Biopeptides are synthesised as large pro-protein precursors that have to undergo proteolytic cleavage at positively charged amino acids (Lys and Arg) in order to become active. This cleavage is mediated by a family of subtilin/kexin related calcium dependent serine endoproteinases named prohormone convertases. The present thesis focuses on the endocrine member of the family named PC1/3. PC1/3 is expressed in the regulated secretory pathway of endocrine and neuroendocrine cells, where it was shown to activate various peptide hormones such as proopiomelanocortin (POMC), pro-insulin and pro-glucagon. PC1/3 is synthesized as a large precursor containing a signal peptide, a propeptide, a catalytic domain, a P domain and a C-terminal domain. The activation of the enzyme requires the sequential removal of the signal peptide, the propeptide and ultimately the C-terminal domain. / The structural characterisation of the enzyme is compromised by the difficulty in producing a sufficient amount of recombinant PC1/3. In this thesis it is clearly demonstrated that the production of PC1/3 using Baculovirus technology can be greatly improved by modifying the expression vector in insect cells (Spodoptera frugiperda). In addition, the intracoelemic injection of insect larvae (Tricoplusia ni) with the Baculovirus encoding the recombinant PC1/3 is shown to be a very efficient method for the production of a large amount of prohormone convertases. / It was previously demonstrated that the propeptide is essential for the folding of the enzyme and act as a tight binding inhibitor of the enzyme until the latter reaches the appropriate compartment for substrate cleavage. To assess the role of certain residues within the propeptide in the inhibition of the cognate enzyme, a mutational analysis by alanine scan was conducted. The results demonstrate that the substitution of a single amino acid can affect markedly the inhibition behavior, potency and selectivity of the propeptide towards the enzyme. Moreover, this mutational analysis allowed the first experimental mapping of the sequence involved in propeptide degradation once its function is achieved. / However, PC1/3 also possesses a C-terminal domain which must also be cleaved to allow the full activation of the enzyme. Previous studies showed that this domain is implicated in the sorting of the enzyme to secretory granules. In addition, over expression experiments showed that the C-terminal domain can inhibit the cleavage of certain substrates by PC1/3. The results, presented here, suggest that the CT-peptide acts as a non-essential activator of PC1/3, in vitro, which adds a supplementary level of complexity to the activation process of the enzyme. / Finally, based upon our results, it can be proposed that PC1/3 is a very complex enzyme capable of controlling its enzymatic activity through the coordinate action of its various domains. This exceptional mode of self-regulation is unique among all protease families.

No description available.

Thesis (Ph.D.). / Title from title page of PDF (viewed 2008/01/30). Written for the Dept. of Medicine, Division of Experimental Medicine. Includes bibliographical references.

A hipercolesterolemia familiar (HF) é uma alteração de origem genética comum que pode se manifestar clinicamente desde o nascimento e provoca um aumento nos níveis plasmáticos de LDL-colesterol (LDL-c), xantomas e doença coronária prematura. Sua detecção e tratamento precoce reduzem a morbidade e mortalidade coronária. A identificação e rastreamento em cascata familiar usando níveis de LDL-c e detecção genética é a estratégia mais aconselhável e rentável para descoberta de novos casos. O tratamento crônico com estatinas reduz o risco cardiovascular da população em geral, contudo, estudos clínicos com estatinas revelam risco cardiovascular residual mesmo após correção das concentrações de LDL-c. Com o surgimento de novas drogas e mais recentemente um inibidor da enzima pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), este estudo enfatizou na investigação específica para aqueles acometidos com defeitos genéticos nessa enzima, por ser de frequência ainda mais rara e pouco estudada, necessitando de melhor investigação na população em estudo a fim de rastrear a ocorrência de mutações patológicas na PCSK9. O objetivo desse estudo foi identificar e caracterizar mutações e/ou deleções patológicas no gene PCSK9 em pacientes com Hipercolesterolemia Familiar provenientes do Hospital das Clínicas de Ribeirão Preto da FMRP/USP selecionados para o teste genético. Foi feito o rastreamento de mutações pelo método Hight Resolution Melting (HRM), de forma prática, rápida e eficiente, onde mutações detectadas foram seqüenciadas. Foram identificadas 7 mutações não patogênicas, caracterizando que a população estudada não apresenta Hipercolesterolemia Familiar associada a mutações no gene PCSK9, fato que não exclui o diagnóstico por outros defeitos genéticas associados a doença. / Familial hypercholesterolemia (FH) is an alteration of common genetic origin that can manifest clinically from birth and which causes an increase in the LDL-cholesterol plasma