Self-Interaction of Human Pex11pβ during Peroxisomal Growth and Division

Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.     

The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD⁺ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.     

Generation of resolving memory neutrophils through pharmacological training with 4-PBA or genetic deletion of TRAM

Neutrophils are the dominant leukocytes in circulation and the first responders to infection and inflammatory cues. While the roles of neutrophils in driving inflammation have been widely recognized, the contribution of neutrophils in facilitating inflammation resolution is under-studied. Here, through single-cell RNA sequencing analysis, we identified a subpopulation of neutrophils exhibiting pro-resolving characteristics with greater Cd200r and Cd86 expression at the resting state. We further discovered that 4-PBA, a peroxisomal stress-reducing agent, can potently train neutrophils into the resolving state with enhanced expression of CD200R, CD86, as well as soluble pro-resolving mediators Resolvin D1 and SerpinB1. Resolving neutrophils trained by 4-PBA manifest enhanced phagocytosis and bacterial-killing functions. Mechanistically, the generation of resolving neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuit modulated by TLR4 adaptor molecule TRAM. We further demonstrated that genetic deletion of TRAM renders the constitutive expansion of resolving neutrophils, with an enhanced signaling circuitry of PPARγ/LMO4/STAT3. These findings may have profound implications for the effective training of resolving neutrophils with therapeutic potential in the treatment of both acute infection as well as chronic inflammatory diseases.Subject terms: Innate immune cells, Inflammation     

Major Facilitator Superfamily Domain-Containing Protein 2a (MFSD2A) Has Roles in Body Growth,Motor Function,and Lipid Metabolism

The metabolic adaptations to fasting in the liver are largely controlled by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARα), where PPARα upregulates genes encoding the biochemical pathway for β-oxidation of fatty acids and ketogenesis. As part of an effort to identify and characterize nutritionally regulated genes that play physiological roles in the adaptation to fasting, we identified Major facilitator superfamily domain-containing protein 2a (Mfsd2a) as a fasting-induced gene regulated by both PPARα and glucagon signaling in the liver. MFSD2A is a cell-surface protein homologous to bacterial sodium-melibiose transporters. Hepatic expression and turnover of MFSD2A is acutely regulated by fasting/refeeding, but expression in the brain is constitutive. Relative to wildtype mice, gene-targeted Mfsd2a knockout mice are smaller, leaner, and have decreased serum, liver and brown adipose triglycerides. Mfsd2a knockout mice have normal liver lipid metabolism but increased whole body energy expenditure, likely due to increased β-oxidation in brown adipose tissue and significantly increased voluntary movement, but surprisingly exhibited a form of ataxia. Together, these results indicate that MFSD2A is a nutritionally regulated gene that plays myriad roles in body growth and development, motor function, and lipid metabolism. Moreover, these data suggest that the ligand(s) that are transported by MFSD2A play important roles in these physiological processes and await future identification.     

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H₂O₂ and β-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein–protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.     

Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis

We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p. All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers. Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy. The partial growth defect on fatty acids of a pex25Δ mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids. Moreover, a severe peroxisomal protein import defect was observed in the pex11Δpex25Δpex27Δ triple mutant strain. This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation. When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed. Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number.     

Identification, separation, and characterization of acyl-coenzyme A dehydrogenases involved in mitochondrial beta-oxidation in higher plants

The existence in higher plants of an additional β-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of β-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial β-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial β-oxidation in higher plants.     

Reverse crosstalk of TGFβ and PPARβ/δ signaling identified by transcriptional profiling

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and transforming growth factor-β (TGFβ) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFβ, which is counteracted by PPARβ/δ activation. This is mediated, at least in part, by the TGFβ-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARβ/δ ligands, indicating that TGFβ and PPARβ/δ signals are integrated by chromatin-associated complexes. A second class represents TGFβ-induced genes that are downregulated by PPARβ/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARβ/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARβ/δ is able to influence the expression of distinct sets of both TGFβ-repressed and TGFβ-activated genes in both directions.     

Mechanism of Butyrate Stimulation of Triglyceride Storage and Adipokine Expression during Adipogenic Differentiation of Porcine Stromovascular Cells

Short chain fatty acids (SCFA), products of microbial fermentation of dietary fiber, exert multiple metabolic effects in cells. Previously, we had demonstrated that soluble fiber influenced fat mass accumulation, gut microbial community structure and SCFA production in pigs. The current study was designed to identify effects of SCFA treatment during adipogenic differentiation of porcine stromovascular cells on lipid metabolism and adipokine expression. Differentiating cells were treated with varying concentrations of butyrate. Results show that butyrate treatment enhanced adipogenesis and lipid accumulation, perhaps through upregulation of glucose uptake and de novo lipogenesis and other mechanisms that include induction of SREBP-1c, C/EBPα/β, GLUT4, LPL, PPARγ, GPAT4, DGAT1 and DGAT2 expression. In addition, butyrate induced adiponectin expression, resulting in activation of downstream target genes, such as AMPK and AKT. Activation of AMPK by butyrate led to phosphorylation of ACC. Although increased ACO gene expression was seen with butyrate treatment, experiments with the peroxisomal fatty acid inhibitor, thioridazine, suggest that butyrate may have an inhibitory effect on peroxisomal fatty acid oxidation. Our studies also provide evidence that butyrate may inhibit lipolysis, perhaps in an FFAR3-dependent manner. Therefore, this study presents a novel paradigm for butyrate action in adipocytes and shows that adipocytes are capable of utilizing butyrate, leading to increased expression of adiponectin for enhanced glucose uptake and improved insulin sensitivity.     

