Despite the progress made in cancer research, cancer remains one of the leading causes of death worldwide. Although the development of new cancer treatments has improved cancer patients' survival rate, a significant number of patients experience refractory and recurrence events with serious side effects. It is known that the immune system actively participates in eliminating cancer. However, cancer cells can develop mechanisms to evade the immune system resulting in immunotolerance. Immunotherapy aids the patient's immune system's ability to recognize and eliminate cancer cells. During the last three decades, immunotherapy has gradually emerged as an effective and more specific approach to treat cancer. Particularly monoclonal antibodies and adoptive cell therapies such as chimeric antigen receptor (CAR) T-cells have proven highly effective. Nevertheless, the success of these novel therapies depends on discovering suitable tumor targets. Recently, we reported localization of Thymidine Kinase 1 (TK1) to the plasma membrane of certain cancer cells but have not found such localization on normal cells. Similarly, another nucleotide salvage pathway enzyme Hypoxanthine Guanine Phosphoribosyltransferase (HPRT), has also been reported to be localized to the plasma membrane of certain cancer cells. Thus, TK1 and HPRT membrane-associated forms can be potential tumor targets for cancer immunotherapy. This dissertation describes the immunotargeting of TK1 for the selective elimination of tumor cells and the surface localization of HPRT on the plasma membrane of cancer cells. Using hybridoma and phage display technologies, we developed monoclonal antibodies (mAb) and isolated human single domain antibodies (sdAb) specific to human TK1. We confirmed that antibodies and sdAbs could target TK1 on the plasma membrane of lung, breast and colon cancer cells, but not on healthy cells. In addition, we demonstrated that cancer cells expressing membrane-associated TK1 (mTK1) co-cultured with human mononuclear cells (MNC) were selectively eliminated through antibody-dependent cell-mediated cytotoxicity (ADCC) when anti-TK1 mAbs were added. Furthermore, we designed novel TK1 specific tumor targeting receptors and expressed them in human T cells and human macrophages. Finally, we proposed using both TK1 and HPRT as biomarkers for the early detection and monitoring of follicular lymphoma (FL), a disease that is usually detected at advanced stages. The knowledge generated from the data presented in this dissertation indicates that TK1 and HPRT may be suitable immunotherapeutic targets for antibody-based and adoptive cell-based therapies against both liquid and solid malignancies. It also proposes the incorporation of TK1 and HPRT as molecular biomarkers for the early detection and monitoring of FL.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate Mr 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.

The SRP and SRP receptor have long been recognized as essential components of the protein translocation machinery in higher eukaryotes. The biochemical studies discussed in this thesis demonstrate that the signal recognition particle (SRP) mediated transport of proteins across the mammalian endoplasmic reticulum requires the participation of guanine ribonucleotides, in a capacity distinct from their role in polypeptide elongation. The requirement for guanine ribonucleotides during translocation was detected by experimentally separating the synthesis and transport phases of the translocation reaction. Here, the initial targeting of ribosomes to the membrane required SRP and an SRP receptor, but not GTP. However, the insertion of the nascent chain into the membrane required the presence of both SRP and SRP receptor, as well as, GTP. Further biochemical characterization of the initially targeted translocation intermediate demonstrated that SRP remains bound to targeted nascent signal sequences, unless GTP is present. The SRP-receptor catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor preparations. GTP specific binding localized to the α subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. In addition, an analysis of the α subunit primary sequence revealed elements which are similar, yet not identical, to guanine ribonucleotide binding site consensus sequence elements. These results, taken together, indicate that the SRP receptor represents a novel class of GTP binding protein and is responsible for the guanine ribonucleotide mediated displacement of SRP from nascent signal sequences. A more detailed biochemical investigation of the GTP hydrolysis cycle of the SRP receptor demonstrated that the affinity between SRP and the SRP receptor is substantially greater in the presence of bound GTP and that the subsequent hydrolysis of bound GTP by SRα is necessary to recycle SRP to the cytoplasm. Purified SRP receptor was shown to hydrolyze GTP slowly. However, the GTP hydrolysis rate was substantially increased when both the SRP receptor and SRP were present in equimolar quantity. SRP does not hydrolyze GTP under these assay conditions. Moreover, free SRP was found not to compete effectively with SRP-ribosome complexes for the receptor, implying that the conformation of SRP is altered upon binding to a signal sequence. This result suggests that the affinity between SRP and the SRP receptor may be exquisitely regulated in order to prevent futile GTP hydrolysis cycles from occurring in the absence of secretory protein synthesis. Furthermore, the demonstration that the SRP receptor is a GTP binding protein provides fundamental insight into the mechanism of protein translocation. The displacement of SRP appears to be tightly coupled to the membrane insertion of nascent signal sequences. The membrane inserted intermediate in nascent chain translocation can be characterized by i) a resistance to extraction from the membrane with either EDTA or 0.5M KOAc; ii) an insensitivity to protease digestion, even after dissolution of the membrane with nonionic detergent. These results indicate that SRP displacement allows the nascent chain to interact with an additional membrane bound, protein component of the cellular translocation apparatus. Once in contact with this additional component, the nascent chain is shown to be capable to transverse the membrane bilayer in the absence of ribonucleotide hydrolysis or the continued elongation of the polypeptide. Thus, the results are incompatible with postulated mechanisms of protein translocation requiring that energy be derived from the continued elongation of the nascent polypeptide or from the direct interaction of a hydrophobic signal sequence with the lipid bilayer.

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

No description available.

No description available.

