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PEX19 binding sites are essential parts of the targeting signals of peroxisomal membrane proteins (mPTS). In this study, we characterized PEX19 binding sites of PEX11, the most abundant peroxisomal and glycosomal membrane protein from Trypanosoma brucei and Saccharomyces cerevisiae. TbPEX11 contains two PEX19 binding sites, one close to the N-terminus (BS1) and a second in proximity to the first transmembrane domain (BS2). The N-terminal BS1 is highly conserved across different organisms and is required for maintenance of the steady-state concentration and efficient targeting to peroxisomes and glycosomes in both baker’s yeast and Trypanosoma brucei. The second PEX19 binding site in TbPEX11 is essential for its glycosomal localization. Deletion or mutations of the PEX19 binding sites in TbPEX11 or ScPEX11 results in mislocalization of the proteins to mitochondria. Bioinformatic analysis indicates that the N-terminal region of TbPEX11 contains an amphiphilic helix and several putative TOM20 recognition motifs. We show that the extreme N-terminal region of TbPEX11 contains a cryptic N-terminal signal that directs PEX11 to the mitochondrion if its glycosomal transport is blocked.

Despite the large amounts of H2O2 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 H2O2 than peroxisomal catalase itself.

Abstract Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post‐translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell‐content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid‐dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule‐based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGCR) is one of the rate-limiting enzymes in the mevalonate pathway required for cholesterol biosynthesis. It is an integral membrane protein of the endoplasmic reticulum (ER) but has occasionally been described in peroxisomes. By co-immunofluorescence microscopy using different HMGCR antibodies, we present evidence for a dual localization of HMGCR in the ER and peroxisomes in differentiated human monocytic THP-1 cells, primary human monocyte-derived macrophages and human primary skin fibroblasts under conditions of low cholesterol and statin treatment. Using density gradient centrifugation and Western blot analysis, we observed a truncated HMGCR variant of 76 kDa in the peroxisomal fractions, while a full-length HMGCR of 96 kDa was contained in fractions of the ER. In contrast to primary human control fibroblasts, peroxisomal HMGCR was not found in fibroblasts from patients suffering from type-1 rhizomelic chondrodysplasia punctata, who lack functional PEX7 and, thus, cannot import peroxisomal matrix proteins harboring a type-2 peroxisomal targeting signal (PTS2). Moreover, in the N–terminal region of the soluble 76 kDa C-terminal catalytic domain, we identified a PTS2-like motif, which was functional in a reporter context. We propose that under sterol-depleted conditions, part of the soluble HMGCR domain, which is released from the ER by proteolytic processing for further turnover, remains sufficiently long in the cytosol for peroxisomal import via a PTS2/PEX7-dependent mechanism. Altogether, our findings describe a dual localization of HMGCR under combined lipid depletion and statin treatment, adding another puzzle piece to the complex regulation of HMGCR.

Magnaporthe oryzae, the pathogen responsible for rice blast disease, utilizes specialized infection structures known as appressoria to breach the leaf cuticle and establish intracellular, infectious hyphae. Our study demonstrates that the peroxin MoPex22 is crucial for appressorium function, specifically for the development of primary penetration hyphae. The ∆Mopex22 mutant exhibited slow growth, reduced aerial hyphae, and almost complete loss of virulence. Specifically, despite the mutant’s capability to form appressoria, it showed abnormalities during appressorium development, including reduced turgor, increased permeability of the appressorium wall, failure to form septin rings, and significantly decreased ability to penetrate host cells. Additionally, there was a delay in the degradation of lipid droplets during conidial germination and appressorium development. Consistent with these findings, the ΔMopex22 mutant showed an inefficient utilization of long-chain fatty acids and defects in cell wall integrity. Moreover, our findings indicate that MoPex22 acts as an anchor for MoPex4, facilitating the localization of MoPex4 to peroxisomes. Together with MoPex4, it affects the function of MoPex5, thus regulating the import of peroxisomal matrix proteins. Overall, these results highlight the essential role of MoPex22 in regulating the transport of peroxisomal matrix proteins, which affect fatty acid metabolism, glycerol accumulation, cell wall integrity, growth, appressorium development, and the pathogenicity of M. oryzae. This study provides valuable insights into the significance of peroxin functions in fungal biology and appressorium-mediated plant infection.

Abstract Peroxisomes are organelles that play essential roles in many metabolic processes, but also play roles in innate immunity, signal transduction, aging and cancer. One of the main functions of peroxisomes is the processing of very-long chain fatty acids into metabolites that can be directed to the mitochondria. One key family of enzymes in this process are the peroxisomal acyl-CoA oxidases (ACOX1, ACOX2 and ACOX3), the expression of which has been shown to be dysregulated in some cancers. Very little is however known about the expression of this family of oxidases in non-small cell lung cancer (NSCLC). ACOX2 has however been suggested to be elevated at the mRNA level in over 10% of NSCLC, and in the present study using both standard and bioinformatics approaches we show that expression of ACOX2 is significantly altered in NSCLC. ACOX2 mRNA expression is linked to a number of mutated genes, and associations between ACOX2 expression and tumour mutational burden and immune cell infiltration were explored. Links between ACOX2 expression and candidate therapies for oncogenic driver mutations such as KRAS were also identified. Furthermore, levels of acyl-CoA oxidases and other associated peroxisomal genes were explored to identify further links between the peroxisomal pathway and NSCLC. The results of this biomarker driven study suggest that ACOX2 may have potential clinical utility in the diagnosis, prognosis and stratification of patients into various therapeutically targetable options.

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.