CMHX008, a Novel Peroxisome Proliferator-Activated Receptor γ Partial Agonist,Enhances Insulin Sensitivity In Vitro and In Vivo

The peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in adipocyte differentiation and insulin sensitivity. Its ligand rosiglitazone has anti-diabetic effect but is frequently accompanied with some severe unwanted effects. The aim of the current study was to compare the anti-diabetic effect of CMHX008, a novel thiazolidinedione-derivative, with rosiglitazone. A luciferase assay was used to evaluate in vitro PPARγ activation. 3T3-L1 cells were used to examine adipocyte differentiation. High fat diet (HFD) mice were used to examine in vivo insulin sensitivity. The mRNA levels were evaluated by real-time RT-PCR. Serum biochemical and hormonal variables were assessed using a clinical chemistry analyser. CMHX008 displayed a moderate PPARγ agonist activity, and promoted 3T3-L1 preadipocyte differentiation with lower activity than rosiglitazone. CMHX008 regulated the expression of PPARγ target genes in a different manner from rosiglitazone. CMHX008 increased the expression and secretion of adiponectin with the similar efficacy as rosiglitazone, but only 25% as potent as rosiglitazone for the induction of adipocyte fatty acid binding protein. Treatment of CMHX008 and rosiglitazone protected mice from high fat diet (HFD)-induced glucose intolerance, hyperinsulinemia and inflammation. CMHX008 reduced the mRNA expression of M1 macrophage markers, and significantly increased the expressions of M2 markers. In conclusion, CMHX008 shared the comparable insulin-sensitizing effects as rosiglitazone with lower adipogenic capacity and might potentially be developed into an effective agent for the treatment of diabetes and metabolic disorders.     

Oleoylethanolamide enhances β-adrenergic-mediated thermogenesis and white-to-brown adipocyte phenotype in epididymal white adipose tissue in rat

β-adrenergic receptor activation promotes brown adipose tissue (BAT) β-oxidation and thermogenesis by burning fatty acids during uncoupling respiration. Oleoylethanolamide (OEA) can inhibit feeding and stimulate lipolysis by activating peroxisome proliferator-activating receptor-α (PPARα) in white adipose tissue (WAT). Here we explore whether PPARα activation potentiates the effect of β3-adrenergic stimulation on energy balance mediated by the respective agonists OEA and {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243. The effect of this pharmacological association on feeding, thermogenesis, β-oxidation, and lipid and cholesterol metabolism in epididymal (e)WAT was monitored. {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243 (1 mg/kg) and OEA (5 mg/kg) co-administration over 6 days enhanced the reduction of both food intake and body weight gain, increased the energy expenditure and reduced the respiratory quotient (VCO₂/VO₂). This negative energy balance agreed with decreased fat mass and increased BAT weight and temperature, as well as with lowered plasma levels of triglycerides, cholesterol, nonessential fatty acids (NEFAs), and the adipokines leptin and TNF-α. Regarding eWAT, {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243 and OEA treatment elevated levels of the thermogenic factors PPARα and UCP1, reduced p38-MAPK phosphorylation, and promoted brown-like features in the white adipocytes: the mitochondrial (Cox4i1, Cox4i2) and BAT (Fgf21, Prdm16) genes were overexpressed in eWAT. The enhancement of the fatty-acid β-oxidation factors Cpt1b and Acox1 in eWAT was accompanied by an upregulation of de novo lipogenesis and reduced expression of the unsaturated-fatty-acid-synthesis enzyme gene, Scd1. We propose that the combination of β-adrenergic and PPARα receptor agonists promotes therapeutic adipocyte remodelling in eWAT, and therefore has a potential clinical utility in the treatment of obesity.KEY WORDS: Peroxisome proliferator-activated receptor alpha, β3-adrenergic receptor, Thermogenesis, β-oxidation, Adipocyte     

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves of Arabidopsis during Extended Darkness

Fatty acid β-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core β-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal β-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly α-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable α-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of α-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and β-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal β-oxidation plays a major role in dark-treated plants after depletion of starch reserves.     

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.     

Increasing Adipocyte Lipoprotein Lipase Improves Glucose Metabolism in High Fat Diet-induced Obesity

Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure.