Thesis (Ph.D.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on July 22, 2009). Research advisor: Edwards A. Park. Document formatted into pages (xi, 94 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 69-89).

Peroxisomes are dynamic and multifunctional organelles, which are essential for human health and development. They are remarkably diverse, with functions that vary significantly between cells and organisms, and can dramatically change their size, shape and dynamics in response to cellular cues. In the past few years, several studies have significantly increased our understanding of the basic principles that enable peroxisome biogenesis and degradation, as well as their pivotal role in cellular signalling and homeostasis. However, several of these processes are still poorly understood. In this thesis we initially studied the peroxisome targeting mechanism of a group of C-terminally anchored membrane proteins, known as tail-anchored (TA) proteins. In order to investigate the molecular signals that enable TA protein targeting to cellular organelles, we analysed the physicochemical properties of a cohort of TA proteins both in silico and in vivo, and show that a combination of transmembrane domain (TMD) hydrophobicity and C-terminal tail charge determines organelle-specific targeting. Focusing on peroxisomes, we demonstrate that a balance between TMD hydrophobicity and high positive tail charge directs TA proteins to this organelle, and enables binding to the peroxisomal chaperone PEX19. These results allowed us to create a bioinformatical tool to predict the targeting of uncharacterised TA proteins and further develop our understanding of the molecular mechanisms involved in the targeting of this protein group. From our initial TA protein screen, we identified the TA protein MIRO1 at peroxisomes and looked at its role in the regulation of peroxisome motility. We show that endogenous MIRO1 localises to mitochondria and peroxisomes, and that dual targeting depends on the C-terminal tail. MIRO1 expression significantly increased peroxisome motility in several cell lines, and revealed a role for motility in peroxisome dynamics, by inducing organelle proliferation and elongation. These results reveal a new molecular complex at peroxisomes and provide us with a tool to further dissect the role of motility on peroxisome function.

Background: Peroxisome proliferator-activated receptor δ (PPARδ) is a versatile regulator of distinct biological processes and overexpression of PPARδ in cancer may be partially related to its suppression of its own co-regulators. Aims: To determine whether recruited suppressor proteins bind to and regulate PPARδ expression, activity and PPARδ-dependent cholangiocarcinoma proliferation. Methods: Yeast two-hybrid assays were done using murine PPARδ as bait. PPARδ mRNA expression was determined by qPCR. Protein expression was measured by western blot. Immunohistochemistry and fluorescence microscopy were used to determine PPARδ expression and co-localization with NDP Kinase alpha (NM23-H2). Cell proliferation assays were performed to determine cell numbers. Results: Yeast two-hybrid screening identified NM23-H2 as a PPARδ binding protein and their interaction was confirmed. Overexpressed PPARδ or treatment with the agonist GW501516 resulted in increased cell proliferation. NM23-H2 siRNA activated PPARδ luciferase promoter activity, upregulated PPARδ RNA and protein expression and increased GW501516-stimulated CCA growth. Overexpression of NM23-H2 inhibited PPARδ luciferase promoter activity, downregulated PPARδ expression and AKT phosphorylation and reduced GW501516-stimulated CCA growth. Conclusions: We report the novel association of NM23-H2 with PPARδ and the negative regulation of PPARδ expression by NM23-H2 binding to the C-terminal region of PPARδ. These findings provide evidence that the metastasis suppressor NM23-H2 is involved in the regulation of PPARδ-mediated proliferation.