Abstract Peroxisomes are very important organelles in eukaryotic cells and participate in various biological processes, including pathogen–host interactions. A variety of proteins involved in peroxisome proliferation and metabolic activity within peroxisomes have been shown to be essential for full virulence of several phytopathogenic fungi. However, the effects of changes in the number of peroxisomes and proteins involved in the peroxisome pathway on the pathogenicity of Botrytis cinerea have rarely been reported. In this study, by analysing transcriptome data and RT-qPCR validation, we found that more than half of the genes annotated to the peroxisome pathway in B. cinerea were upregulated more than twofold between mycelial samples cultured in medium with tomato leaves and without tomato leaves. A strain of B. cinerea with fluorescently labelled peroxisomes was obtained by overexpression of GFP fused to peroxisomal targeting signal 1 (the tripeptide ‘SKL’). The addition of tomato leaves to the liquid medium induced a significant increase in the number of peroxisomes, β-oxidation level, H2O2 content, and acetyl-CoA level in B. cinerea mycelia. When B. cinerea was cultured with oleic acid as the sole carbon source, the formation of infection-related structures and their penetration into plant cells were found to be associated with peroxisome pathway activity. Furthermore, peroxisome proliferation and lipid metabolism increased in response to different extracellular stresses in B. cinerea. Taken together, our results confirmed that activation of the peroxisome pathway in B. cinerea contributes to the initial infection and the ability to cope with environmental stress.

Sperm are terminally differentiated cells that lack most of the membranous organelles, resulting in a high abundance of ether glycerolipids found across different species. Ether lipids include plasmalogens, platelet activating factor, GPI-anchors and seminolipid. These lipids play important roles in sperm function and performance, and thus are of special interest as potential fertility markers and therapeutic targets. In the present article, we first review the existing knowledge on the relevance of the different types of ether lipids for sperm production, maturation and function. To further understand ether-lipid metabolism in sperm, we then query available proteomic data from highly purified sperm, and produce a map of metabolic steps retained in these cells. Our analysis pinpoints the presence of a truncated ether lipid biosynthetic pathway that would be competent for the production of precursors through the initial peroxisomal core steps, but devoid of subsequent microsomal enzymes responsible for the final synthesis of all complex ether-lipids. Despite the widely accepted notion that sperm lack peroxisomes, the thorough analysis of published data conducted herein identifies nearly 70% of all known peroxisomal resident proteins as part of the sperm proteome. In view of this, we highlight open questions related to lipid metabolism and possible peroxisomal functions in sperm. We propose a repurposed role for the truncated peroxisomal ether-lipid pathway in detoxification of products from oxidative stress, which is known to critically influence sperm function. The likely presence of a peroxisomal-derived remnant compartment that could act as a sink for toxic fatty alcohols and fatty aldehydes generated by mitochondrial activity is discussed. With this perspective, our review provides a comprehensive metabolic map associated with ether-lipids and peroxisomal-related functions in sperm and offers new insights into potentially relevant antioxidant mechanisms that warrant further research.

Microglial cells ensure essential roles in brain homeostasis. In pathological condition, microglia adopt a common signature, called disease-associated microglial (DAM) signature, characterized by the loss of homeostatic genes and the induction of disease-associated genes. In X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disease, microglial defect has been shown to precede myelin degradation and may actively contribute to the neurodegenerative process. We previously established BV-2 microglial cell models bearing mutations in peroxisomal genes that recapitulate some of the hallmarks of the peroxisomal β-oxidation defects such as very long-chain fatty acid (VLCFA) accumulation. In these cell lines, we used RNA-sequencing and identified large-scale reprogramming for genes involved in lipid metabolism, immune response, cell signaling, lysosome and autophagy, as well as a DAM-like signature. We highlighted cholesterol accumulation in plasma membranes and observed autophagy patterns in the cell mutants. We confirmed the upregulation or downregulation at the protein level for a few selected genes that mostly corroborated our observations and clearly demonstrated increased expression and secretion of DAM proteins in the BV-2 mutant cells. In conclusion, the peroxisomal defects in microglial cells not only impact on VLCFA metabolism but also force microglial cells to adopt a pathological phenotype likely representing a key contributor to the pathogenesis of peroxisomal disorders.

Protein phosphatase 2A (PP2A) is targeted to the plant peroxisome via a C-terminal SSL sequence on its regulatory B′ theta (θ) subunit. To date the substrates of peroxisomal PP2A are unknown but are thought to be recruited by the regulatory B'θ subunit. Employing yeast two hybrid screening, we have identified Arabidopsis E3 ligase SINA-like 10 as a B'θ binding partner. The E3 ligase SINA-like 10 was found to harbor the PP2A B′-binding Short Linear interaction Motif or SLiM, LxxIxE. This interaction was further verified both in vitro and in vivo using direct pulldown assays and bimolecular fluorescence complementation. Utilizing peroxisomal targeted and a cytosolic version of B'θ (lacking its C-terminal peroxisomal targeting sequence SSL>) bimolecular fluorescence complementation suggests an interaction to occur in the cytosol followed by piggybacking E3 ligase SINA-like 10 into peroxisomes. These results identify a first peroxisomal PP2A interactor, which also obtains a PP2A B′-binding SLiM. • Protein phosphatase 2A is targeted to the plant peroxisome via a C-terminal PTS1 (SSL>) on its regulatory B'θ subunit. • Y2H screening identified Arabidopsis E3 ligase SINA-like 10 as an interactor for PP2A B'θ. • Interaction between SINA-like 10 and PP2A B'θ was confirmed by direct pulldown and bimolecular fluorescence complementation. • Targeting SINA-like 10 to the peroxisome was dependent on PP2A B'θ. [ABSTRACT FROM AUTHOR]

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported in a posttranslational manner. Intricate protein import machineries have evolved that catalyze the different stages of translocation. In humans, PEX5L was found to be an essential component of the peroxisomal translocon. PEX5L is the main receptor for substrate proteins carrying a peroxisomal targeting signal (PTS). Substrates are bound by soluble PEX5L in the cytosol after which the cargo-receptor complex is recruited to peroxisomal membranes. Here, PEX5L interacts with the docking protein PEX14 and becomes part of an integral membrane protein complex that facilitates substrate translocation into the peroxisomal lumen in a still unknown process. In this study, we show that PEX5L containing complexes purified from human peroxisomal membranes constitute water-filled pores when reconstituted into planar-lipid membranes. Channel characteristics were highly dynamic in terms of conductance states, selectivity and voltage- and substrate-sensitivity. Our results show that a PEX5L associated pore exists in human peroxisomes, which can be activated by receptor-cargo complexes. [ABSTRACT FROM AUTHOR]

For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles. [ABSTRACT FROM AUTHOR]

Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs. [ABSTRACT FROM AUTHOR]

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynaminrelated protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) - a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used humanderived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1. [ABSTRACT FROM AUTHOR]

Inherited deficiency in ether lipids, a subgroup of glycerophospholipids with unique biochemical and biophysical properties, evokes severe symptoms in humans resulting in a multi-organ syndrome. Mouse models with defects in ether lipid biosynthesis have widely been used to understand the pathophysiology of human disease and to study the roles of ether lipids in various cell types and tissues. However, little is known about the function of these lipids in cardiac tissue. Previous studies included case reports of cardiac defects in ether-lipid-deficient patients, but a systematic analysis of the impact of ether lipid deficiency on the mammalian heart is still missing. Here, we utilize a mouse model of complete ether lipid deficiency (Gnpat KO) to accomplish this task. Similar to a subgroup of human patients with rhizomelic chondrodysplasia punctata (RCDP), a fraction of Gnpat KO fetuses present with defects in ventricular septation, presumably evoked by a developmental delay. We did not detect any signs of cardiomyopathy but identified increased left ventricular end-systolic and end-diastolic pressure in middle-aged ether-lipid-deficient mice. By comprehensive electrocardiographic characterization, we consistently found reduced ventricular conduction velocity, as indicated by a prolonged QRS complex, as well as increased QRS and QT dispersion in the Gnpat KO group. Furthermore, a shift of the Wenckebach point to longer cycle lengths indicated depressed atrioventricular nodal function. To complement our findings in mice, we analyzed medical records and performed electrocardiography in ether-lipid-deficient human patients, which, in contrast to the murine phenotype, indicated a trend towards shortened QT intervals. Taken together, our findings demonstrate that the cardiac phenotype upon ether lipid deficiency is highly heterogeneous, and although the manifestations in the mouse model only partially match the abnormalities in human patients, the results add to our understanding of the physiological role of ether lipids and emphasize their importance for proper cardiac development and function. [ABSTRACT FROM AUTHOR]

Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms, from growth and development to stress response. Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles, including peroxisomes, single‐membrane and highly dynamic organelles ubiquitous in eukaryotes. Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism, detoxification of reactive oxygen species (ROS), biosynthesis of vital hormones and cofactors, and photorespiration. Plant peroxisomes possess a complex proteome with functions varying among different tissue types and developmental stages, and during plant response to distinct environmental cues. However, how these diverse functions are regulated at the post‐translational level is poorly understood, especially in plants. In this review, we summarized current knowledge of the involvement of protein ubiquitination in peroxisome protein import, remodeling, pexophagy, and metabolism, focusing on plants, and referencing discoveries from other eukaryotic systems when relevant. Based on previous ubiquitinomics studies, we compiled a list of 56 ubiquitinated Arabidopsis peroxisomal proteins whose functions are associated with all the major plant peroxisomal metabolic pathways. This discovery suggests a broad impact of protein ubiquitination on plant peroxisome functions, therefore substantiating the need to investigate this significant regulatory mechanism in peroxisomes at more depths. [ABSTRACT FROM AUTHOR]

On the basis of findings that cultured rat hepatocytes secrete lipoprotein with a high plasmalogen content and the occurrence of this lipid in human serum, it has been suggested that hepatocytes play a role in the supply of plasmalogens to tissues. We tested this hypothesis in a mouse with a hepatocyte-specific defect in peroxisomes, an organelle essentially required for plasmalogen biosynthesis. We analyzed plasmalogens in lipid extracts of forebrain, liver and five further tissues and in plasma by reaction with dansylhydrazine in hydrochloric acid, which cleaves the vinyl ether of plasmalogens and forms a fluorescent dansylhydrazone, which we quantified by reversed phase high performance liquid chromatography. Reaction with dansylhydrazine in acetic acid was used to quantify free aldehydes as a control. Our results show normal levels of plasmalogens in plasma and in all tissues examined, including forebrain and the liver, irrespective of the inactivation of hepatic peroxisomes. None of the selected ether lipids analyzed by mass spectrometry in plasma and liver was decreased in the mice deficient in liver peroxisomes. In contrast, we found three plasmenylcholine species which were even significantly increased in the livers of these animals. Quantification of mRNA expression of plasmalogen biosynthetic enzymes revealed particularly low expression of fatty acyl-CoA reductase, the key regulatory enzyme of plasmalogen biosynthesis, in liver, with and without hepatic peroxisome deficiency. Our results do not support the suggested role of hepatocytes in supplying plasmalogens to tissues. [ABSTRACT FROM AUTHOR]

Polyamines (PAs) are positively charged amines that are present in all organisms. In addition to their functions specific to growth and development, they are involved in responding to various biotic and abiotic stress tolerance functions. The appropriate concentration of PA in the cell is maintained by a delicate balance between the catabolism and anabolism of PAs, which is primarily driven by two enzymes, namely diamine oxidase and polyamine oxidase (PAO). PAOs have been found to be localized in multiple subcellular locations, including peroxisomes. This paper presents a holistic account of peroxisomal PAOs. PAOs are flavin adenine dinucleotide-dependent enzymes with varying degrees of substrate specificity. They are expressed differentially upon various abiotic stress conditions, namely heat, cold, salinity, and dehydration. It has also been observed that in a particular species, the various PAO isoforms are expressed differentially with a spatial and temporal distinction. PAOs are targeted to peroxisome via a peroxisomal targeting signal (PTS) type 1. We conducted an extensive bioinformatics analysis of PTS1s present in various peroxisomal PAOs and present a consensus peroxisome targeting signal present in PAOs. Furthermore, we also propose an evolutionary perspective of peroxisomal PAOs. PAOs localized in plant peroxisomes are of potential importance in abiotic stress tolerance since peroxisomes are one of the nodal centers of reactive oxygen species (ROS) homeostasis and an increase in ROS is a major indicator of the plant being in stress conditions; hence, in the future, PAO enzymes could be used as a key candidate for generating abiotic stress tolerant crops. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum stress (ERS) occurs when cellular demand for protein folding exceeds the capacity of the organelle. Adaptation and cell survival in response to ERS requires a critical contribution by mitochondria and peroxisomes. During ERS responses, mitochondrial respiration increases to ameliorate reactive oxygen species (ROS) accumulation. We now show in yeast that peroxisome abundance also increases to promote an adaptive response. In pox1Δ cells, which are defective in peroxisomal ß-oxidation of fatty acids, the respiratory response to ERS is impaired and ROS accrues. However, the respiratory response to ERS is rescued and ROS production is mitigated in pox1Δ cells overexpressing Mpc1, the mitochondrial pyruvate carrier that provides another source of acetyl CoA to fuel the tricarboxylic acid cycle and oxidative phosphorylation. Using proteomics, select mitochondrial proteins were identified that undergo upregulation upon ERS to remodel the respiratory machinery. The abundance of several peroxisome-based proteins was also increased, corroborating the role of peroxisomes in ERS adaptation. Finally, ERS stimulates assembly of respiratory complexes into higher-order supercomplexes, underlying increased electron transfer efficiency. Our results highlight peroxisomal and mitochondrial support for ERS adaptation to favor cell survival. [ABSTRACT FROM AUTHOR]