levels (LDL-c), xanthomas and premature coronary disease. Its early detection and treatment reduce morbidity and coronary mortality. The identification and tracking in familial cascade using levels of LDL-c and genetic detection is the most advisable and profitable strategy to find new cases. The chronic treatment with statins reduces the cardiovascular risk in the population in general. However, clinic studies on statins show a residual cardiovascular risk even after the correction of LDL-c concentrations. With the appearance of new drugs and, more recently, of a proprotein convertase subtilisin/kexin type 9 enzyme inhibitor (PCSK9), this study highlighted the specific investigation for those stricken by genetic defects in this enzyme, once it is even rarer and understudied and needs further investigation in the study\'s population aiming at tracking the occurrence of a pathological mutation in the PCSK9. This study aimed at identifying and characterizing mutations and/or pathological deletions in the PCSK9 gene in patients with Familial Hypercholesterolemia from the RPMS/USP Ribeirão Preto Clinical Hospital which were selected for the genetic test. We performed the mutation tracking by using the High Resolution Melting (HRM) method in a practical, fast and efficient way, where the mutations detected were sequenced. We identified 7 non-pathogenic mutations, showing that the population studied does not present Familial Hypercholesterolemia associated to mutations in the PCSK9 gene, which doesn\'t exclude the diagnosis by other genetic defects associated to the disease.

A hipercolesterolemia familiar (HF) é uma alteração de origem genética comum que pode se manifestar clinicamente desde o nascimento e provoca um aumento nos níveis plasmáticos de LDL-colesterol (LDL-c), xantomas e doença coronária prematura. Sua detecção e tratamento precoce reduzem a morbidade e mortalidade coronária. A identificação e rastreamento em cascata familiar usando níveis de LDL-c e detecção genética é a estratégia mais aconselhável e rentável para descoberta de novos casos. O tratamento crônico com estatinas reduz o risco cardiovascular da população em geral, contudo, estudos clínicos com estatinas revelam risco cardiovascular residual mesmo após correção das concentrações de LDL-c. Com o surgimento de novas drogas e mais recentemente um inibidor da enzima pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), este estudo enfatizou na investigação específica para aqueles acometidos com defeitos genéticos nessa enzima, por ser de frequência ainda mais rara e pouco estudada, necessitando de melhor investigação na população em estudo a fim de rastrear a ocorrência de mutações patológicas na PCSK9. O objetivo desse estudo foi identificar e caracterizar mutações e/ou deleções patológicas no gene PCSK9 em pacientes com Hipercolesterolemia Familiar provenientes do Hospital das Clínicas de Ribeirão Preto da FMRP/USP selecionados para o teste genético. Foi feito o rastreamento de mutações pelo método Hight Resolution Melting (HRM), de forma prática, rápida e eficiente, onde mutações detectadas foram seqüenciadas. Foram identificadas 7 mutações não patogênicas, caracterizando que a população estudada não apresenta Hipercolesterolemia Familiar associada a mutações no gene PCSK9, fato que não exclui o diagnóstico por outros defeitos genéticas associados a doença. / Familial hypercholesterolemia (FH) is an alteration of common genetic origin that can manifest clinically from birth and which causes an increase in the LDL-cholesterol plasma levels (LDL-c), xanthomas and premature coronary disease. Its early detection and treatment reduce morbidity and coronary mortality. The identification and tracking in familial cascade using levels of LDL-c and genetic detection is the most advisable and profitable strategy to find new cases. The chronic treatment with statins reduces the cardiovascular risk in the population in general. However, clinic studies on statins show a residual cardiovascular risk even after the correction of LDL-c concentrations. With the appearance of new drugs and, more recently, of a proprotein convertase subtilisin/kexin type 9 enzyme inhibitor (PCSK9), this study highlighted the specific investigation for those stricken by genetic defects in this enzyme, once it is even rarer and understudied and needs further investigation in the study\'s population aiming at tracking the occurrence of a pathological mutation in the PCSK9. This study aimed at identifying and characterizing mutations and/or pathological deletions in the PCSK9 gene in patients with Familial Hypercholesterolemia from the RPMS/USP Ribeirão Preto Clinical Hospital which were selected for the genetic test. We performed the mutation tracking by using the High Resolution Melting (HRM) method in a practical, fast and efficient way, where the mutations detected were sequenced. We identified 7 non-pathogenic mutations, showing that the population studied does not present Familial Hypercholesterolemia associated to mutations in the PCSK9 gene, which doesn\'t exclude the diagnosis by other genetic defects associated to the disease.

