋白酶啟動系統在沒有內部失活結構域的RIP中，也能通過HIV蛋白酶的切割而活化並取得抗病毒活性。我們還揭示玉米RIP活性體可以有效抑制猿免疫缺陷病毒（SIV）在感染恒河猴體內的複制。此外，我們嘗試通過與聚乙二醇（PEG）融合來優化玉米RIP的免疫原性和半衰期，成功製備了兩種融合突變體，MOD-PEG₂₀ₖ顯示較不容易被抗體識別且延長了血液半衰期。 / 綜上所述，我們的研究表明玉米RIP作為抗HIV抑制劑具有良好的研發前景，而RTA的改造展示蛋白酶啟動系統可應用於其他RIP，同時我們還證明了PEG修飾可以很好的應用於蛋白類藥物的研發。 / Au, Ka Yee. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Title from PDF title page (viewed on 17, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Erythromycin has long been recognized for its ability to inhibit protein synthesis by interfering with mRNA translation on the bacterial ribosome. We have recently shown that erythromycin also inhibits the assembly of the 50S ribosomal subunit in growing bacterial cells. The nature of this assembly inhibition has been investigated using 3H-uridine pulse-chase labeling of control and erythromycin treated E. coli cells. Subunit assembly was examined by sucrose gradient centrifugation of labeled cell lysates. Normal assembly kinetics of subunit assembly were observed in control cells at 37°C. Formation of the 30S subunit was completed by 7.5 minutes and assembly of the 50S subunit was finished by 15 minutes after an unlabeled uridine chase. At 37°C, in the presence of erythromycin, 30S subunit assembly was unaffected but 50S assembly was greatly reduced. When the assembly kinetics were examined at 27°C, the assembly of both subunits was slowed and 30-32S precursor particle was seen to accumulate. This particle was found to bind 14C-erythromycin in vivo and in vitro. RNase E has been implicated in the normal degradation and turnover of rRNA. A RNase E-mutant accumulated substantially more precursor to the 50S subunit than did control cells at either 37°C or 27°C. This precursor particle was also found to bind 14C-erythromycin. Specific 50S proteins and the 23S and 5SrRNAs were found in the 30S gradient region from lysates of cells grown at 27°C, confirming the presence of a 50S subunit precursor co-sedimenting with 30S subunits under these conditions. The precursor particle in the RNase E-mutant had a larger number of associated 50S proteins thandid the precursor from SK901. These data are consistent with our model of 50S subunit inhibition by erythromycin in which a fraction of 50S precursor particles undergo degraded.

Les organismes cellulaires appartiennent à l'un des trois domaines du vivant : Archaea, Bacteria, Eucarya. Les Archaea sont des organismes unicellulaires avec un phénotype bactérien mais qui possèdent de nombreux caractères moléculaires eucaryotes. En particulier, la machinerie de réplication archéenne est une version homologue et simplifiée de celle des eucaryotes. Au cours de cette thèse, j'ai étudié la réplication de l'ADN chez les Archaea en combinant des approches in vitro et in silico.<br />Premièrement, j'ai essayé de purifier la protéine initiatrice de la réplication Cdc6/Orc1, sous une forme native, dans l'espoir de mettre au point le premier système de réplication de l'ADN in vitro chez les Archaea. Malheureusement, cette approche a été infructueuse en raison de l'instabilité et des propriétés d'agrégation de la protéine.<br />Deuxièmement, j'ai réalisé une analyse comparative du contexte génomique des gènes de réplication dans les génomes d'Archaea. Cette analyse nous a permis d'identifier une association très conservée entre des gènes de la réplication et des gènes liés au ribosome. Cette organisation suggère l'existence d'un mécanisme de couplage entre la réplication de l'ADN et la traduction. De manière remarquable, des données expérimentales obtenues chez des modèles bactériens et eucaryotes appuient cette idée. J'ai ensuite mis au point des outils expérimentaux qui permettront d'éprouver la pertinence biologique de certaines des prédictions effectuées.<br />Finalement, j'ai examiné la distribution taxonomique des gènes de la réplication dans les génomes d'Archaea afin de prédire la composition probable de la machinerie de réplication de l'ADN chez le dernier ancêtre commun des Archaea. Dans leur ensemble, les profils phylétiques des gènes de la réplication suggèrent que la machinerie ancestrale était plus complexe que celle des organismes archéens contemporains.

Cell replication and division are central to the proliferation of life, and have implications for normal growth and development as well as disease state. Assembly of a complete picture of the systems which control this process requires identification of individual genetic components, but the identity and complete sequence of events that trigger initiation of cell division, at a point called START in yeast, remain unknown. Here, we evaluated panels of non-essential single gene deletion strains and tested the effects of FDA-approved drugs on cell-cycle progression, using flow cytometry to detect altered DNA content. Previous studies relied mainly on cell size changes to systematically identify genes required for the timely completion of START. This analysis revealed that most gene deletions that altered cell-cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell-cycle control mechanisms. We found that cystathionine-beta-synthase (CBS) advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function which does not require that activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, it is generally not known whether the compounds tested alter the timing of particular cell-cycle transitions. Our approach revealed strong cell-cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell-cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation. Our studies not only transform our view of START, but also expand the repertoire of genetic and chemical means to modulate the eukaryotic cell cycle.

La protéine kinase TOR (Target Of Rapamycin) a été identifiée chez la levure et les mammifères comme participant à deux complexes protéiques qui servent de carrefour entre la perception des facteurs endogènes et exogènes et la stimulation de la croissance cellulaire. Depuis la découverte de la kinase AtTOR chez Arabidopsis thaliana, des études ont été menées afin de mieux caractériser son rôle chez les plantes et l'influence de son niveau d'expression sur la régulation du métabolisme et du développement.Au cours de ce travail, j'ai contribué à l'étude de cette kinase en étudiant l'influence de l'inactivation de TOR sur la composition du ribosome au niveau protéique et sur le niveau de phosphorylation de ces protéines, ainsi que sur l'organisation du méristème au niveau moléculaire et cytologique Au cours de cette étude, j'ai montré que certaines protéines constitutives du ribosome pourraient être des cibles de l'activité TOR au niveau de leur abondance et/ou de leur état de phosphorylation. Ainsi, l'inactivation de TOR entraine une diminution du niveau de phosphorylation des protéines RPS6 et pourrait influencer l'abondance des protéines acides constitutives du stalk ribosomal, une structure importante dans la régulation de la traduction. Les résultats obtenus suggèrent également que l'activité TOR est nécessaire au maintien du méristème à l'état fonctionnel en régulant les voies importantes contrôlant la division et la différentiation au sein de cette structure.