Peroxisomes are essential metabolic organelles, well known for their roles in the metabolism of complex lipids and reactive ionic species. In the past 10 years, peroxisomes have also been cast as central regulators of immunity. Lipid metabolites of peroxisomes, such as polyunsaturated fatty acids (PUFAs), are precursors for important immune mediators, including leukotrienes (LTs) and resolvins. Peroxisomal redox metabolism modulates cellular immune signaling such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Additionally, peroxisomal β-oxidation and ether lipid synthesis control the development and aspects of the activation of both innate and adaptive immune cells. Finally, peroxisome number and metabolic activity have been linked to inflammatory diseases. These discoveries have opened avenues of investigation aimed at targeting peroxisomes for therapeutic intervention in immune disorders, inflammation, and cancer. Peroxisomes are important metabolic regulators of immune cell development and function. Peroxisomes produce distinct lipids that control core cellular immune responses, including phagocytosis and secretion of inflammatory cytokines. Peroxisomes regulate cellular reactive oxygen and nitrogen species to modulate immune and inflammatory signaling. Intracellular pathogens interfere with peroxisomes in different ways to evade the peroxisome-dependent immune response and support their survival. Manipulation of peroxisome plasticity and metabolism may lead to novel therapeutics to treat immune disorders and antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

With global warming, drought has become one of the major environmental pressures that threaten the development of global agricultural and forestry production. Cenococcum geophilum (C. geophilum) is one of the most common ectomycorrhizal fungi in nature, which can form mycorrhiza with a large variety of host trees of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. In this study, six C. geophilum strains with different drought tolerance were selected to analyze their molecular responses to drought stress with treatment of 10% polyethylene glycol. Our results showed that drought-sensitive strains absorbed Na and K ions to regulate osmotic pressure and up-regulated peroxisome pathway genes to promote the activity of antioxidant enzymes to alleviate drought stress. However, drought-tolerant strains responded to drought stress by up-regulating the functional genes involved in the ubiquinone and other terpenoid-quinone biosynthesis and sphingolipid metabolism pathways. The results provided a foundation for studying the mechanism of C. geophilum response to drought stress. [ABSTRACT FROM AUTHOR]

The chlorophyte algae are a dominant group of photosynthetic eukaryotes. Although many are photoautotrophs, there are also mixotrophs, heterotrophs, and even parasites. The physical characteristics of green algae are also highly diverse, varying greatly in size, shape, and habitat. Given this morphological and trophic diversity, we postulated that diversity may also exist in the protein components controlling intracellular movement of material by vesicular transport. One such set is the multisubunit tethering complexes (MTCs)—components regulating cargo delivery. As they span endomembrane organelles and are well‐conserved across eukaryotes, MTCs should be a good proxy for assessing the evolutionary dynamics across the diversity of Chlorophyta. Our results reveal that while green algae carry a generally conserved and unduplicated complement of MTCs, some intriguing variation exists. Notably, we identified incomplete sets of TRAPPII, exocyst, and HOPS/CORVET components in all Mamiellophyceae, and what is more, not a single subunit of Dsl1 was found in Cymbomonas tetramitiformis. As the absence of Dsl1 has been correlated with having unusual peroxisomes, we searched for peroxisome biogenesis machinery, finding very few components in Cymbomonas, suggestive of peroxisome degeneration. Overall, we demonstrate conservation of MTCs across green algae, but with notable taxon‐specific losses suggestive of unusual endomembrane systems. [ABSTRACT FROM AUTHOR]

Vancomycin (VCM)-induced nephrotoxicity impedes its treatment applications. Thus, it is important to clarify the relevant mechanism. This study investigated phosphoprotein changes attributable to the VCM nephrotoxicity mechanisms. Biochemical, pathological and phosphoproteomic analyses based on C57BL/6 mice were performed to explore the mechanisms.VCM-treated mice showed increased levels of blood urea nitrogen and creatinine, and signs of acute tubular necrotic lesions. Phosphoproteomic profiling identified 3025 differentially phosphorylated phosphopeptides between the model and control group. Gene Ontology enrichment analysis demonstrated that Molecular Function "oxidoreductase activity" and Cellular Component "peroxisome" were markedly enriched. KEGG pathway analysis identified an enrichment in peroxisome pathway and PPAR (peroxisome proliferator-activated receptor) signaling pathways. Parallel reaction monitoring analysis revealed a significant downregulation of CAT, SOD-1, AGPS, DHRS4, and EHHADH at phosphorylation level by VCM. Notably, the phosphorylation of ACO, AMACR, and SCPX was downregulated by VCM, which are the fatty acid β-oxidation-related proteins involved in PPAR signaling pathways. The phosphorylated PEX5 involved in peroxisome biogenesis was upregulated by VCM. Collectively, these findings indicated that VCM-induced nephrotoxicity is closely associated with peroxisome pathway and PPAR signaling pathways. The current study provides important insight into the mechanisms of VCM nephrotoxicity and will aid in the development of preventive and therapeutic strategies against this nephropathy. [ABSTRACT FROM AUTHOR]

