Markus Islinger

Fission and proliferation of peroxisomes

Peroxisomes are remarkably dynamic, multifunctional organelles, which react to physiological changes in their cellular environment and adopt their morphology, number, enzyme content and metabolic functions accordingly. At the organelle level, the key molecular machinery controlling peroxisomal membrane elongation and remodeling as well as membrane fission is becoming increasingly established and defined. Key players in peroxisome division are conserved in animals, plants and fungi, and key fission components are shared with mitochondria. However, the physiological stimuli and corresponding signal transduction pathways regulating and modulating peroxisome maintenance and proliferation are, despite a few exceptions, largely unexplored. There is emerging evidence that peroxisomal dynamics and proper regulation of peroxisome number and morphology are crucial for the physiology of the cell, as well as for the pathology of the organism. Here, we discuss several key aspects of peroxisomal fission and proliferation and highlight their association with certain diseases. We address signaling and transcriptional events resulting in peroxisome proliferation, and focus on novel findings concerning the key division components and their interplay. Finally, we present an updated model of peroxisomal growth and division. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.

Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish

In order to monitor the influence of estrogenic compounds on the reproductive physiology of fish, molecular markers for zebrafish vitellogenin, estrogen receptor and ZP2 were developed. For this purpose, sequence information about the zebrafish estrogen receptor and vitellogenin had to be obtained. By means of RT-PCR, a sequence fragment of the zebrafish estrogen receptor alpha was cloned and sequenced. Continuous cDNAs of two zebrafish vitellogenin-like gene products (zfvg1 and zfvg3) were constructed by the help of expressed sequence tags of zebrafish and completely sequenced. The sequences of the estrogen receptor and of the vitellogenins showed significant similarities to corresponding cDNAs of other fish species. Expression of these gene products was measured following exposure to 17alpha-ethinylestradiol and compared with histological endpoints. RT-PCR was used as a semiquantitative technique to record gene expression in adult male zebrafish, which were exposed to 17alpha-ethinylestradiol in time-and dose-response experiments. As for time-dependent expression, all hepatic genes investigated were expressed at considerable amounts from 24 h after onset of exposure to 50 ng/l 17alpha-ethinylestradiol to the end of experiment (17 days). In testes, expression of the estrogen receptor- as well as ZP2-mRNA remained unchanged for the entire experiment, except for the individuals exposed for 17 days, which displayed elevated expression levels of ZP2. In the dose-response experiment, male zebrafish were exposed to 17alpha-ethinylestradiol in concentrations from 0.25-85 ng/l for 4 and 21 days. LOECs for vitellogenin as well as estrogen receptor alpha expression were found to be 2.5 ng/l already after 4 d of exposure. Extension of the exposure time to 21 days resulted in enhanced transcription of vitellogenin-mRNAs at 2.5 ng/l 17alpha-ethinylestradiol, whereas the detection limit could not be lowered. In contrast, in testes no induction of both ZP2 as well as estrogen receptor expression was detected at any concentration tested. To examine estrogen-caused alterations at the ultrastructural level, liver and testes of males exposed to 25 ng/l 17alpha-ethinylestradiol were analysed. Male livers responded with a feminisation reflected by the proliferation of rough endoplasmatic reticulum and Golgi apparatus typical of female hepatocytes during vitellogenesis. However, in testes no signs of feminisation were detectable; rather, destructive phenomena like phagocytosis of sperm cells by Sertoli cells were observed. Thus, in sexually differentiated males no reorganisation of the gonadal tissue towards an ovary could be definitely detected at any level investigated.

The peroxisome: an update on mysteries

Peroxisomes contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, which render them indispensable to human health and development. Peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. In recent years, the interest in peroxisomes and their physiological functions has significantly increased. This review intends to highlight recent discoveries and trends in peroxisome research, and represents an update as well as a continuation of a former review article. Novel exciting findings on the biological functions, biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross-talk of peroxisomes with other subcellular compartments are addressed. Furthermore, recent findings on the role of peroxisomes in the brain are discussed.

Rat liver peroxisomes after fibrate treatment: A survey using quantitative mass spectrometry

Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptorα-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22–C26) but rather metabolize preferentially long chain fatty acids (C16–18).

