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Significance: Innate immune cells adopt distinct memory states during the pathogenesis of acute and chronic inflammatory diseases. Intracellular generations of reactive oxygen species (ROS) play key roles during the programming dynamics of innate immune cells such as monocytes and macrophages. Recent Advances: ROS modulate the adaptation of innate leukocytes to varying intensities and durations of inflammatory signals, facilitate fundamental reprogramming dynamics such as priming, tolerance, and exhaustion, in addition to fundamental processes of proliferation, differentiation, phagocytosis, chemotaxis, as well as expression of pro- and anti-inflammatory mediators. ROS can be generated at distinct subcellular compartments including cellular membrane, mitochondria, and peroxisome. Complex inflammatory signals may finely regulate ROS generation within distinct subcellular compartments, which in turn may differentially facilitate innate memory dynamics. Critical Issues: Complex inflammatory signals with varying strengths and durations may differentially trigger ROS generation at peroxisome, mitochondria, and other subcellular organelles. Peroxisomal or mitochondrial ROS may facilitate the assembly of distinct signaling platforms involved in the programming of memory innate leukocytes. Despite the emerging connection of subcellular ROS with innate immune memory, underlying mechanisms are still not well defined. Future Directions: Recent important discoveries linking subcellular ROS and innate memory as critically reviewed here hold novel translational relevance related to acute and chronic inflammatory diseases. Capitalizing on these novel findings, future systems studies that use next-generation single-cell dynamic analyses in response to complex inflammatory environments are urgently needed to comprehensively decipher the programming dynamics of innate immune memory, finely modulated by subcellular ROS. Antioxid. Redox Signal. 39, 1027–1038. [ABSTRACT FROM AUTHOR]

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% H 2 O 2 . 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. [ABSTRACT FROM AUTHOR]

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic and debilitating disease characterized by unexplained physical fatigue, cognitive and sensory dysfunction, sleeping disturbances, orthostatic intolerance, and gastrointestinal problems. People with ME/CFS often report a prodrome consistent with infections. Using regression, Bayesian and enrichment analyses, we conducted targeted and untargeted metabolomic analysis of plasma from 106 ME/CFS cases and 91 frequency-matched healthy controls. Subjects in the ME/CFS group had significantly decreased levels of plasmalogens and phospholipid ethers (p < 0.001), phosphatidylcholines (p < 0.001) and sphingomyelins (p < 0.001), and elevated levels of dicarboxylic acids (p = 0.013). Using machine learning algorithms, we were able to differentiate ME/CFS or subgroups of ME/CFS from controls with area under the receiver operating characteristic curve (AUC) values up to 0.873. Our findings provide the first metabolomic evidence of peroxisomal dysfunction, and are consistent with dysregulation of lipid remodeling and the tricarboxylic acid cycle. These findings, if validated in other cohorts, could provide new insights into the pathogenesis of ME/CFS and highlight the potential use of the plasma metabolome as a source of biomarkers for the disease.

Copyright of Biology Teaching is the property of East China Normal University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Salinity is a crucial environmental factor influencing the survival, growth, development, and reproduction of aquatic animals. However, the underlying molecular mechanisms of the shrimp’s response to salinity stress are not yet fully understood. Therefore, we used the Illumina NovaSeq 6000 platform to perform transcriptome sequencing of the hepatopancreas of Litopenaeus vannamei under high-salinity (30 PSU), medium-salinity (10 PSU), and low-salinity (0.5 PSU) conditions. We obtained 63.23 Gb of high-quality data and identified 3589 differentially expressed genes (DEGs), including 1638 upregulated and 1951 downregulated genes. Notably, a comparison between the control group (30 PSU) and the low-salinity group (0.5 PSU) revealed that the BBOX1 and CHE1 genes were significantly upregulated, while the ACOX1, MPV, CYP2L1, GCH, MVK, TREt1, and XDH genes were significantly downregulated. These genes are primarily involved in key metabolic pathways, such as fatty acid oxidation, cholesterol metabolism, and hormone synthesis and metabolism. The key genes involved in fatty acid β-oxidation, such as ACOX1, ACAD, HADH, HSD17B4, PECR, CROT, PIPOX, and CG5009, all showed a downward trend, suggesting that L. vannamei may respond to salt stress by regulating fatty acid oxidative metabolism, optimizing energy utilization, and maintaining cell homeostasis under low-salinity conditions. Functional annotation of gene ontology (GO) and KEGG pathway enrichment analysis highlighted the roles of these significant DEGs in the adaptation of L. vannamei to environments of varying salinity, underscoring the importance of metabolic pathways in their adaptive physiological responses. This study provides a crucial molecular biological basis for understanding the molecular mechanisms and physiological protection strategies of L. vannamei under salinity stress.

The peroxisomal biogenesis factor 19 (PEX19) is necessary for early peroxisomal biogenesis. PEX19 has been implicated in the replication of a variety of viruses, but the details pertaining to the mechanisms of how PEX19 engages in the life cycle of these viruses still need to be elucidated. Here, we demonstrated that the C terminus of PEX19 interacted with the cytoplasmic tail region of the M2 protein of the influenza A virus (IAV) and inhibited the viral growth titers. IAV infection or PEX19 knockdown triggered a reduction in the peroxisome pool and led to the accumulation of ROS and cell damage, thereby creating favorable conditions for IAV replication. Moreover, a reduction in the peroxisome pool led to the attenuation of early antiviral response mediated by peroxisome MAVS and downstream type III interferons. This study also showed that the interaction between IAV M2 and PEX19 affected the binding of PEX19 to the peroxisome-associated protein PEX14 and peroxisome membrane protein 24 (PMP24). Collectively, our data demonstrate that host factor PEX19 suppresses the replication of the IAV, and the IAV employs its M2 protein to mitigate the restricting role of PEX19.

