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The Journal of Biological Chemistry Aug 2023Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid...
Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.
Topics: Humans; Adaptor Proteins, Signal Transducing; Endoplasmic Reticulum; HEK293 Cells; Lipid Metabolism; Membrane Proteins; Mitochondrial Membranes; Peroxisomes
PubMed: 37414147
DOI: 10.1016/j.jbc.2023.105013 -
Proceedings of the National Academy of... Jul 2023Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed...
Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino ., , 1476 (2013); H. G. Lee and P. J. Seo, , 12525 (2016)] but had not been implicated in lipid droplet dynamics. transcript levels were unchanged in mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks ., , 1515 (2022)]. When expressed in , human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.
Topics: Humans; Arabidopsis; Arabidopsis Proteins; Lipid Droplets; Lipid Mobilization; Peroxisomes; Seedlings; Transcription Factors; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases
PubMed: 37410814
DOI: 10.1073/pnas.2304870120 -
Nature Cell Biology Aug 2023Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts...
Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.
Topics: Animals; Mitochondria; Peroxisomes; Fatty Acids; Oxidation-Reduction; Lipid Metabolism; Homeostasis; Mammals
PubMed: 37400497
DOI: 10.1038/s41556-023-01180-2 -
Free Radical Biology & Medicine Sep 2023Reduced (NADH) and oxidized (NAD) nicotinamide adenine dinucleotides are ubiquitous hydride-donating/accepting cofactors that are essential for cellular bioenergetics....
Reduced (NADH) and oxidized (NAD) nicotinamide adenine dinucleotides are ubiquitous hydride-donating/accepting cofactors that are essential for cellular bioenergetics. Peroxisomes are single-membrane-bounded organelles that are involved in multiple lipid metabolism pathways, including beta-oxidation of fatty acids, and which contain several NAD(H)-dependent enzymes. Although maintenance of NAD(H) homeostasis in peroxisomes is considered essential for peroxisomal beta-oxidation, little is known about the regulation thereof. To resolve this issue, we have developed methods to specifically measure intraperoxisomal NADH levels in human cells using peroxisome-targeted NADH biosensors. By targeted CRISPR-Cas9-mediated genome editing of human cells, we showed with these sensors that the NAD/NADH ratio in cytosol and peroxisomes are closely connected and that this crosstalk is mediated by intraperoxisomal lactate and malate dehydrogenases, generated via translational stop codon readthrough of the LDHB and MDH1 mRNAs. Our study provides evidence for the existence of two independent redox shuttle systems in human peroxisomes that regulate peroxisomal NAD/NADH homeostasis. This is the first study that shows a specific metabolic function of protein isoforms generated by translational stop codon readthrough in humans.
Topics: Humans; NAD; Codon, Terminator; Peroxisomes; Protein Biosynthesis; Oxidation-Reduction; Homeostasis
PubMed: 37355054
DOI: 10.1016/j.freeradbiomed.2023.06.020 -
Cellular and Molecular Life Sciences :... Jun 2023Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in...
Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in mitochondria are a function of preserving cellular function or a response to cellular damage caused by the absence of peroxisomes. To address this, we developed conditional hepatocyte-specific Pex16 deficient (Pex16 KO) mice that develop peroxisome loss and subjected them to a low-protein diet to induce metabolic stress. Loss of PEX16 in hepatocytes led to increased biogenesis of small mitochondria and reduced autophagy flux but with preserved capacity for respiration and ATP capacity. Metabolic stress induced by low protein feeding led to mitochondrial dysfunction in Pex16 KO mice and impaired biogenesis. Activation of PPARα partially corrected these mitochondrial disturbances, despite the absence of peroxisomes. The findings of this study demonstrate that the absence of peroxisomes in hepatocytes results in a concerted effort to preserve mitochondrial function, including increased mitochondrial biogenesis, altered morphology, and modified autophagy activity. Our study underscores the relationship between peroxisomes and mitochondria in regulating the hepatic metabolic responses to nutritional stressors.
Topics: Mice; Animals; Peroxisomes; Organelle Biogenesis; Mitochondria; Liver; Autophagy
PubMed: 37338571
DOI: 10.1007/s00018-023-04827-3 -
The Journal of Biological Chemistry Jul 2023Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified...
Whereas it is known that p53 broadly regulates cell metabolism, the specific activities that mediate this regulation remain partially understood. Here, we identified carnitine o-octanoyltransferase (CROT) as a p53 transactivation target that is upregulated by cellular stresses in a p53-dependent manner. CROT is a peroxisomal enzyme catalyzing very long-chain fatty acids conversion to medium chain fatty acids that can be absorbed by mitochondria during β-oxidation. p53 induces CROT transcription through binding to consensus response elements in the 5'-UTR of CROT mRNA. Overexpression of WT but not enzymatically inactive mutant CROT promotes mitochondrial oxidative respiration, while downregulation of CROT inhibits mitochondrial oxidative respiration. Nutrient depletion induces p53-dependent CROT expression that facilitates cell growth and survival; in contrast, cells deficient in CROT have blunted cell growth and reduced survival during nutrient depletion. Together, these data are consistent with a model where p53-regulated CROT expression allows cells to be more efficiently utilizing stored very long-chain fatty acids to survive nutrient depletion stresses.