Les oxydations sur les bases nucléiques constituent l’une des sources principale d’apparition de lésions sur l’ADN, qui peuvent être mutagènes ou létales pour les cellules en l’absence de réparation de l’ADN. La Formamidopyrimidine-ADN glycosylase (Fpg), une enzyme procaryote du système de réparation de l’ADN par excision de base (BER), initie la réparation d’un large panel de lésions de ce type via ses activités ADN glycosylase (excision de la base oxydée) et AP lyase (clivage du site abasique par ß,d-élimination). Nous avons réalisé des études fonctionnelles par des techniques biochimiques et structurales par cristallographie des rayons X afin de préciser la spécificité de substrat et le mécanisme catalytique de Fpg. Ainsi, nous avons pu mettre en évidence des déterminants structuraux permettant à cette enzyme d’accommoder des lésions de tailles très différentes dans son site actif, en l’occurrence des résidus 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) substitués ou non en N7 par des adduits encombrants. D’autre part, nous avons caractérisé structuralement et fonctionnellement la reconnaissance et l’excision par Fpg d’une lésion pyrimidique, la 5-hydroxy-5-méthyle-hydantoïne (Hyd). Ainsi, nous avons montré que cette lésion appariée à une cytosine était un bon substrat pour l’enzyme, et nous avons précisé structuralement le mode de reconnaissance de l’Hyd par Fpg. D’autre part, nous avons mis en évidence un comportement inattendu de l’enzyme sur ce substrat. En l’occurrence, nous avons montré biochimiquement et structuralement qu’un pontage covalent se formait en quantités non négligeables entre Fpg et l’Hyd dans des conditions physiologiques. / Oxidations on nucleic bases constitute one of the major sources of DNA lesions appearance, which can be mutagenic or lethal for cells in the absence of DNA repair. The prokaryotic Formamidopyrimidine-DNA glycosylase (Fpg), a base excision DNA repair (BER) enzyme, initiate the repair of a wide range of such lesions via its DNA glycosylase (excision of the oxidized base) and AP lyase (cleavage of the AP site by ß,d-elimination) activities. We carried out functional studies by biochemical techniques and structural studies by X-ray crystallography so as to state Fpg’s substrate specificity and catalytic mechanism. Thus, we have been able to underline the structural determinants enabling this enzyme to accommodate lesions of very different sizes in its active site, in this case 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) residues N7-substituted or not by bulky adducts. On the other hand, we structurally and functionally characterized the recognition and excision by Fpg of a pyrimidic lesion, the 5-hydroxy-5-methyl-hydantoin (Hyd). Thus, we have shown that this lesion paired with a cytosine was a good substrate for the enzyme, and stated structurally the recognition mode of Hyd by Fpg. On the other hand, we have underlined an unexpected behaviour of the enzyme on this substrate. In this case, we have biochemically and structurally shown that a covalent link was formed in sizeable quantities between Fpg and Hyd in physiological conditions.

O presente trabalho teve como objetivos a clonagem, expressão e purificação da proteína HGPRT de Leishimania tarentolae, para a caracterização e cristalização dessa enzima, a fim do seu estudo estrutural e funcional. O gene da HGPRT foi amplificado a partir de uma biblioteca genômica de Leishmania tarentolae da cepa UC Lambda ZAP Express BamHI-Sal3A I. Esse gene foi clonado no vetor de expressão pET29a(+) e usado na transformação da bactéria Escherichia coli BL21 (DE3). Esse sistema de expressão apresentou uma superexpressão da proteína recombinante, que foi purificada em cromatografia de forma iônica, com o uso da coluna de troca anônica POROS 20HQ. A proteína purificada foi utilizada para sua caracterização, como a determinação das constantes cinéticas (Km, Vmax e Kcat) para os diferentes substratos. A proteína foi encontrada como dímero em solução e o pI de aproximadamente 8,2. Além disso, essa proteína foi utilizada nos ensaios de cristalização pelo método de difusão de vapor em gotas suspensas. Os cristais obtidos foram utilizados nos testes da difração de raios-X sendo coletado um conjunto de dados no Laboratório Nacional de Luz Síncroton. Os dados de difração de raios-X foram processados com o uso do pacote de programa HKL. A resolução da estrutura cristalográfica foi obtida pelo método de substituição molecular através do programa AmoRe. Para o refinamento da estrutura foram utilizados os programas de refinamento automático CNS e REFMAC, e a manipulação na estação gráfica foi realizada através do programa O. A estrutura da HGPRT foi resolvida a 2,1 &#197 de resolução com os fatores de concordância Rwork e Rfree finais de 17,34 e 21,53%, respectivamente. / The aims of this work were the cloning, expression and purification of the protein HGPRT from Leishimania tarentolae, for the characterization and crystallization of the enzyme for it structural and functional study. The full-length hgprt gene was isolated from a leishmania tarentolae UC strain Lambda ZAP Express BamHI-Sal3A I genomic library. This gene was cloned in the expression vector pET29a+, and used in the transformation of the bacteria Escherichia coli BL21(DE3). This expression system presented a super-expression of the recombinant protein, which was purified by ionic change chromatography, utilizing the anionic change column POROS 20HQ. The purified protein was utilized for its characterization, including its kinetics constants (Km, Vmax e Kcat) for different substrates. The protein was found to be a dimmer in solution with pI close to 8.2. Besides, this protein was used in the crystallization assays by the steam diffusion in suspended drop technique. The obtained crystals were utilized in the x-ray diffraction studies. The data collection was performed in the Laboratório Nacional de Luz Síncroton. The X-ray diffraction data were processed utilizing the package program HKL. The crystallographic structure resolution was obtained by the molecular replacement method from the AmoRe program. For the structure refinement, the automatic refinement programs CNS and REFMAC were used, and the graphic manipulation was made through the O program. The structure of the HGPRT was resolved with 2,1 &#197 of resolution and final agreement factors Rwork and Rfree of 17,34% and 21,53%, respectively.

O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos. / Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.

O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos. / Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.