Transfer of metabolites across the peroxisomal membrane

AbstractPeroxisomes perform a large variety of metabolic functions that require a constant flow of metabolites across the membranes of these organelles. Over the last few years it has become clear that the transport machinery of the peroxisomal membrane is a unique biological entity since it includes nonselective channels conducting small solutes side by side with transporters for ‘bulky’ solutes such as ATP. Electrophysiological experiments revealed several channel-forming activities in preparations of plant, mammalian, and yeast peroxisomes and in glycosomes of Trypanosoma brucei. The properties of the first discovered peroxisomal membrane channel – mammalian Pxmp2 protein – have also been characterized. The channels are apparently involved in the formation of peroxisomal shuttle systems and in the transmembrane transfer of various water-soluble metabolites including products of peroxisomal β-oxidation. These products are processed by a large set of peroxisomal enzymes including carnitine acyltransferases, enzymes involved in the synthesis of ketone bodies, thioesterases, and others. This review discusses recent data pertaining to solute permeability and metabolite transport systems in peroxisomal membranes and also addresses mechanisms responsible for the transfer of ATP and cofactors such as an ATP transporter and nudix hydrolases. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease

On the role of microsomal aldehyde dehydrogenase in metabolism of aldehydic products of lipid peroxidation

AbstractTo elucidate a possible role of membrane-bound aldehyde dehydrogenase in the detoxication of aldehydic products of lipd peroxidation, the substrate specificity of the highly purified microsomal enzyme was investigated. The aldehyde dehydrogenase was active with different aliphatic aldehydes including 4-hydroxyalkenals, but did not react with malonic dialdehyde. When Fe/ADP-ascorbate-induced lipid peroxidation of arachidonic acid was carried out in an in vitro system, the formation of products which react with microsomal aldehyde dehydrogenase was observed parallel with malonic dialdehyde accumulation

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

AbstractTo understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice.The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders

Trim37-deficient mice recapitulate several features of the multi-organ disorder Mulibrey nanism

Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37(-/-)) model for MUL. Trim37(-/-) mice were viable and had normal weight development until approximately 12 months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37(-/-) mice as compared with wildtype. Both male and female Trim37(-/-) mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37(-/-) mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37(-/-) mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5 years Trim37(-/-) mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37(-/-) mice. The most consistently seen phenotypes in Trim37(-/-) mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37(-/-) mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis.Peer reviewe

Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal β-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed

The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential

Abstract The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel’s selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17−/− mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions

Electron microscopy of cellular organelles separated by Optiprep gradient centrifugation.

<p>Fractions enriched in glycosomes (fractions 2–5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>), fragments of flagella (fractions 8–11) or mitochondria and other organelles (fractions 15–18) were combined and processed for EM examination (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). (<b>A</b> and <b>B</b>) Isolated glycosomes shown at lower (<b>A</b>) and higher (<b>B</b>) magnifications. The fraction consists mostly of glycosomes. Some contamination by fragments of flagella is also visible. Importantly, fragments of flagella (paraflagellar rods and axonemes) show no sign of attachment to the flagellar membrane. Note the presence of intact glycosomes as electron-dense vesicles surrounded by a single membrane (marked by arrows in panel <b>B</b>). (<b>C</b> and <b>D</b>) Fractions enriched in flagella at low (<b>C</b>) and high (<b>D</b>) magnifications. One can see many paraflagellar rods in longitudinal section (<b>C</b>) and recognize flagellar axonemes (marked by arrows in panel <b>D</b>). Some glycosomes are also visible in panel <b>C</b>. (<b>E</b> and <b>F</b>) Composition of the fraction from the top of the Optiprep gradient that is enriched with mitochondria. Several types of organelles – mitochondria, lysosomes, lipid droplets, clathrin-coated vesicles, and components from the flagellar apparatus – can be observed. Note the shrinking of the mitochondrial inner membrane (see panel <b>F</b>) apparently due to osmotic misbalance. Scale bars: 2 µm (<b>C</b> and <b>E</b>); 1 µm (<b>A</b>); 0.5 µm (<b>D</b> and <b>F</b>), and 0.1 µm (<b>B</b>).</p

Detection of channel-forming activities in subcellular fractions.

<p>Fractions 2–4 (glycosomes), 8–11 (fragments of flagella), and 15–18 (mitochondria) from Optiprep density gradients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>) were combined and treated with Genapol X-080 to solubilize membrane proteins (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). After sedimentation of insoluble material, aliquots of the resulting supernatants were used for MCR (<b>A</b>–<b>C</b>) or SCA (<b>D</b>). (<b>A</b>) Traces of the current monitoring in the presence of glycosomal (upper panel) or mitochondrial (lower panel) preparations. The middle trace represents a timescale-expanded current recording of the upper trace. The bath solution contained 3 M KCl and the applied voltage was +10 mV. (<b>B</b>) Histograms of insertion events registered in subcellular fractions (see panel <b>A</b>). Bin size is 4.0 pA. The total number of insertion events (I.e.) is indicated. Here and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3</a> C (upper panel) all insertion events with current increments over 180 pA (for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3C</a>, lower panel −90 pA) are combined in one bin (180 pA or 90 pA, respectively). Note that the amount of insertion events in the flagella fraction (see <b>B</b>, middle panel) is lower than that observed in other fractions. This is mainly due to low channel-forming activity (per protein content) in the preparations of this fraction. For the sake of compatibility we used the same amounts of protein for measurements in different fractions. (<b>C</b>) Histograms of insertion events detected for glycosomal preparations using NH₄Cl as the electrolyte. Bin size: 4 pA (upper panel) or 2 pA (lower panel). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figures 3A and 3B</a> for other details. (<b>D</b>) Trace of the current monitoring using the glycosomal fraction (initial holding potential +10 mV) indicating the insertion (marked by one asterisk) of a large-conductance channel that spontaneously closed (marked by two asterisks) after stepwise (each step is +10 mV) increase in the holding potential up to 50 mV.</p