Oxidative stress and inflammation are the key players in neuroinflammation, in which microglia dysfunction plays a central role. Previous studies suggest that argan oil attenuates oxidative stress, inflammation, and peroxisome dysfunction in mouse brains. In this study, we explored the effects of two major argan oil (AO) phytosterols, Schottenol (Schot) and Spinasterol (Spina), on oxidative stress, inflammation, and peroxisomal dysfunction in two murine microglial BV-2 cell lines, wild-ype (Wt) and Acyl-CoA oxidase 1 (Acox1)-deficient cells challenged with LPS treatment. Herein, we used an MTT test to reveal no cytotoxicity for both phytosterols with concentrations up to 5 µM. In the LPS-activated microglial cells, cotreatment with each of these phytosterols caused a significant decrease in intracellular ROS production and the NO level released in the culture medium. Additionally, Schot and Spina were able to attenuate the LPS-dependent strong induction of Il-1β and Tnf-α mRNA levels, as well as the iNos gene and protein expression in both Wt and Acox1−/− microglial cells. On the other hand, LPS treatment impacted both the peroxisomal antioxidant capacity and the fatty acid oxidation pathway. However, both Schot and Spina treatments enhanced ACOX1 activity in the Wt BV-2 cells and normalized the catalase activity in both Wt and Acox1−/− microglial cells. These data suggest that Schot and Spina can protect cells from oxidative stress and inflammation and their harmful consequences for peroxisomal functions and the homeostasis of microglial cells. Collectively, our work provides a compelling argument for the protective mechanisms of two major argan oil phytosterols against LPS-induced brain neuroinflammation. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum stress (ERS) occurs when cellular demand for protein folding exceeds the capacity of the organelle. Adaptation and cell survival in response to ERS requires a critical contribution by mitochondria and peroxisomes. During ERS responses, mitochondrial respiration increases to ameliorate reactive oxygen species (ROS) accumulation. We now show in yeast that peroxisome abundance also increases to promote an adaptive response. In pox1Δ cells, which are defective in peroxisomal β-oxidation of fatty acids, the respiratory response to ERS is impaired and ROS accrues. However, the respiratory response to ERS is rescued and ROS production is mitigated in pox1Δ cells overexpressing Mpc1, the mitochondrial pyruvate carrier that provides another source of acetyl CoA to fuel the tricarboxylic acid cycle and oxidative phosphorylation. Using proteomics, select mitochondrial proteins were identified that undergo upregulation upon ERS to remodel the respiratory machinery. The abundance of several peroxisome-based proteins was also increased, corroborating the role of peroxisomes in ERS adaptation. Finally, ERS stimulates assembly of respiratory complexes into higher-order supercomplexes, underlying increased electron transfer efficiency. Our results highlight peroxisomal and mitochondrial support for ERS adaptation to favor cell survival. [ABSTRACT FROM AUTHOR]

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function. [ABSTRACT FROM AUTHOR]

Peroxisomes are very important organelles in eukaryotic cells and participate in various biological processes, including pathogen–host interactions. A variety of proteins involved in peroxisome proliferation and metabolic activity within peroxisomes have been shown to be essential for full virulence of several phytopathogenic fungi. However, the effects of changes in the number of peroxisomes and proteins involved in the peroxisome pathway on the pathogenicity of Botrytis cinerea have rarely been reported. In this study, by analysing transcriptome data and RT-qPCR validation, we found that more than half of the genes annotated to the peroxisome pathway in B. cinerea were upregulated more than twofold between mycelial samples cultured in medium with tomato leaves and without tomato leaves. A strain of B. cinerea with fluorescently labelled peroxisomes was obtained by overexpression of GFP fused to peroxisomal targeting signal 1 (the tripeptide ‘SKL’). The addition of tomato leaves to the liquid medium induced a significant increase in the number of peroxisomes, β-oxidation level, H2O2 content, and acetyl-CoA level in B. cinerea mycelia. When B. cinerea was cultured with oleic acid as the sole carbon source, the formation of infection-related structures and their penetration into plant cells were found to be associated with peroxisome pathway activity. Furthermore, peroxisome proliferation and lipid metabolism increased in response to different extracellular stresses in B. cinerea. Taken together, our results confirmed that activation of the peroxisome pathway in B. cinerea contributes to the initial infection and the ability to cope with environmental stress. [ABSTRACT FROM AUTHOR]

Classical theory of fusion considers the fusion of bilayer membranes as a unification of the material of the membranes themselves and the water volumes surrounded by them. It has been shown that membrane fusion is accompanied by significant deformation of lipid monolayers. The optimal trajectory of the process passes through several intermediate structures characterized by local minima of the free energy of the system; the minima are separated by energy barriers. The key fusion intermediate is stalk, where the contacting membrane monolayers have already fused, but the distal monolayers have not yet, and hemifusion diaphragm, a structure with an extended lipid bilayer formed by two distal monolayers of merging membranes located in the center between the radially displaced fused contact monolayers. In this work, we consider fusion of a bilayer membrane and a lipid monolayer located at the water–triolein interface from the standpoint of the classical theory of fusion. An intermediate π-shaped structure, formed as a result of a lipid droplet monolayer and a peroxisome bilayer fusion, was considered, and the dependence of its energy on the geometric parameters and elastic characteristics of the system was analyzed. In particular, it was shown that the π‑shaped structure is similar to the hemifusion diaphragm of the classical theory of bilayer membrane fusion: an increase in the radial dimensions of both structures becomes more energetically favorable with a decrease in the spontaneous curvature of the membrane monolayers. This result is consistent with the available experimental data on the fusion of lipid droplets with peroxisomes. [ABSTRACT FROM AUTHOR]

Abstract α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals. Graphical Abstract

