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.

No description available.

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Peroxisomes are organelles originating from the endoplasmic reticulum. Peroxisome biogenesis requires multiple peroxins, including PEX19, a prenylated protein that helps deliver peroxisomal membrane proteins in yeast and mammals. Arabidopsis thaliana PEX19 is encoded by two isogenes, PEX19A and PEX19B. I demonstrate that pex19A and pex19B insertional mutants lack obvious abberant physiological phenotypes. I provide evidence that pex19A pex19B double mutants are inviable, that PEX19B is more abundant than PEX19A in young seedlings, that Arabidopsis PEX19 is farnesylated in vivo, and that YFP-PEX19 predominantly associates with what appears to be a subcellular membrane regardless of its prenylation state. I show that farnesyltransferase mutants apparently contain only non-prenylated PEX19 and lack phenotypes that would indicate inefficient peroxisome activity. My analysis of PEX19 suggests that PEX19 prenylation is dispensable for peroxisome biogenesis, and has generated tools for future studies of the earliest steps in peroxisome biogenesis in plants.

No description available.

Peroxisomes are indispensable organelles that perform many essential metabolic activities. Thus, eukaryotic cells have evolved molecular mechanisms to ensure the inheritance of peroxisomes from mother cell to daughter cell at cell division. In the budding yeast Saccharomyces cerevisiae, the class V myosin motor, Myo2p, interacts with its peroxisomal receptor, Inp2p, to move peroxisomes along actin from mother cell to bud, while the peroxisomal membrane protein Inp1p functions to tether peroxisomes to the cell cortex. In this thesis, I report the results of investigations of peroxisome inheritance using the dimorphic yeast Yarrowia lipolytica as a model system. We showed that peroxisome mobility and inheritance are dependent on actin in Y. lipolytica. Interrogation of the Y. lipolytica genome revealed one class V myosin. This myosin V is involved in transporting peroxisomes from mother cell to bud. We characterized YlInp1p, the othologue of S. cerevisiae Inp1p, as the first peroxisomal protein required for peroxisome inheritance in Y. lipolytica. We demonstrated that YlInp1p functions to anchor peroxisomes in both mother cell and bud. YlInp1p has an additional role in the dimorphic transition from the yeast form to the hyphal form in Y. lipolytica. We identified Pex3Bp, a paralogue of Pex3p, as the peroxisome-specific receptor for myosin V in Y. lipolytica. Pex3Bp interacts directly with the globular tail of myosin V. Pex3Bp also interacts with itself and with Pex3p. In cells lacking Pex3Bp, peroxisomes are preferentially retained in the mother cell, while the majority of peroxisomes gather and are transferred to the bud in cells overproducing Pex3Bp. Overexpression of PEX3 can partially complement the phenotype of pex3B cells, while overexpression of PEX3B cannot complement the phenotype of pex3 cells. Interestingly, Pex3p, which has been shown previously to function in the de novo formation of peroxisomes from the ER, also interacts directly with the globular tail of myosin V. Therefore, Pex3p is involved in peroxisome inheritance. In addition, cells lacking Pex3Bp contain hyperelongated, tubulo-reticular peroxisomes, indicating that Pex3Bp has a role in peroxisome morphology. Our findings suggest that both Pex3Bp and Pex3p are multifunctional proteins that are involved in different steps of the peroxisome biogenic cascade.

No description available.

Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of α-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and α-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.

Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of α-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and α-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.

The influence of pharmacokinetic parameters on the hepatic biochemical responses to the peroxisome proliferators, ciprofibrate, bezafibrate and clofibrate, was studied in the Fischer rat following a 26-week treatment period. With once daily dosing, the induction profiles of these compounds were dissimilar and the order of response was ciprofibrate > bezafibrate > clofibrate. By adjusting the frequency of dosing with respect to drug half life, i. e. clofibrate and bezafibrate twice daily and ciprofibrate once every 48 hours, the differences in response were ablated. The effect of short term ciprofibrate administration on hepatic enzyme parameters was studied in different rat strains and species. The rat (all strains), mouse, hamster and rabbit were termed responsive due to a coordinate induction of cytochrome P-452, carnitine acetyltransferase and peroxisomal beta-oxidation. No treatment related changes were observed in the guinea pig. In the marmoset a slight increase in peroxisomal beta-oxidation was demonstrated with no induction of cytochrome P-452 and carnitine acetyltransferase. In responsive species increased 12-hydroxylation of lauric acid correlated with an increase in mRNA hybridising to a cytochrome P-452 cDNA probe. The guinea pig and marmoset were designated non-responsive. In the marmoset and Fischer rat the hepatic enzyme responses to a 14-day and 26-week ciprofibrate administration were similar. A 4-week recovery group was included in the marmoset chronic study and the increase in peroxisomal beta-oxidation was found to be readily reversible whereas mitochondrial and microsomal changes were not. Electron microscopy revealed no peroxisome proliferation in the marmoset. Renal enzyme parameters were examined in ciprofibrate treated animals and considerable rat strain and species differences were observed. Renal enzyme changes were minimal. The response to ciprofibrate appeared to be largely specific for the liver in responsive species. From these results it is clear that the rat is not a suitable animal model to predict the hepatic response in the marmoset. If it is assumed that the marmoset resembles man more closely than the rat, extrapolation would indicate that peroxisome proliferators are not a toxicological hazard to man.