Peroxisomal Content Research Articles

SH-SY5Y cells undergo changes in peroxisomal metabolism when exposed to decanoic acid.

Medium-chain fatty acids (MCFAs), particularly decanoic acid (C10) and octanoic acid (C8), have garnered attention in recent years for their potential antiepileptic properties. A previous study from our laboratory demonstrated that C10 targets the PPARγ nuclear receptor, increasing the activity of the antioxidant enzyme catalase and thereby possibly modulating peroxisomal content. Here, we examined markers of peroxisomal content and activity in response to C10 and C8 exposure in neuronal-like SH-SY5Y cells. SH-SY5Y were treated with 250 mM C10 or C8 for a period of 6 days. Following this, biochemical markers of peroxisomal content and function were assessed, including acyl-coA oxidase activity, peroxisomal gene expression and peroxisomal VLCFA β-oxidation. Our findings revealed that C10 treatment augments acyl-CoA oxidase 1 (ACOx1) activity by 129% in comparison to control cells. An exploration into genes related to peroxisomal biosynthesis showed 23% increased expression of PEX11α upon C10 exposure, implying peroxisomal proliferation. Furthermore, it was observed that C10 exposure not only elevated ACOx1 activity but also enhanced peroxisomal β-oxidation of docosanoic acid (C22). Our findings bolster the premise that C10 functions as a peroxisome proliferator, influencing peroxisomal content and function. Further investigations are required to fully understand the mechanistic details as to how this may be beneficial in epilepsy and the potential implications with regards to peroxisomal disease.

Chronic acidosis rewires cancer cell metabolism through PPARα signaling.

The mechanisms linking tumor microenvironment acidosis to disease progression are not understood. Here, we used mammary, pancreatic, and colon cancer cells to show that adaptation to growth at an extracellular pH (pHe ) mimicking acidic tumor niches is associated with upregulated net acid extrusion capacity and elevated intracellular pH at physiological pHe , but not at acidic pHe . Using metabolic profiling, shotgun lipidomics, imaging and biochemical analyses, we show that the acid adaptation-induced phenotype is characterized by a shift toward oxidative metabolism, increased lipid droplet-, triacylglycerol-, peroxisome content and mitochondrial hyperfusion. Peroxisome proliferator-activated receptor-α (PPARA, PPARα) expression and activity are upregulated, at least in part by increased fatty acid uptake. PPARα upregulates genes driving increased mitochondrial and peroxisomal mass and β-oxidation capacity, including mitochondrial lipid import proteins CPT1A, CPT2 and SLC25A20, electron transport chain components, peroxisomal proteins PEX11A and ACOX1, and thioredoxin-interacting protein (TXNIP), a negative regulator of glycolysis. This endows acid-adapted cancer cells with increased capacity for utilizing fatty acids for metabolic needs, while limiting glycolysis. As a consequence, the acid-adapted cells exhibit increased sensitivity to PPARα inhibition. We conclude that PPARα is a key upstream regulator of metabolic changes favoring cancer cell survival in acidic tumor niches.

BNIP3L/NIX regulates both mitophagy and pexophagy

Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX‐dependent manner. We reveal that the outer mitochondrial membrane‐anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Catalase is required for peroxisome maintenance during adipogenesis

Although obesity contributes to the onset and pathogenesis of metabolic diseases, it has been repeatedly demonstrated that being overweight or mildly obese carries a survival advantage compared with being thin or normal-weight. This relationship is called the obesity paradox. Hence, it is necessary to clarify the underlying mechanism of obesity onset for the prevention and treatment of these diseases. Catalase is distributed in peroxisomes under normal redox conditions and catalase activity is increased during the differentiation of 3T3-L1 preadipocytes to adipocytes. Although peroxisomes are responsible for lipid metabolism, the role of peroxisomal catalase in the process of lipid accumulation remains unclear. The present study aimed to investigate the relationships among catalase activity, peroxisome content, and lipid accumulation during the differentiation of 3T3-L1 preadipocytes to adipocytes. Increased catalase activity and lipid accumulation were observed during the differentiation of preadipocytes. Silencing of catalase by small interfering RNA or treatment with 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, resulted in reduced lipid accumulation. Inhibition of catalase activity in peroxisomes increases hydrogen peroxide (H2O2) levels, which results in a reduction of peroxisome content. Extracellular H2O2 had no influence on lipid accumulation during differentiation. The occurrence of autophagy was clearly enhanced in cells treated with 3-AT. Spautin-1, an inhibitor of autophagy flux, protected against a reduction in lipid accumulation by treatment with 3-AT. Our data provide evidence that catalase protects against the degradation of peroxisomes via the occurrence of autophagy triggered by the generation of H2O2 in peroxisomes. These results suggest that catalase in peroxisomes is crucial to adipogenesis.

