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Peroxisomes represent crucial subcellular compartments for human life and health. They are remarkably dynamic organelles which respond to stimulation by adapting their structure, abundance, and metabolic functions according to cellular needs. Peroxisomes can form from pre-existing organelles by membrane growth and division, which results in peroxisome multiplication/proliferation. Growth and division in mammalian cells follows a well-defined multi-step process of morphological alterations including elongation/remodeling of the peroxisomal membrane (by PEX11β), constriction and recruitment of division factors (e.g. Fis1, MFF), and final membrane scission (by the dynamin-related GTPase Drp1) (Chapter 1). Although our understanding of the mechanisms by which peroxisomes proliferate is increasing, our knowledge on how the division/multiplication process is linked to extracellular signals is limited, in particular in humans. The classical pathway involved in peroxisome proliferation is mediated by a family of ligand-activated transcription factors known as peroxisome proliferator activated receptors (PPARs) (Chapter 1). This project focused on identifying novel signaling pathways and associated factors involved in peroxisome proliferation in humans. In this study, a cell-based peroxisome proliferation assay using the HepG2 cell model with spherical peroxisomal forms has been developed to investigate different stimuli and their ability to induce peroxisome proliferation (Chapters 2 and 3). In this system, peroxisome elongation has been used as the read-out for peroxisome 4 proliferation. We also showed that the number of peroxisomes increased after division of elongated peroxisomes indicating peroxisome proliferation. Different stimuli, such as fatty acids, PPAR agonists and antagonists, have been used in this study. PPAR agonists and antagonists had no stimulatory or inhibitory effect on peroxisome elongation in our assay, suggesting PPAR-independent regulatory processes. However, arachidonic acid and linoleic acid were able to induce peroxisome elongation, whereas palmitic acid and oleic acid were not effective. These findings indicate that general stimulation of fatty acid β-oxidation is not sufficient to induce peroxisome elongation/proliferation in HepG2 cells. Moreover, mRNA expression levels of peroxismal genes have been monitored during a time course in the HepG2 cell-based assay by qPCR. This analysis shows a regulation of expression of peroxins during peroxisome proliferation in human cells and suggests differences in the regulation pattern of PEX11α and PEX11β. In Chapter 4, motif binding sites for transcription factors in peroxisomal genes were analyzed. An initial map of candidate regulatory motif sites across the human peroxisomal genes has been developed (Secondment at the University of Sevilla, Spain with Prof. D. Devos). This analysis also revealed the presence of different transcription factor binding sites in the promoter regions of PEX11α and PEX11β, supporting different regulatory mechanisms. Based on the computational analysis, PEX11β contained a putative SMAD2/3 binding site suggesting a novel link between the canonical TGFβ signaling pathway and expression of PEX11β, a key regulator of peroxisome dynamics and proliferation. 5 Addition of TGFβ to HepG2 cells cultured under serum-free conditions induced elongation/growth of peroxisomes as well as peroxisome proliferation supporting a role for TGFβ signalling in peroxisomal growth and division (Chapter 5). Furthermore, to demonstrate that this induction is through a direct effect of TFGβ on the SMAD binding site found in PEX11β, we performed functional studies using a dual luciferase reporter assay with PEX11β wild type and mutated promoter regions (Secondment at Amsterdam Medical Center, Netherlands with Prof. H. Waterham). Whereas luciferase activity was induced by TGFβ stimulation with the PEX11β wild type promoter, mutation of the SMAD binding site abolished activation. In summary, this study revealed a new signaling pathway involved in peroxisome proliferation in humans and provided a tool to monitor peroxisome morphology and gene expression upon treatment with defined stimuli. Furthermore, I contributed to a study revealing that ER-peroxisome contacts are important for peroxisome elongation (Chapter 6). Our group identified peroxisomal acyl-CoA binding domain protein 5 (ACBD5), ACBD4 and VABP as a molecular linker between peroxisomes and the ER (Costello et al., 2017). Motif analysis of ACBD4 and ACBD5 promoter regions revealed that unlike PEX11β, these genes do not contain a binding site for SMAD, suggesting they are not co-regulated. Also, ACBD4 and ACBD5 do not share any common transcription factor binding sites suggesting different regulation. An interesting binding motif within the ACBD4 promoter is a glucocorticoid receptor binding site. In our study, we found potential glucocorticoid response elements (GRE) in other peroxisomal genes encoding β-oxidation enzymes. This may suggest an important role for glucocorticoid receptors in activating expression of peroxisomal genes resulting in the stimulation of fatty acid breakdown and energy production.

Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos. / Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos. / Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.