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a negative regulator of the low-density lipoprotein receptor, and PCSK9 inhibition has become an important cholesterol-lowering therapeutic strategy. PCSK9 also associates with LDL particles, and evidence suggests that the activity of PCSK9 may be regulated by LDL binding. We have investigated the biochemistry of the interaction between PCSK9 and lipoproteins. Through mutagenesis and in-vitro binding assays, we found conserved motifs in the PCSK9 N-terminus that play a role in LDL binding. Through secondary structure studies using circular dichroism and computational modelling, we determined that the N-terminal region of the PCSK9 prodomain undergoes an environment-dependent structural shift that affects the ability of PCSK9 to bind LDL. We also found that the commonly found loss-of-function polymorphism R46L is capable of modulating this structural shift. Importantly, we found a surface-exposed region of the PCSK9 prodomain that maps a cluster of gain-of-function mutations (L108R, S127R, and D129G) that severely disrupt LDL binding. Through gel shift assays and density gradient centrifugation, we observed that PCSK9 shows remodeling-dependent ability to bind different classes of lipoprotein particles in vitro, binding strongly to LDL and IDL but showing barely detectable association to VLDL. Further, in human plasma, we found that lipoprotein-bound populations of PCSK9 shifted in response to differences in lipoprotein profiles between normolipidemic and hypercholesterolemic or hypertriglyceridemic subjects. Overall, elucidation of how lipoproteins regulate PCSK9 activity will reveal new targets for designing cholesterol-lowering therapeutics.

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/25). Includes bibliographical references.

Mondialement, le cancer du sein est celui le plus fréquent et mortel chez les femmes. Malgré un programme de dépistage national et de nouveaux traitements, le taux de rechute demeure haut. Ceci illustre le besoin de nouvelles thérapies. Même dans un pays industrialisé comme le Canada, ce cancer est toujours le deuxième plus mortel chez les femmes. Nos travaux précédents ont identifié PACE4, un membre de la famille des proprotéines convertases (PCs), comme étant une nouvelle cible thérapeutique dans le cancer de la prostate. Dans cette étude, nous avons vérifié si PACE4 pourrait aussi être une cible dans le cancer du sein. À l’aide d’échantillons clinique de cancer du sein, nous avons premièrement démontré que PACE4 est spécifiquement surexprimé dans le sous-type moléculaire positif aux récepteurs hormonaux. Par la suite, nous avons sélectionné une lignée cellulaire pour poursuivre nos expériences. Nous avons choisi la lignée de cancer du sein ZR-75-1 parce qu’elle provient du sous-type positif aux récepteurs hormonaux et qu’elle exprime PACE4. En culture cellulaire, nous avons comparé la croissance de cellules ZR-75-1 modifiées qui sous-exprimaient furine, PACE4 ou PC7. Lors de ces expériences, PACE4 était la seule PC importante pour la prolifération cellulaire. L’importance de PACE4 pour la croissance tumorale a aussi été confirmée in vivo à l’aide d’un modèle de xénogreffes de cellules ZR-75-1 chez des souris immunosupprimées. Les inhibiteurs peptidiques spécifiques à PACE4 C23 et Multi-Leu (ML) ont aussi été testés en culture cellulaire où ils étaient en mesure de diminuer