Peroxisomes and mitochondria play a key role in oxygen metabolism. It was widely accepted that the evolutionary adaptation of eukaryotes to anaerobiosis is reflected by the metabolic reduction of mitochondria, and concomitant loss of peroxisomes. The anaerobic protists Mastigamoeba balamuthi (Mb), Pelomyxa schiedti (Ps), and Entamoeba histolytica (Eh) contradict this paradigm. They possess anaerobic types of mitochondria (hydrogenosomes, mitosomes) but also host "anaerobic peroxisomes". Mb/Ps peroxisomes contain a common set of 13 peroxins (Pexs) that retain the core members of each functional category including components of both PTS1 and PTS2 machinery for matrix protein import. However, Eh peroxisomes harbour a reduced set of 7 known Pexs and lacks several components that are highly conserved among most eukaryotic lineages, including components of PTS2 machinery (Pex7), the RING complex (Pex2/10/12), docking complex (Pex13), and peroxisomal membrane protein import receptor (Pex3). Concerning the functional annotation, no clear biochemical context has been found in these anaerobic peroxisomes. They are diverse in enzymatic contents and are involved in various metabolic reactions, while catalase and typical peroxisomal enzymes of fatty acid beta-oxidation are absent. Mb peroxisomes appear to be involved in...

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18327号 / 農博第2052号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4834(農学部図書室) / 31185 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

No / Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is activated by several compounds, including the thiazolidinediones. In addition to being a therapeutic target for obesity, hypolipidaemia and diabetes, perturbation of PPARgamma signalling is now believed to be a strategy for treatment of several cancers, including breast. Although differential expression of PPARgamma is observed in tumours compared to normal tissues and PPARgamma agonists have been shown to inhibit tumour cell growth and survival, the interdependence of these observations is unclear. This study demonstrated that the potent, irreversible and selective PPARgamma antagonist GW9662 prevented activation of PPARgamma and inhibited growth of human mammary tumour cell lines. Controversially, GW9662 prevented rosiglitazone-mediated PPARgamma activation, but enhanced rather than reversed rosiglitazone-induced growth inhibition. As such, these data support the existence of PPARgamma-independent pathways and question the central belief that PPARgamma ligands mediate their anticancer effects via activation of PPARgamma.

The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.

No description available.

Chez les plantes, le RNA silencing (RNAi) est le principal mécanisme de défense antivirale. Il est opéré par de petites molécules d’ARN (siRNA), de 21-22nt de long, générées à partir de l’ARN viral par DCL4 et DCL2, respectivement. Ces siRNA confèrent la séquence-spécificité des réactions de défense intracellulaire et peuvent se déplacer à longue distance pour immuniser les cellules saines. En conséquence, les virus ont développé des protéines (VSRs) capables de supprimer ces deux aspects du RNAi. Au cours de cette thèse, j’ai pu démontrer in vivo que la protéine P15 du Peanut clump virus (PCV) est capable de séquestrer les siRNA de 21 et 22nt et qu’elle bloque le mouvement de ces derniers plus efficacement que ceux de 21nt. Pour compenser cette faiblesse, au cours de l’infection par le PCV, P15 est transportée à l’intérieur des peroxisomes en association avec les siRNA qu’elle séquestre. Le confinement des siRNA mobiles de 21nt à l’intérieur de ces organelles conduit à une inhibition du RNAi systémique et stimule fortement la propagation du PCV à travers la plante. Ces travaux définissent une nouvelle stratégie de pathogénèse virale au cours de laquelle une organelle est utilisé pour neutraliser des molécules de défense produites par l’hôte. / In plants, RNA interference (RNAi) is the main antiviral defense mechanism. It is initiated through the processing of viral RNA into 21-22nt long siRNA by DCL4 and DCL2, respectively. These siRNA can mediate sequence-specific local defense reactions (cell-autonomous RNAi) or move to distant tissues to prime defenses in naive cells (systemic RNAi). Consequently, viruses have evolved proteins (VSRs) to suppress both aspects of RNAi. In this in vivo study, I show that P15, the VSR of Peanut clump virus (PCV), binds and sequesters both 21nt and 22nt siRNA. Importantly, it stops the movement of 22nt siRNA more efficiently than 21nt siRNA. During infection, P15 is shuttled into peroxisomes, and is able to « piggyback » siRNA into these organelles. By confining mobile DCL4-dependent antiviral 21nt siRNA within peroxisomes, P15 is able to shut down systemic RNAi and strongly promote PCV movement. This work describes a novel pathogenic strategy in which an organelle is used to neutralize host defensive molecules.