Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)     

PEX11 promotes peroxisome division independently of peroxisome metabolism

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Temperature sensitivity in peroxisome assembly processes characterizes milder forms of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) contain various clinical phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), decreasing in the clinical severity in this order. We found that all IRD cell lines and some NALD lines belonging to several different complementation groups are temperature-sensitive in peroxisome assembly; that is, they lacked catalase-positive peroxisomes at 37°C, but do gain the peroxisomes at 30°C. We identified heterozygous mutations E55K/R119Stop in the PEX2 gene of an IRD patient of complementation group F. The E55K mutation was the direct cause of the temperature-sensitivity because similar phenotypes could be transferred to PEX2-defective CHO cells by transfecting the mutant gene. Thus, temperature-sensitive peroxisome assembly is representative of milder forms of PBDs. The main part of this study was published by Imamura et al. (1).     

The life cycle of the peroxisome

Peroxisomes are highly adaptable organelles that carry out oxidative reactions. Distinct cellular machineries act together to coordinate peroxisome formation, growth, division, inheritance, turnover, movement and function. Soluble and membrane-associated components of these machineries form complex networks of physical and functional interactions that provide supramolecular control of the precise dynamics of peroxisome biogenesis.     

An ER‐peroxisome tether exerts peroxisome population control in yeast

Eukaryotic cells compartmentalize biochemical reactions into membrane‐enclosed organelles that must be faithfully propagated from one cell generation to the next. Transport and retention processes balance the partitioning of organelles between mother and daughter cells. Here we report the identification of an ER‐peroxisome tether that links peroxisomes to the ER and ensures peroxisome population control in the yeast Saccharomyces cerevisiae. The tether consists of the peroxisome biogenic protein, Pex3p, and the peroxisome inheritance factor, Inp1p. Inp1p bridges the two compartments by acting as a molecular hinge between ER‐bound Pex3p and peroxisomal Pex3p. Asymmetric peroxisome division leads to the formation of Inp1p‐containing anchored peroxisomes and Inp1p‐deficient mobile peroxisomes that segregate to the bud. While peroxisomes in mother cells are not released from tethering, de novo formation of tethers in the bud assists in the directionality of peroxisome transfer. Peroxisomes are thus stably maintained over generations of cells through their continued interaction with tethers.     

Molecular insights into peroxisome homeostasis and peroxisome biogenesis disorders

Peroxisomes are single-membrane organelles essential for cell metabolism including the β-oxidation of fatty acids, synthesis of etherlipid plasmalogens, and redox homeostasis. Investigations into peroxisome biogenesis and the human peroxisome biogenesis disorders (PBDs) have identified 14 PEX genes encoding peroxins involved in peroxisome biogenesis and the mutation of PEX genes is responsible for the PBDs. Many recent findings have further advanced our understanding of the biology, physiology, and consequences of a functional deficit of peroxisomes. In this Review, we discuss cell defense mechanisms that counteract oxidative stress by 1) a proapoptotic Bcl-2 factor BAK-mediated release to the cytosol of H₂O₂-degrading catalase from peroxisomes and 2) peroxisomal import suppression of catalase by Ser232-phosphorylation of Pex14, a docking protein for the Pex5–PTS1 complex. With respect to peroxisome division, the important issue of how the energy-rich GTP is produced and supplied for the division process was recently addressed by the discovery of a nucleoside diphosphate kinase-like protein, termed DYNAMO1 in a lower eukaryote, which has a mammalian homologue NME3. In regard to the mechanisms underlying the pathogenesis of PBDs, a new PBD model mouse defective in Pex14 manifests a dysregulated brain-derived neurotrophic factor (BDNF)-TrkB pathway, an important signaling pathway for cerebellar morphogenesis. Communications between peroxisomes and other organelles are also addressed.     

Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris

Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers. A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris. The PHA synthase was expressed under the control of the promoter of the P. pastoris acyl-CoA oxidase gene. Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium. PHA accumulated as inclusions within the peroxisomes. P. pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plants.     

Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups.     

The peroxisome in oxidative stress

Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for proper functioning of cells and has been implicated in aging and carcinogenesis. We will give an overview of the peroxisomal processes involved in the oxygen radical homeostasis and its implications for the cell.     

过氧化物酶体生物发生研究进展

过氧化物酶体是存在于真核细胞中的一种亚细胞器,主要功能是参与脂肪酸等脂质的代谢过程和氧化应激的调节。近年来研究发现,多种疾病都与过氧化物酶体的生物发生异常有关。过氧化物酶体的生物发生指过氧化物酶体的形成过程,包括从头合成和分裂增殖两条途径。两条途径中,参与过氧化物酶体生物发生的蛋白质,即peroxin(PEX)的基因发生突变,会导致过氧化物酶体生成障碍,引起疾病的发生。因此,就过氧化物酶体生物发生的研究进展进行综述,有助于为相关疾病的诊断和治疗提供参考和依据。     

Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.     

Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motility and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells.     

On the role of the peroxisome in cell differentiation and carcinogenesis

This article reviews the currently available data on the role of peroxisomal function in relation to the processes of cell differentiation and carcinogenesis. In regard to tumourigenesis, both genotoxic and non-genotoxic processes have been considered, and the peroxisomal relationships with these phenomena and with differentiation are described at the level of organelle characteristics, enzyme contents, and the involvement of retinoids, steroid hormones, oxygen free radicals, growth factors, apoptosis, omega-3 polyunsaturated fatty acids and the cellular signalling networks. Overall these data serve to illustrate the unique and distinctive role of the peroxisome in differentiation and carcinogenesis, and point to the advantages of considering the peroxisomal involvement in the holistic context of the differentiation dedifferentiation continuum rather than the narrower focus of non-genotoxic carcinogenesis. The review also outlines the potential for medical benefit arising from a fuller understanding of these peroxisomal affiliations.     

Ring finger in the peroxisome assembly factor-1.

The peroxisome assembly factor-1 (PAF-1) is reported here to contain the signature subsequence for a ring finger motif in its carboxyl-terminus. This conserved subsequence in PAF-1 may be the key to a gene expression regulatory pathway important in peroxisome biogenesis.     

On the mechanism of the hepatocarcinogenicity of peroxisome proliferators

The absence of a genotoxic action in the rat of several peroxisome proliferators (PP) has been confirmed by measuring gross degradation, unscheduled DNA-synthesis (UDS), as well as by measurement of single strand breaks using alkali unwinding in absence and presence of inhibitors of DNA-repair. Similar results were obtained even after drastically lowering the glutathione content of liver. Further, after oral administration of ciprofibrate, no potentiating effect was found in vivo on the generation of micronuclei in hepatocytes by ionizing radiation. The metabolically inert PP, perfluorooctanoic acid, was found to act as a promoter of liver tumors in the rat induced by diethylnitrosamine in an initiation-selection-promotion protocol. The results are discussed in light of available information concerning the mechanism of action of PPs.