la prolifération des cellules ZR-75-1 et MCF-7. Nous avons aussi démontré, à l’aide d’un peptide ML radiomarqué, que l’expression de PACE4 est nécessaire pour que les peptides inhibiteurs pénètrent dans la cellule. De plus, le peptide C23, lorsqu’administré systémiquement à des souris immunosupprimées portant des tumeurs de cancer du sein, était en mesure de diminuer la croissance tumorale. De manière intéressante, les tumeurs sous-exprimant PACE4 et celles traitées avec le peptide C23 démontraient un ralentissement de la croissance tumorale similaire. De plus, lorsque ces tumeurs ont été analysées en immunohistochimie, elles possédaient une diminution du marqueur de prolifération Ki67 et une augmentation de cellules positives aux marqueurs d’arrêt du cycle cellulaire p27KIP1 et p21Waf1/Cip1 comparativement aux tumeurs contrôles. Pour conclure, la sous-expression de PACE4 et l’administration systémique d’un inhibiteur de PACE4 en culture cellulaire ainsi que dans un modèle de xénogreffe résultent en une diminution de la prolifération néoplasique. Nos résultats suggèrent que PACE4 est une cible intéressante dans le cancer du sein positif aux récepteurs hormonaux. / Abstract: Breast cancer is the most frequent and deadly malignancy in women worldwide. Despite national screening prog rams combined with new treatments relapse rate remain high and new therapies are needed. Even in industrialized country like Canada, it is still the second most deadly cancer in women. From previous work, we identified PACE4, a member of the proprotein convertases (PCs) family of endoproteases, as a novel therapeutic target in prostate cancer. In the present study, we examined whether PACE4 could also be a potential target in breast cancer. In clinical samples of breast adenocarcinoma, we observed a specific overexpression of PACE4 in the estrogen-receptor-positive (ER+) subtype. We therefore looked for a breast cancer cell line model which would be representative and thus focused on ZR-75-1 since it both expresses PACE4 and is estrogen-receptor-positive. We compared stable knockdowns of furin, PACE4 and PC7 in the estrogen-receptor-positive cell line ZR-75-1 to evaluate their respective contribution to cell growth and tumor progression. PACE4 was found to be the only PC displaying a decrease in cell growth. The impact of PACE4 on tumor growth was also confirmed in vivo with xenograft of the ZR-75-1 cell line in athymic nude mice. PACE4 specific peptide-based inhibitors (C23 and Multi-Leu) were tested and shown to decrease prolife ration of ZR-75-1 cells in cell-based assays. We also confirmed that PACE4 expression was necessary for its specific peptide-based inhibitors to penetrates in the cell using a radiolabeled Multi-Leu peptide. Moreover, the systemic administration of C23 had a potent effect on tumor growth on xenografts of the ZR-75-1 cell line. Interestingly, PACE4-silencing and systemic administration of the PACE4 inhibitor C23 resulted in similar slowed tumor growth in vivo. Furthermore, these tumors demonstrated lower Ki67 proliferative indices with increased cell quiescence assessed with p27 KIP1 and p21 Waf1/Cip1 biomarkers. To conclude, PACE4-silencing and systemic administration of a PACE4 inhibitor result in hindered tumor progression and slower cellular proliferation. Our results suggest that PACE4 is a promising target for estrogen-receptor-positive breast cancer.