[Display omitted] • Heterozygous diploid Pichia pastoris was first used for fatty alcohol production. • Highest fatty alcohol titer in methanol minimal medium to date. • Dual pathway with cytosolic and peroxisomal engineering. Microbial cell factories provide an efficient approach for the green manufacturing of chemicals. However, the excessive use of sugars increases the potential risk of food crisis. Methanol, an abundant feedstock, holds promise in facilitating low-carbon production processes. However, the current methanol bioconversion is hindered by limited regulatory strategies and relatively low conversion efficiency. Here, a yeast biocatalyst was extensively engineered for efficient biosynthesis of fatty alcohols through reinforcement of precursor supply and methanol assimilation in Pichia pastoris. Furthermore, the dual cytoplasmic and peroxisomal biosynthetic pathways were constructed by mating and exhibited robust production of 5.6 g/L fatty alcohols by using methanol as the sole carbon source. This study provides a heterozygous diploid P. pastoris strain with dual cytoplasmic and peroxisomal biosynthetic pathways, which achieved the highest fatty alcohol production from one-carbon feedstocks to date. [ABSTRACT FROM AUTHOR]

[Display omitted] • Introducing the squalene synthesis pathway into the peroxisomes. • The rate-limiting steps in the accumulation of 2,3-oxidosqualene have been overcome. • Introducing the lupeol synthase and optimizing its expression. • The highest lupeol titer (414.83 ± 1.75 mg/L) reported to date in shake flasks. • The betulinic acid titer reached 205.74 mg/L in a 5-L fermenter. Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae , and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla -derived lupeol C-28 oxidase (BPLO) and Arabidopsis -derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae. [ABSTRACT FROM AUTHOR]

In this study, we report the rejuvenation of aged muscle fibers facilitated by heterochronic parabiosis, underscored by a notable upregulation in peroxisomal gene expression. Our findings highlight the pivotal role of dysregulation in peroxisomal biogenesis and consequential impairment of fatty acid β-oxidation in myoblast differentiation and muscle regeneration. Notably, we demonstrate the novel therapeutic strategy that involves the administration of peroxisome-COS-FITC complexes and nanozymes that mimic peroxisomal function, effectively rejuvenating aged muscle tissues. This innovative approach paves the way for new methods to mitigate or reverse sarcopenia, offering new avenues of the treatment of age-related muscle atrophy and a significant advancement in our understanding for the molecular underpinnings in muscle aging. [Display omitted] • Peroxisome dysfunction linked to sarcopenia and muscle degeneration. • Innovative peroxisomal transplant therapy rejuvenates aged muscles. • Nanozyme technology mimics peroxisomal β-oxidation for muscle health. • Interplay between peroxisomes and mitochondria restores muscle function. [ABSTRACT FROM AUTHOR]

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

Quinone outside inhibitor fungicides (QoIs) are crucial fungicides for controlling plant diseases, but resistance, mainly caused by G143A, has been widely reported with the high and widespread use of QoIs. However, two phenotypes of Corynespora casiicola (RI and RII) with the same G143A showed significantly different resistance to QoIs in our previous study, which did not match the reported mechanisms. Therefore, transcriptome analysis of RI and RII strains after trifloxystrobin treatment was used to explore the new resistance mechanism in this study. The results show that 332 differentially expressed genes (DEGs) were significantly up-regulated and 448 DEGs were significantly down-regulated. The results of GO and KEGG enrichment showed that DEGs were most enriched in ribosomes, while also having enrichment in peroxide, endocytosis, the lysosome, autophagy, and mitophagy. In particular, mitophagy and peroxisome have been reported in medicine as the main mechanisms of reactive oxygen species (ROS) scavenging, while the lysosome and endocytosis are an important organelle and physiological process, respectively, that assist mitophagy. The oxidative stress experiments showed that the oxidative stress resistance of the RII strains was significantly higher than that of the RI strains: specifically, it was more than 1.8-fold higher at a concentration of 0.12% H2O2. This indicates that there is indeed a significant difference in the scavenging capacity of ROS between the two phenotypic strains. Therefore, we suggest that QoIs’ action caused a high production of ROS, and that scavenging mechanisms such as mitophagy and peroxisomes functioned in RII strains to prevent oxidative stress, whereas RI strains were less capable of resisting oxidative stress, resulting in different resistance to QoIs. In this study, it was first revealed that mitophagy and peroxisome mechanisms available for ROS scavenging are involved in the resistance of pathogens to fungicides.

Peroxisomes are single-membrane-bound organelles that play critical roles in eukaryotic cellular functions. Peroxisome quantity is a key factor influencing the homeostasis and pathogenic processes of pathogenic fungi. The aim of the present study was to investigate the underlying mechanisms of the reduction in number of peroxisomes in Fusarium graminearum consequent to FgPex4 and FgPex22-like deletion. The number of peroxisomes decreased by 40.55% and 39.70% when FgPex4 and FgPex22-like, respectively, were absent. Peroxisome biogenesis-related proteins, as well as inheritance- and division-related dynamin-like proteins were reduced at the transcriptional level in the mutant strains. In addition, the degree of pexophagy was intensified and the accumulation of ubiquitinated FgPex5 was also increased in F. graminearum when FgPex4 or FgPex22-like was absent. The findings suggest that FgPex4 and FgPex22-like influence the number of peroxisomes by influencing peroxisome biogenesis and pexophagy.

Emerging evidence suggests that peroxisomes play a role in the regulation of tumorigenesis and cancer progression. However, the prognostic value of peroxisome-related genes has been rarely investigated. This study aimed to establish a peroxisome-related gene signature for overall survival (OS) prediction in patients with hepatocellular carcinoma (HCC). First, univariate Cox regression analysis was employed to identify prognostic peroxisome-related genes in The Cancer Genome Atlas liver cancer cohort, and least absolute shrinkage and selection operator Cox regression analysis was used to construct a 10-gene signature. The risk score based on the signature was positively correlated with poor prognosis (HR = 4.501, 95% CI = 3.021–6.705, P = 1.39e−13). Second, multivariate Cox regression incorporating additional characteristics revealed that the signature was an independent predictor. Time-dependent ROC curves demonstrated good performance of the signature in predicting the OS of HCC patients. The prognostic performance was validated using International Cancer Genome Consortium HCC cohort data. Gene set enrichment analysis revealed that the signature-related alterations in biological processes mainly involved peroxisomal functions. Finally, we developed a nomogram model based on the gene signature and TNM stage, which showed a superior prognostic power (C-index = 0.702). Thus, our study revealed a novel peroxisome-related gene signature that may help improve personalized OS prediction in HCC patients.