Topics: 5' Untranslated Regions; Carnitine; Carnitine Acyltransferases; Cell Growth Processes; Cell Respiration; Cell Survival; Fatty Acids; Mitochondria; Mutation; Nutrients; Oxidation-Reduction; Peroxisomes; Response Elements; Stress, Physiological; Transcriptional Activation; Tumor Suppressor Protein p53
PubMed: 37307919
DOI: 10.1016/j.jbc.2023.104908 -
Redox Biology Jul 2023Despite the large amounts of HO generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry...
Despite the large amounts of HO generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by HO than peroxisomal catalase itself.
Topics: Rats; Animals; Glutathione Disulfide; Peroxisomes; Cysteine; Protein Disulfide Reductase (Glutathione); Hydrogen Peroxide; Glutathione; Oxidation-Reduction; Proteins; Mammals; Homeostasis
PubMed: 37257275
DOI: 10.1016/j.redox.2023.102764 -
Advanced Science (Weinheim,... Jul 2023The peroxisome is a ubiquitous organelle in rodent cells and plays important roles in a variety of cell types and tissues. It is previously indicated that peroxisomes...
The peroxisome is a ubiquitous organelle in rodent cells and plays important roles in a variety of cell types and tissues. It is previously indicated that peroxisomes are associated with auditory function, and patients with peroxisome biogenesis disorders (PBDs) are found to have hearing dysfunction, but the specific role of peroxisomes in hearing remains unclear. In this study, two peroxisome-deficient mouse models (Atoh1-Pex5 and Pax2-Pex5 ) are established and it is found that peroxisomes mainly function in the hair cells of cochleae. Furthermore, peroxisome deficiency-mediated negative effects on hearing do not involve mitochondrial dysfunction and oxidative damage. Although the mammalian target of rapamycin complex 1 (mTORC1) signaling is shown to function through peroxisomes, no changes are observed in the mTORC1 signaling in Atoh1-Pex5 mice when compared to wild-type (WT) mice. However, the expression of large-conductance, voltage-, and Ca2 -activated K (BK) channels is less in Atoh1-Pex5 mice as compared to the WT mice, and the administration of activators of BK channels (NS-1619 and NS-11021) restores the auditory function in knockout mice. These results suggest that peroxisomes play an essential role in cochlear hair cells by regulating BK channels. Hence, BK channels appear as the probable target for treating peroxisome-related hearing diseases such as PBDs.
Topics: Mice; Animals; Large-Conductance Calcium-Activated Potassium Channels; Peroxisomes; Hair Cells, Auditory; Hearing Loss; Mice, Knockout; Mechanistic Target of Rapamycin Complex 1; Mammals
PubMed: 37171794
DOI: 10.1002/advs.202300402 -
Acta Neuropathologica Communications May 2023Peroxisomes are eukaryotic organelles that rapidly change in number depending on the metabolic requirement of distinct cell types and tissues. In the brain, these...
Peroxisomes are eukaryotic organelles that rapidly change in number depending on the metabolic requirement of distinct cell types and tissues. In the brain, these organelles are essential for neuronal migration and myelination during development and their dysfunction is associated with age-related neurodegenerative diseases. Except for one study analysing ABCD3-positive peroxisomes in neurons of the frontal neocortex of Alzheimer disease (AD) patients, no data on other brain regions or peroxisomal proteins are available. In the present morphometric study, we quantified peroxisomes labelled with PEX14, a metabolism-independent peroxisome marker, in 13 different brain areas of 8 patients each either with low, intermediate or high AD neuropathological changes compared to 10 control patients. Classification of patient samples was based on the official ABC score. During AD-stage progression, the peroxisome density decreased in the area entorhinalis, parietal/occipital neocortex and cerebellum, it increased and in later AD-stage patients decreased in the subiculum and hippocampal CA3 region, frontal neocortex and pontine gray and it remained unchanged in the gyrus dentatus, temporal neocortex, striatum and inferior olive. Moreover, we investigated the density of catalase-positive peroxisomes in a subset of patients (> 80 years), focussing on regions with significant alterations of PEX14-positive peroxisomes. In hippocampal neurons, only one third of all peroxisomes contained detectable levels of catalase exhibiting constant density at all AD stages. Whereas the density of all peroxisomes in neocortical neurons was only half of the one of the hippocampus, two thirds of them were catalase-positive exhibiting increased levels at higher ABC scores. In conclusion, we observed spatiotemporal differences in the response of peroxisomes to different stages of AD-associated pathologies.
Topics: Humans; Alzheimer Disease; Peroxisomes; Catalase; Pilot Projects; Neocortex
PubMed: 37170361
DOI: 10.1186/s40478-023-01567-0 -
Nature May 2023Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction. Among...
Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes. Current models postulate a large pore formed by transmembrane proteins; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels.
Topics: Intracellular Membranes; Membrane Proteins; Peroxins; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Phase Transition; Protein Binding; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Intrinsically Disordered Proteins
PubMed: 37165185
DOI: 10.1038/s41586-023-06044-1