The liver regulates inflammation during sepsis, and most liver functions are carried out by hepatocytes. Bacterial lipids, including lipopolysaccharide (LPS) and lipoteichoic acid (LTA), can be cleared by hepatocytes, but the underlying mechanisms are uncertain. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates uptake of LPS by hepatocytes, but it is unknown whether LTA uptake is similarly regulated. Therefore, our objectives were to characterize the PCSK9-regulated pathway of bacterial lipid uptake by hepatocytes by identifying whether low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein 1 (LRP1) are the target receptors, and by determining which lipoproteins are involved. To study this pathway, we assessed the uptake of fluorescently-labeled LPS or LTA by human HepG2 hepatocytes using flow cytometry. We pre-treated HepG2 cells with PCSK9, alone or in combination with anti-LDLR or anti-LRP1 antibodies, in order to identify the PCSK9-regulated receptors that are involved, and utilized media containing normal serum or lipoprotein-deficient serum to investigate the lipoprotein- dependence of this pathway. We also determined the roles of LDL and HDL in bacterial lipid uptake through a series of add-back experiments to lipoprotein-deficient serum, and blocked LDLR to confirm that LDLR mediates LDL-dependent uptake. The HepG2 cell response to variable degrees of bacterial lipid uptake was also assessed in a subset of experiments by measuring several cytokines and extracellular alanine aminotransferase (ALT) activity in the cell culture supernatant. We found that PCSK9 regulates LDLR-mediated uptake of both LPS and LTA through an LDL-dependent mechanism, while LRP1 is not involved. Increased bacterial lipid uptake did not result in any hepatocellular injury or cytokine production, as measured by ALT activity and interleukin (IL)-6, IL-8, IL-10, and IL-17 concentrations. In conclusion, we completed our objective of characterizing the PCSK9-regulated pathway of bacterial lipid uptake, and provide supporting evidence for targeting PCSK9 as a novel therapeutic avenue in sepsis. / Thesis / Master of Science (MSc) / Bacterial compounds stimulate inflammation that can be overwhelming during sepsis. Understanding the processes behind uptake and clearance of these compounds may lead to better sepsis treatments. Therefore, our goal was to understand how uptake of two bacterial compounds, lipopolysaccharide and lipoteichoic acid, occurs by liver cells called hepatocytes. Hepatocytes are naturally equipped to clear foreign compounds, so understanding their role in clearing bacterial compounds is important. Another goal was to identify the role of the protein PCSK9 in this uptake process, as treatments targeting PCSK9 could be applied to sepsis once we understand its role in this disease. Our research demonstrates the negative role of PCSK9 in regulating uptake of lipopolysaccharide and lipoteichoic acid through a lipoprotein receptor called LDLR, and identifies the role of lipoproteins in this process. These findings further our understanding of the hepatocyte response to bacterial compounds in relation to sepsis, and identify PCSK9 as a potential target for new sepsis therapies.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction. The aims/objectives of this study were achieved by utilizing two approaches, which included animal intervention with niacin followed by genetic screening of five prioritized genes involved in cholesterol synthesis and regulation. For animal intervention, 16 vervet monkeys were divided into two groups of eight animals consisting of a control and an experimental (niacin) group. The control group was given a normal standard diet of pre-cooked maize meal throughout the study, while the experimental group received the same diet supplemented with 100 mg/kg of niacin (SR) for 12 weeks. During the niacin intervention, blood was collected at baseline, every four weeks during the treatment period and the end of the washout period. The collected blood was used for biochemical analysis (total cholesterol, triglycerides, LDL-C, and HDL-C) and downstream genetic applications. The second phase included the screening of PCSK9, LDLR, SREBP-2, CETP and APOB-100 using genotyping and gene expression. Niacin administration produced statistically significant increases in plasma HDL-C at fourtime points (T1, T2, T3 and T4), which resulted in an overall increase in plasma HDL-C. Additionally, niacin administration resulted in a slight reduction in LDL-C and total cholesterol levels. Furthermore, the genotyping analysis revealed 13 sequence variants identified in PCSK9, LDLR, SREBP-2, CETP and APOB-100 genes. Five of these variants were predicted to be disease-causing and correlated with gene expression patterns. Three identified PCSK9 variants (H177N, R148S, G635G) were categorized as LOF mutations, and this was supported by a decline in gene expression in animals harbouring these variants. The LDLR also had LOF variants that were the reason for its decreased mRNA expression. Additionally, SREBP-2 proved to be a key mediator of cholesterol pathways. Therefore, the findings of the study conclusively suggest that niacin does increase HDL-C and decrease LDL-C and total cholesterol. Moreover, an interaction between niacin administration and PCSK9 was observed which resulted in decreased gene expression.