Abstract Background Peroxisomes are pivotal metabolic organelles that exist in almost all eukaryote cells. A reduction in numbers and enzymatic activities of peroxisomes was found in colon adenocarcinomas. However, the role of peroxisomes or the peroxisome pathway in colorectal cancer (CRC) is not defined. Methods In the current study, a peroxisome score was calculated to indicate the activity of the peroxisome pathway using gene set variant analysis based on transcriptomic datasets. CIBERSORTx was chosen to infer enriched immune cells for tumors among subgroups. The SubMap algorithm was applied to predict its sensitivity to immunotherapy. Results The patients with a relatively low peroxisome score and high level of T-cell immunoglobulin and mucin domain 3 (TIM-3) presented the worse overall survival than others. Moreover, low peroxisome scores were associated with high infiltration of lymphocytes and poor prognosis in those CRC patients. Thus, a PERLowTIM3High CRC risk subpopulation was identified and characterized by high immune infiltration. The results also showed that CD8 T cells and macrophages highly infiltrated tumors of the PERLowTIM3High group, regardless of consortium molecular subtype and microsatellite instability status. This subgroup had the highest tumor mutational burden and overexpression of immune checkpoint genes. Further, the PERLowTIM3High group showed a higher probability of responding to programmed cell death protein-1-based immunotherapy. In addition, genes involved in peroxisomal metabolic processes in CRC were also investigated since peroxisome is a rather pleiotropic and highly metabolic organelle in cell. The results indicated that only those genes involved in fatty acid alpha oxidation could be used to stratify CRC patients as similar as peroxisome pathway genes. Conclusions We revealed the favorable prognostic value of the peroxisome pathway in CRC and provided a new CRC stratification based on peroxisomes and TIM3, which might be helpful for CRC diagnostics and personalized treatment.

Macroautophagy/autophagy is a highly conserved process in which subcellular components destined for degradation are sequestered within autophagosomes. The selectivity of autophagy is determined by autophagy receptors, such as Pichia pastoris Atg30 (autophagy-related 30), which controls the selective degradation of peroxisomes (pexophagy) through the assembly of a receptor-protein complex (RPC). Previously, we proved that the peroxisomal acyl-CoA-binding protein, Atg37, and the highly conserved peroxin, Pex3, are required for RPC formation and efficient pexophagy. Here, we describe how Atg37 and Pex3 regulate the assembly and activation of the pexophagic RPC. We demonstrate that Atg30 requires both Atg37 and Pex3 to recruit Atg8 and Atg11 to the pexophagic RPC, because Atg37 depends on Pex3 for its localization at the peroxisomal membrane. We establish that due to close proximity of Atg37- and Pex3-binding sites in the middle domain of Atg30, the binding of these proteins to Atg30 is mutually exclusive within this region. We also show that direct binding of Pex3 and Atg37 to Atg30 regulates its phosphorylation by the Hrr25 kinase, negatively and positively, respectively. Based on these results we present a model that clarifies the assembly and activation of the pexophagic RPC through the phosphoregulation of Atg30.

Peroxisomes are actively involved in the metabolism of various lipids including fatty acids, ether phospholipids, bile acids as well as the processing of reactive oxygen and nitrogen species. Recent studies show that peroxisomes can regulate cholesterol homeostasis by mediating cholesterol transport from the lysosomes to the endoplasmic reticulum and towards primary cilium as well. Disruptions of peroxisome biogenesis or functions lead to peroxisomal disorders that usually involve neurological deficits. Peroxisomal dysfunction is also linked to several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In many peroxisomal disorders and neurodegenerative diseases, aberrant cholesterol accumulation is frequently encountered yet largely neglected. This review discusses the current understanding of the mechanisms by which peroxisomes facilitate cholesterol trafficking within the cell and the pathological conditions related to impaired cholesterol transport by peroxisomes, with the hope to inspire future development of the treatments for peroxisomal disorders and neurodegenerative diseases.

Summary: The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well‐known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD‐endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD‐peroxisome connections, that facilitate the degradation of LD‐stored triacylglycerols (TAGs), and the more recently discovered LD‐plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s). [ABSTRACT FROM AUTHOR]

Kinetoplastea and Diplonemea possess peroxisome‐related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but also the organelles display remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well‐studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here, we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane. [ABSTRACT FROM AUTHOR]

Brown leaf spot on tobacco is a serious fungal disease caused by Alternaria alternata. Peroxisomes are organelles playing an important role in the development and infection of plant pathogenic fungi. But, until now, there is no report on the peroxisome dynamics during the conidia germination of A. alternata. To evaluate the roles of peroxisome in the development of the fungus, in the present work, an enhanced green fluorescent protein (eGFP) cassette tagged with peroxisome targeting signal 2 (PTS2) was integrated into A. alternata to label the organelles, and an eGFP cassette carrying a nuclear located signal (NLS) was performed parallelly. The transformants containing the fusions emitted fluorescence in punctate patterns. The fluorescence of eGFP-PTS2 was distributed exactly in the peroxisomes while those of eGFP-NLS were located in the nucleus. Typical AaGB transformants were selected to be investigated for the peroxisome dynamics. The results showed that during spore germination, the number of peroxisomes in the spores decreased gradually, but increased in the germ tubes. In addition, when the transformants were cultured on lipid media, the numbers of peroxisomes increased significantly, and in a larger portion, present in striped shapes. These findings give some clues for understanding the peroxisomal functions in the development of A. alternata. [ABSTRACT FROM AUTHOR]