Measurement of vitellogenin-mRNA expression in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blot/RNAse protection-assay

The induction of vitellogenin synthesis both in vivo and in vitro has proven to be a reliable biomarker for assessing the estrogenic activity of individual substances and the more complex effluents of sewage treatment plants. However, due to the requirement of radioactively labelled nucleotides, the measurement of vitellogenin-mRNA has not been widely used in routine testing--even though this technique promises elevated sensitivity. In order to develop a practicable, reliable and cost-effective bioassay suitable for routine testing, a combined dot-blot/RNAse protection assay, utilising digoxigenin-labelled cRNA transcripts of plasmid psg5Vg1.1 was used for the quantification of vitellogenin-mRNA in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes. By re-cloning the Vg1.1 insert into a pGemZf7(-)-vector, the sense-transcript of Vg1.1 was utilized as a standard for the quantification of vitellogenin-mRNA concentrations. Male rainbow trout hepatocytes were cultured as monolayers in pure M199 medium. The addition of serum supplements did not result in increased expression of vitellogenin-mRNA following 17 beta-estradiol administration. This indicates that for this assay no supplementation of the culture medium is necessary. After addition of 17 beta-estradiol, hepatocytes exhibited an exponential time-dependent expression of vitellogenin-mRNA over a period of 144 h. The dot blot system was sufficiently sensitive to detect vitellogenin-mRNA following addition of 1 microM 17 beta-estradiol after 6 h of incubation. However, the amount of vitellogenin-mRNA expressed was found to be a function of both incubation time and inducer concentration. Prolonged incubation times were therefore required to enhance the sensitivity of the system. After a 96-h incubation, detection limits for 17 beta-estradiol were between 100 pM and 1 nM. Vitellogenin-mRNA could not be detected in untreated hepatocytes. The vitellogenin-mRNA dot blot/RNAse protection assay was further used as a tool for assessing the estrogenic potential of the xenoestrogens nonylphenol and bisphenol A, which exhibited estrogenic activities approximately 2000-fold less than the natural inducer 17 beta-estradiol. The vitellogenin-mRNA response to 17 alpha-ethinylestradiol reached maximum efficacy down to the lowest tested concentration of 10(-9) M. The assay also successfully identified estrogenic activity in selected waste water samples.

Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes – Evidence for a Natural Piggyback Import Mechanism in Mammals

Most newly synthesized peroxisomal proteins are imported in a receptor‐mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so‐called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner ‘copper chaperone of SOD1’ (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D‐liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1‐dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS‐mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co‐import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.

The different facets of organelle interplay-an overview of organelle interactions.

Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy—a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction.

Peroxisomes and reactive oxygen species, a lasting challenge.

Oxidases generating and enzymes scavenging H2O2 predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H2O2 generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H2O2 and O2– radicals. The latter are converted to O2 and H2O2 by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein.

Peroxisome interactions and cross-talk with other subcellular compartments in animal cells.

Peroxisomes are remarkably plastic and dynamic organelles, which fulfil important functions in hydrogen peroxide and lipid metabolism rendering them essential for human health and development. Despite great advances in the identification and characterization of essential components and molecular mechanisms associated with the biogenesis and function of peroxisomes, our understanding of how peroxisomes are incorporated into metabolic pathways and cellular communication networks is just beginning to emerge. Here we address the interaction of peroxisomes with other subcellular compartments including the relationship with the endoplasmic reticulum, the peroxisome-mitochondria connection and the association with lipid droplets. We highlight metabolic cooperations and potential cross-talk and summarize recent findings on peroxisome-peroxisome interactions and the interaction of peroxisomes with microtubules in mammalian cells.

Free-flow electrophoresis in the proteomic era: A technique in flux

Since its introduction five decades ago, free‐flow electrophoresis (FFE) has been mainly employed for the isolation and fractionation of cells, cell organelles and protein mixtures. In the meantime, the growing interest in the proteome of these bio‐particles and biopolymers has shed light on two further facets in the potential of FFE, namely its applicability as an analytical tool and sensor. This review is intended to outline recent innovations, FFE has gained in the proteomic era, and to point out the valuable contributions it has made to the analysis of the proteome of cells, sub‐cellular organelles and functional protein networks.