Slc25a17 Gene Trapped Mice: PMP34 Plays a Role in the Peroxisomal Degradation of Phytanic and Pristanic Acid.

Mice lacking PMP34, a peroxisomal membrane transporter encoded by Slc25a17, did not manifest any obvious phenotype on a Swiss Webster genetic background, even with various treatments designed to unmask impaired peroxisomal functioning. Peroxisomal α- and β-oxidation rates in PMP34 deficient fibroblasts or liver slices were not or only modestly affected and in bile, no abnormal bile acid intermediates were detected. Peroxisomal content of cofactors like CoA, ATP, NAD+, thiamine-pyrophosphate and pyridoxal-phosphate, based on direct or indirect data, appeared normal as were tissue plasmalogen and very long chain fatty acid levels. However, upon dietary phytol administration, the knockout mice displayed hepatomegaly, liver inflammation, and an induction of peroxisomal enzymes. This phenotype was partially mediated by PPARα. Hepatic triacylglycerols and cholesterylesters were elevated and both phytanic acid and pristanic acid accumulated in the liver lipids, in females to higher extent than in males. In addition, pristanic acid degradation products were detected, as wells as the CoA-esters of all these branched fatty acids. Hence, PMP34 is important for the degradation of phytanic/pristanic acid and/or export of their metabolites. Whether this is caused by a shortage of peroxisomal CoA affecting the intraperoxisomal formation of pristanoyl-CoA (and perhaps of phytanoyl-CoA), or the SCPx-catalyzed thiolytic cleavage during pristanic acid β-oxidation, could not be proven in this model, but the phytol-derived acyl-CoA profile is compatible with the latter possibility. On the other hand, the normal functioning of other peroxisomal pathways, and especially bile acid formation, seems to exclude severe transport problems or a shortage of CoA, and other cofactors like FAD, NAD(P)+, TPP. Based on our findings, PMP34 deficiency in humans is unlikely to be a life threatening condition but could cause elevated phytanic/pristanic acid levels in older adults.

Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification (ECAR) but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into TCA cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation (FAO) was functionally important, as drug-mediated and genetic dampening of FAO resulted in a selective reduction GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.

Peroxisomal gene and protein expression increase in response to a high-lipid challenge in human skeletal muscle

Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM). AimsWe hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply. Materials and methodsHSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene (PEX) expression were assessed using western blotting and realtime qRT-PCR respectively. 1-14C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively. Results1) Under fasting conditions, HSkM tissue demonstrated a significant correlation (P ≪ 0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes (Pex19, PEX11A, and PEX5) were significantly (P ≪ 0.05) upregulated. 3) Increments in PMP70, carnitine octanoyl transferase (CrOT), PGC-1α, and ERRα mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly (P ≪ 0.05) elevated 48-h post lipid treatment. ConclusionsThese results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue.

Fungal Peroxisomes Proteomics.

Peroxisomes in fungi are involved in a huge number of different metabolic processes. In addition, non-metabolic functions have also been identified. The proteins that are present in a particular peroxisome determine its metabolic function, whether they are the matrix localized enzymes of the different metabolic pathways or the membrane proteins involved in transport of metabolites across the peroxisomal membrane. Other peroxisomal proteins play a role in organelle biogenesis and dynamics, such as fission, transport and inheritance. Hence, obtaining a complete overview of which proteins are present in peroxisomes at a given time or under a given growth condition provides invaluable insights into peroxisome biology. Bottom up approaches are ideal to follow one or a few proteins at a time but they are not able to give a global view of the content of peroxisomes. To gain such information, top down approaches are required and one that has provided valuable insights into peroxisome function is mass spectrometry based organellar proteomics. Here, we discuss the findings of several such studies in yeast and filamentous fungi and outline new insights into peroxisomal function that were gained from these studies.