Organelles contribute to plant growth via their movements and interactions, which ensure efficient metabolic flow and help plants adapt to environmental stress. Live-cell imaging of the interactions of organelles has been performed in yeast, plant, and animal cells. However, high-throughput quantitative methods are needed to simultaneously analyze the interactions of many organelles in living plant cells. Here, we developed a semi-automatic high-throughput method to quantitatively evaluate the interactions between peroxisomes and chloroplasts using a distance transformation algorithm and high-resolution 3D fluorescent images taken by confocal laser scanning microscopy. Using this method, we measured the 3D distance between the center of peroxisome and chloroplast surface in Arabidopsis thaliana. We then compared the distances between these organelles in leaf mesophyll cells under light and dark conditions. This distance was shorter in the light than in the dark, which is in agreement with the findings of previous studies. We used our method to evaluate peroxisome–chloroplast (plastid) interactions in different cell types in the light and dark, including guard, stem, and root cells. Like in mesophyll cells, the distance between the peroxisome and chloroplast was shorter in the light in guard and stem cells, but not in root cells, suggesting that photosynthetic plastids (chloroplasts) play important roles in these interactions. When leaf mesophyll cells were incubated under high-intensity light, the frequency of shorter distances between peroxisomes and chloroplasts significantly increased. Our high-throughput, semi-automatic method represents a powerful tool for evaluating peroxisome–chloroplast interactions in different types of plant cells under various environmental conditions. [ABSTRACT FROM AUTHOR]

Fusarium crown rot (FCR) of wheat, an important soil-borne disease, presents a worsening trend year by year, posing a significant threat to wheat production. Fusarium pseudograminearum cv. b was reported to be the dominant pathogen of FCR in China. Peroxisomes are single-membrane organelles in eukaryotes that are involved in many important biochemical metabolic processes, including fatty acid β-oxidation. PEX11 is important proteins in peroxisome proliferation, while less is known in the fungus F. pseudograminearum. The functions of FpPEX11a, FpPEX11b, and FpPEX11c in F. pseudograminearum were studied using reverse genetics, and the results showed that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction. After deleting FpPEX11a and FpPEX11b, cell wall integrity was impaired, cellular metabolism processes including active oxygen metabolism and fatty acid β-oxidation were significantly blocked, and the production ability of deoxynivalenol (DON) decreased. In addition, the deletion of genes of FpPEX11a and FpPEX11b revealed a strongly decreased expression level of peroxisome-proliferation-associated genes and DON-synthesis-related genes. However, deletion of FpPEX11c did not significantly affect these metabolic processes. Deletion of the three protein-coding genes resulted in reduced pathogenicity of F. pseudograminearum. In summary, FpPEX11a and FpPEX11b play crucial roles in the growth and development, asexual reproduction, pathogenicity, active oxygen accumulation, and fatty acid utilization in F. pseudograminearum. [ABSTRACT FROM AUTHOR]

Phospholipid transport from the periphery to the brain is an understudied topic. When certain lipid species are deficient due to impaired synthesis, though, transfer across the blood-brain barrier is essential for replenishing lipids in the brain. For example, the deficiency in plasmalogens, the most abundant ether lipids in mammals, has detrimental effects on the brain, which is a major issue in inherited peroxisomal disorders but also contributes to more common disorders like Alzheimer's disease. Oral administration of alkylglycerols like batyl alcohol, which carry a pre-formed ether bond, enables replenishment of ether lipids in various peripheral tissues. However, plasmalogen deficiency in the brain cannot be overcome by this approach. Here, we tried to increase cerebral plasmalogen uptake by modulating the efflux transport across the blood-brain barrier. We hypothesized, based on previous literature, that at least some ether lipid species readily enter endothelial cells of the barrier through the transporter MFSD2A but are re-exported by ATP-binding cassette (ABC) transporters. By crossbreeding Mdr1a -/- /Mdr1b -/- /Bcrp -/- and ether lipid-deficient Gnpat -/- mice as well as pharmacological inhibition with MK-571 to inactivate the major ABC transporters at the blood-brain barrier, we evaluated the potential of combined ABC transporter inhibition and oral batyl alcohol administration for the treatment of plasmalogen deficiency. We found that even in the absence of the most abundant ABC transporters, batyl alcohol supplementation did not restore plasmalogen levels in the brain, despite the presence of a wide spectrum of ether lipid subspecies in the plasma as demonstrated by lipidomic analysis. Surprisingly, batyl alcohol treatment of pregnant Gnpat +/- dams had beneficial effects on the plasmalogen levels of Gnpat -/- offspring with defective ether lipid biosynthesis, independently of ABC transporter status at the placental barrier. Our results underline the autonomy of brain lipid homeostasis and indicate that peripheral supplementation of ether lipids is not sufficient to supply the brain with larger amounts of plasmalogens. Yet, the findings suggest that alkylglycerol treatment during pregnancy may pose a viable option to ameliorate some of the severe developmental defects of inborn ether lipid deficiency. [ABSTRACT FROM AUTHOR]

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy‐mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP‐AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2O2) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down‐regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium‐like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild‐type toxin levels in axenic culture, the mutant induced a lower level of H2O2 and smaller necrotic lesions on citrus leaves. In addition to H2O2, nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy‐mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions. [ABSTRACT FROM AUTHOR]

Exposure to endotoxins (lipopolysaccharides, LPS) may lead to a potent inflammatory cytokine response and a severe impairment of metabolism, causing tissue injury. The protective effect provided by cactus seed oil (CSO), from Opuntia ficus-indica, was evaluated against LPS-induced inflammation, dysregulation of peroxisomal antioxidant, and β-oxidation activities in the brain and the liver. In both tissues, a short-term LPS exposure increased the proinflammatory interleukine-1β (Il-1β), inducible Nitroxide synthase (iNos), and Interleukine-6 (Il-6). In the brain, CSO action reduced only LPS-induced iNos expression, while in the liver, CSO attenuated mainly the hepatic Il-1β and Il-6. Regarding the peroxisomal antioxidative functions, CSO treatment (as Olive oil (OO) or Colza oil (CO) treatment) induced the hepatic peroxisomal Cat gene. Paradoxically, we showed that CSO, as well as OO or CO, treatment can timely induce catalase activity or prevent its induction by LPS, respectively, in both brain and liver tissues. On the other hand, CSO (as CO) pretreatment prevented the LPS-associated Acox1 gene and activity decreases in the liver. Collectively, CSO showed efficient neuroprotective and hepato-protective effects against LPS, by maintaining the brain peroxisomal antioxidant enzyme activities of catalase and glutathione peroxidase, and by restoring hepatic peroxisomal antioxidant and β-oxidative capacities. [ABSTRACT FROM AUTHOR]