No description available.

Peroxisomes are ubiquitous and are considered to be vital organelles in eukaryotic cells; however, unlike mitochondria and chloroplast, they lack DNA and a protein secretory apparatus. Therefore, peroxisome biogenesis requires a group of proteins called peroxins encoded by the pex genes. Out of the thirty two known peroxins discovered so far, a subset of peroxins including enzyme IDP3 and proteins namely, PEX18, PEX21 and PEX6 were chosen for this research. IDP3 plays a vital role in peroxisomal metabolism where it generates NADPH which in turn is needed by the peroxisomal enzymes to degrade unsaturated fatty acids. PEX18 and PEX21 are mutually redundant but essential for the transport of PTS2 targeted proteins into the peroxisome. PEX6 is involved in the ATP-dependent recycling of the protein receptor from the peroxisomal membrane to the cytosol. Expression plasmids were constructed that encoded each of these proteins in tandem with a histidine tag at either or both the amino and carboxy terminals of the protein. The purification of IDP3 was achieved using affinity chromatography on a nickel resin. After several unsuccessful attempts using ion exchange and size exclusion chromatography, PEX18 and PEX21 were purified by nickel affinity chromatography after denaturation to expose their His tags. The expression of PEX6 was poor by comparison with the other proteins and the low amount of protein precluded a complete purification. Future work will involve crystal screen trials, X-ray diffraction and structure refinement.

Peroxisomes are ubiquitous and are considered to be vital organelles in eukaryotic cells; however, unlike mitochondria and chloroplast, they lack DNA and a protein secretory apparatus. Therefore, peroxisome biogenesis requires a group of proteins called peroxins encoded by the pex genes. Out of the thirty two known peroxins discovered so far, a subset of peroxins including enzyme IDP3 and proteins namely, PEX18, PEX21 and PEX6 were chosen for this research. IDP3 plays a vital role in peroxisomal metabolism where it generates NADPH which in turn is needed by the peroxisomal enzymes to degrade unsaturated fatty acids. PEX18 and PEX21 are mutually redundant but essential for the transport of PTS2 targeted proteins into the peroxisome. PEX6 is involved in the ATP-dependent recycling of the protein receptor from the peroxisomal membrane to the cytosol. Expression plasmids were constructed that encoded each of these proteins in tandem with a histidine tag at either or both the amino and carboxy terminals of the protein. The purification of IDP3 was achieved using affinity chromatography on a nickel resin. After several unsuccessful attempts using ion exchange and size exclusion chromatography, PEX18 and PEX21 were purified by nickel affinity chromatography after denaturation to expose their His tags. The expression of PEX6 was poor by comparison with the other proteins and the low amount of protein precluded a complete purification. Future work will involve crystal screen trials, X-ray diffraction and structure refinement.

Abstract Peroxisomes are small oxidative organelles found in all eukaryotes. They contain a matrix which is surrounded by a single membrane and consists mainly of soluble proteins. Peroxisomal enzymes are involved in a broad spectrum of metabolic pathways including conversion of lipids, amino- and hydroxyacids, purines and reactive oxygen species. The carbon fluxes through peroxisomal pathways require a continuous metabolite crossing of the peroxisomal membrane. A long-standing and still unresolved problem of the physiology of mammalian peroxisomes is the role of the membrane of these organelles as a permeability barrier to solute molecules. In this study, we have shown that the peroxisomal membrane represents a type of biomembrane where channel-forming proteins coexist with solute transporters. Disruption of the mouse Pxmp2 gene, encoding the peroxisomal integral membrane protein Pxmp2 also known as PMP22, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations revealed that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2-/- mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Hence, Pxmp2 is the first peroxisomal pore-forming protein identified, and its existence suggests that the mammalian peroxisomal membrane is permeable to small solutes, while transfer of bulky metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters. In addition, the phenotypic characterisation of Pxmp2-/- mice has revealed a role for Pxmp2 during the development of the epithelia in the mammary glands of female mice. The disruption of Pxmp2 leads to the impairment of ductal outgrowth of mammary glands at puberty, which is followed by the inability of Pxmp2-/- mice to nurse their offspring.

No description available.

No description available.

No description available.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.

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.

Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the Tombusviridae family of positive-strand RNA viruses. Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (pMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epifluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.