Unravelling a role of LRPPRC in peroxisomal lipid metabolism through lipidomic investigations in human and mouse

RationaleLRPPRC (leucine‐rich pentatricopeptide repeat‐containing protein) is commonly described as a regulator of RNA metabolism in both mitochondria and nuclei, but the overall biological impact of this role remains unclear. Integrative genomics analyses have revealed that mutations in the LRPPRC gene were causing the cytochrome c oxidase defect in the French‐Canadian variant of Leigh Syndrome (LSFC) (Mootha et al. 2003). More recently, a targeted metabolomics study in LSFC patients highlighted specific markers reflecting mitochondrial metabolic perturbations for various nutrients including fatty acids (Thompson‐Legault et al. 2015). The role of LRPPRC in lipid metabolism remains, however, to be better deciphered.Goal & MethodsTo dissect the role of LRPPRC in lipid metabolism, we have analyzed plasma from LSFC patients as well as plasma and livers from hepato‐specific KO‐LRPPRC mice using a combination of mass spectrometry (MS)‐based approaches. These include: (1) an untargeted and comprehensive lipidomic workflow, which enables the coverage of > 1300 unique lipid entities, and (2) targeted lipidomic analyses to probe fatty acid (FA) metabolism through acylcarnitines (ACs) profiling (covering >100 species) or cholesterol catabolism into bile acids (BAs; 9 (un)conjugated species).ResultsThe lipidomic profile observed in LSFC patients is typically characteristic of peroxisomal dysfunction as revealed by a: (i) 2‐fold decrease in plasmalogens (Pls; p<0.01), (ii) 2‐fold decrease in conjugate BAs such as glyco‐(G), glycodeoxy‐(GD) (p<0.001) and tauro‐(T) (p<0.05) cholic acids (CA), and (iii) 1.5‐fold increase in specific AC species, namely those with odd chains or > 20 carbons (p<0.05). These results in humans were corroborated in transgenic mice. Indeed, in plasma, there is a 1.5‐fold decrease in Pls (p<0.05). In addition, in liver, several results point out to a remodeling of peroxisomal metabolism. These include: (i) an imbalance in BA conjugation (10‐fold increase in CA, p<0.01; 1.4‐fold decrease in GDCA, p<0.05) and a 4‐ to 10‐fold increase in odd chain and >20 carbons ACs (p<0.01). This notion is further substantiated by changes in gene and/or protein expression levels for: (i) catalase, a classical marker of peroxisome content (+16%; protein, p<0.001), (ii) ACOX1, marker of peroxisomal β‐oxidation (−40%; protein, p<0.05) and (iii) several peroxins (Pex) involved in peroxisome biogenesis and transport, namely Pex11β, Pex14, Pex19, Pex1 and Pex 10 (up to +60%; mRNA; p<0.05).ConclusionCollectively there results highlight a novel role for LRPPRC in the regulation of lipid metabolism beyond mitochondria, namely in peroxisomes. Whether peroxisomal lipid perturbations are specific to LRPPRC gene defect or represent a common feature of mitochondrial dysfunction remain, however, to be further ascertained.Support or Funding InformationThis work was supported by the “Fondation Grand défi Pierre Lavoie”.

Correlation of peroxisome pathway reactive oxygen species oxidative stress gene and its correlation with the antitumor sensitivity of artesunate against pancreatic cancer

Objective To explore the screening of peroxisome pathway reactive oxygen species (ROS) oxidative stress gene and its correlation with the antitumor sensitivity of artesunate against pancreatic cancer. Methods Based on microarray mRNA expressions of 55 tumor cell lines in the National Cancer Institute common database, peroxisome pathway-related key genes which were significant correlation with half-inhibitory concentration (IC50) values of artesunate antitumor activity against human pancreatic cancer were selected by Kendall test. The candidate genes associated with artesunate sensitivity were identified and their mRNA expressions in pancreatic cancer cells were tested using fluorescent quantitative PCR. The contents of peroxidase in pancreatic cancer cells were detected through the DAB staining. Results Thirteen key genes mRNA expressions in peroxidase pathways were significantly correlated with IC50 values for artesunate antitumor activity. Compared with normal liver cells HL-7702 (1.00), CRAT (2.89±0.06), PEX11B (1.90±0.07) and PEX16 (1.35±0.07) mRNA expression levels were significantly increased in pancreatic cancer Panc-1 cells which sensitive to artesunate (t=33.00, P<0.01; t=17.85, P<0.01; t=4.54, P<0.05). While CAT (1.43±0.03), SOD1 (2.07±0.04) and SOD2 (1.15±0.01) mRNA expression levels were also significantly increased in Panc-1 cells which sensitive to artesunate (t=11.71, P<0.01; t=35.85, P<0.01; t=13.22, P<0.01). However, PEX12 (0.51±0.02), CAT (0.47±0.02), PRDX1 (0.43±0.01), and SOD1 (0.44±0.01) mRNA expression levels in pancreatic cancer BXPC-3 cells which resistant to artesunate were significantly lower than that of HL-7702 cells (t=37.53, P<0.01; t=16.52, P<0.01; t=84.20, P<0.01; t=48.24, P<0.01). DAB staining showed that the positive expression rate of peroxisomal content was apparently higher in Panc-1 cells (61.5%) than that of HL-7702 cells (43.8%), with a significant difference (χ2=16.11, P<0.01). Conclusion Peroxisome and its related ROS antioxidant enzymes CAT, PRDX1, SOD gene expression may be the important factors that affect artesunate antitumor activity against human pancreatic cancer. Key words: Pancreatic neoplasms; Peroxisomes; Reactive oxygen species; Antineoplastic agents; Artesunate

Malnutrition-associated liver steatosis and ATP depletion is caused by peroxisomal and mitochondrial dysfunction

Severe malnutrition in young children is associated with signs of hepatic dysfunction such as steatosis and hypoalbuminemia, but its etiology is unknown. Peroxisomes and mitochondria play key roles in various hepatic metabolic functions including lipid metabolism and energy production. To investigate the involvement of these organelles in the mechanisms underlying malnutrition-induced hepatic dysfunction we developed a rat model of malnutrition. Weanling rats were placed on a low protein or control diet (5% or 20% of calories from protein, respectively) for four weeks. Peroxisomal and mitochondrial structural features were characterized using immunofluorescence and electron microscopy. Mitochondrial function was assessed using high-resolution respirometry. A novel targeted quantitative proteomics method was applied to analyze 47 mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle and fatty acid β-oxidation pathways. Low protein diet-fed rats developed hypoalbuminemia and hepatic steatosis, consistent with the human phenotype. Hepatic peroxisome content was decreased and metabolomic analysis indicated peroxisomal dysfunction. This was followed by changes in mitochondrial ultrastructure and increased mitochondrial content. Mitochondrial function was impaired due to multiple defects affecting respiratory chain complex I and IV, pyruvate uptake and several β-oxidation enzymes, leading to strongly reduced hepatic ATP levels. Fenofibrate supplementation restored hepatic peroxisome abundance and increased mitochondrial β-oxidation capacity, resulting in reduced steatosis and normalization of ATP and plasma albumin levels. Malnutrition leads to severe impairments in hepatic peroxisomal and mitochondrial function, and hepatic metabolic dysfunction. We discuss the potential future implications of our findings for the clinical management of malnourished children. Severe malnutrition in children is associated with metabolic disturbances that are poorly understood. In order to study this further, we developed a malnutrition animal model and found that severe malnutrition leads to an impaired function of liver mitochondria which are essential for energy production and a loss of peroxisomes, which are important for normal liver metabolic function.

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.

Enhanced peroxisomal β-oxidation is associated with prevention of obesity and glucose intolerance by fish oil-enriched diets

The effects of different amounts of omega 3-polyunsaturated fatty acids in diets with normal or high content of fat on lipid and carbohydrate metabolism were investigated. Mice were fed for 8 weeks on diets enriched with fish oil or lard at 10% or 60% of energy. Energy balance and energy expenditure were analyzed. Fatty acid (FA) oxidative capacity of the liver and the activity of enzymes involved in this pathway were assessed. Fish oil-fed mice had lower body weight and adiposity compared with lard-fed animals, despite having lower rates of oxygen consumption. Mice fed diets containing fish oil also displayed lower glycemia, reduced fat content in the liver, and improved glucose tolerance compared with lard-fed animals. The fish oil-containing diets increased markers of hepatic peroxisomal content and increased the generation of metabolites derived from FA β-oxidation in liver homogenates. In contrast, no changes were observed in the content of mitochondrial electron transport chain proteins or carnitine palmitoyl transferase-1 in the liver, indicating little direct effect of fish oil on mitochondrial metabolism. Collectively, our findings suggest that the energy inefficient oxidation of FAs in peroxisomes may be an important mechanism underlying the protection against obesity and glucose intolerance of fish oil administration.

Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in Arabidopsis thaliana

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H(2)O(2), which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP-ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.

Interaction of the Aspergillus nidulans Microtubule-Organizing Center (MTOC) Component ApsB with Gamma-Tubulin and Evidence for a Role of a Subclass of Peroxisomes in the Formation of Septal MTOCs

Peroxisomes are a diverse class of organelles involved in different physiological processes in eukaryotic cells. Although proteins imported into peroxisomes carry a peroxisomal targeting sequence at the C terminus (PTS1) or an alternative one close to the N terminus (PTS2), the protein content of peroxisomes varies drastically. Here we suggest a new class of peroxisomes involved in microtubule (MT) formation. Eukaryotic cells assemble MTs from distinct points in the cell. In the fungus Aspergillus nidulans, septum-associated microtubule-organizing centers (sMTOCs) are very active in addition to the spindle pole bodies (SPBs). Previously, we identified a novel MTOC-associated protein, ApsB (Schizosaccharomyces pombe mto1), whose absence affected MT formation from sMTOCs more than from SPBs, suggesting that the two protein complexes are organized differently. We show here that sMTOCs share at least two further components, gamma-tubulin and GcpC (S. pombe Alp6) with SPBs and found that ApsB interacts with gamma-tubulin. In addition, we discovered that ApsB interacts with the Woronin body protein HexA and is targeted to a subclass of peroxisomes via a PTS2 peroxisomal targeting sequence. The PTS2 motif was necessary for function but could be replaced with a PTS1 motif at the C terminus of ApsB. These results suggest a novel function for a subclass of peroxisomes in cytoskeletal organization.

Peroxisome Dynamics in Cultured Mammalian Cells

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants.

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.

Peroxisomal enhancement of mitochondrial function

Background A reduction in skeletal muscle (SKM) mitochondrial oxidation of fatty acids is linked to accumulation of bioactive lipids and insulin resistance. In the liver, the cellular defense against lipotoxic accumulation of fatty acids is the peroxisome, which partially oxidizes very long- (VLCFA) and long-chain (LCFA) fatty acids which, as exported acylcarnitines, are utilized by mitochondria more efficiently because entry into the matrix is CPT-1 independent. To date, little data exists on peroxisomal function in SKM. Purpose To test the direct effects of elevated peroxisomal content on SKM mitochondrial function. Methods Peroxisomes and mitochondria were isolated from rat liver and whole gastrocnemius, respectively. Oxidation rates (as captured 14CO2) were compared under the following conditions: 1) mitochondria plus 1-14C Palmitic Acid (LCFA) ± malonyl-CoA ± increasing peroxisomal content 2) mitochondria plus 1-14C Lignoceric Acid (VLCFA) ± malonyl-CoA ± increasing peroxisomal content. Results SKM mitochondrial oxidation of VLCFA was linearly enhanced in the presence of increasing peroxisomal content. Peroxisomal titration did not alter baseline mitochondrial LCFA oxidation, but did rescue a significant portion of LCFA oxidation when malonyl-CoA was present. Conclusion This study is the first to directly demonstrate that increasing peroxisomal content results in increased SKM mitochondrial oxidation of fatty acids in the inhibited state. These findings suggest a metabolic target for therapeutic intervention that could lower intramuscular lipids suspected to play a major role in the etiology of obesity and diabetes. Support by NIH R15-DK061314

Peroxisomes during development and in distinct cell types.

In normal wild type cells of the same individual enzymatic content and activity of peroxisomes (Px) vary greatly with developmental stage and with cell-and tissue type; so do peroxisomal number, shape and volume. Wcell-and examples in humans are AGT and pipecolic acid oxidase, enzymes which are expressed only in liver parenchyma*, precluding their investigation in cultured fibroblasts and prenatal testing in chorionic villi. Since the genome is identical in all these circumstances, several delicate regulatory processes must be determining the final phenotype. Studying this variability could give insight into the control mechanisms, and possibly create opportunities to modify peroxisomal functions on purpose.