BackgroundMitchell syndrome is a rare, neurodegenerative disease caused by an ACOX1 gain-of-function mutation (c.710A>G; p.N237S), with fewer than 20 reported cases. Affected patients present with leukodystrophy, seizures, and hearing loss. ACOX1 serves as the rate-limiting enzyme in peroxisomal beta-oxidation of very long-chain fatty acids. The N237S substitution has been shown to stabilize the active ACOX1 dimer, resulting in dysregulated enzymatic activity, increased oxidative stress, and glial damage. Mitchell syndrome lacks a vertebrate model, limiting insights into the pathophysiology of ACOX1-driven white matter damage and neuroinflammatory insults.MethodsWe report a patient presenting with rapidly progressive white matter damage and neurological decline, who was eventually diagnosed with an ACOX1 N237S mutation through whole genome sequencing. We developed a zebrafish model of Mitchell syndrome using transient ubiquitous overexpression of the human ACOX1 N237S variant tagged with GFP. We assayed zebrafish behavior, oligodendrocyte numbers, expression of white matter and inflammatory transcripts, and analysis of peroxisome counts.ResultsThe patient experienced progressive leukodystrophy and died 2 years after presentation. The transgenic zebrafish showed a decreased swimming ability, which was restored with the reactive microglia-targeted antioxidant dendrimer-N-acetyl-cysteine conjugate. The mutants showed no effect on oligodendrocyte counts but did display activation of the integrated stress response (ISR). Using a novel SKL-targeted mCherry reporter, we found that mutants had reduced density of peroxisomes.ConclusionsWe developed a vertebrate (zebrafish) model of Mitchell syndrome using transient ubiquitous overexpression of the human ACOX1 N237S variant. The transgenic mutants exhibited motor impairment and showed signs of activated ISR, but interestingly, there were no changes in oligodendrocyte counts. However, the mutants exhibited a deficiency in the number of peroxisomes, suggesting a possible shared mechanism with the Zellweger spectrum disorders.

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal β-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal β-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.

There are two types of autophagy, non-selective (bulk) autophagy, in which substrates are randomly incorporated into autophagosomes, and selective autophagy, in which substrates are specifically targeted. In filamentous fungi, the molecular mechanism underlying selective autophagy remains largely unknown. Recently we identified a novel protein, AoAtg8-interacting protein A (AeiA), in the filamentous fungus Aspergillus oryzae. AeiA was localized to peroxisomes and autophagosomal intermediates, such as phagophore assembly site (PAS) and the phagophore. Moreover, pexophagy flux was reduced in AeiA deletants. Taken together, AeiA is a novel selective autophagy-related protein that contributes to pexophagy in A. oryzae. Our findings provide insight into the molecular mechanisms of selective autophagy including pexophagy in filamentous fungi. Abbreviations: AIM, Atg8-family interacting motifs; Atg8, autophagy-related 8; EGFP, enhanced green fluorescent protein; GABARAP, Gamma aminobutyric acid A receptor associated protein; LC3, Microtubule-associated protein light chain 3; MTS, microbody targeting signal; PD, potato dextrose.

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. [ABSTRACT FROM AUTHOR]

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV- induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The size and shape of organelles can influence the rate of biochemical reactions in cells. Previous studies have suggested that organelle morphology changes due to intra‐ and extracellular environmental responses, affecting the metabolic efficiency of and signal transduction emanating from neighboring organelles. In this study, we tested the possibility that intracellularly distributed organelles exhibit a heterogeneous response to intra‐ and extracellular environments. We detected a high correlation between peroxisome morphology and distance to the nucleus in light‐exposed cells. Moreover, the proximity area between chloroplasts and peroxisomes varied with distance to the nucleus. These results indicate that peroxisome morphology varies with proximity to the nucleus, suggesting the presence of a nucleus–peroxisome signal transduction cascade mediated by chloroplasts. [ABSTRACT FROM AUTHOR]

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae. [ABSTRACT FROM AUTHOR]

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and β-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

Abstract 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. Graphical Abstract

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants.

Gliomas, the most prevalent and lethal form of brain cancer, are known to exhibit metabolic alterations that facilitate tumor growth, invasion, and resistance to therapies. Peroxisomes, essential organelles responsible for fatty acid oxidation and reactive oxygen species (ROS) homeostasis, rely on the receptor PEX5 for the import of metabolic enzymes into their matrix. However, the prognostic significance of peroxisomal enzymes for glioma patients remains unclear. In this study, we elucidate that PEX5 is indispensable for the cell growth, migration, and invasion of glioma cells. We establish a robust prognosis model based on the expression of peroxisomal enzymes, whose localization relies on PEX5. This PEX5-dependent signature not only serves as a robust prognosis model capable of accurately predicting outcomes for glioma patients, but also effectively distinguishes several clinicopathological features, including the grade, isocitrate dehydrogenase (IDH) mutation, and 1p19q codeletion status. Furthermore, we developed a nomogram that integrates the prognostic model with other clinicopathological factors, demonstrating highly accurate performance in estimating patient survival. Patients classified into the high-risk group based on our prognostic model exhibited an immunosuppressive microenvironment. Finally, our validation reveals that the elevated expression of GSTK1, an antioxidant enzyme within the signature, promotes the cell growth and migration of glioma cells, with this effect dependent on the peroxisomal targeting signal recognized by PEX5. These findings identify the PEX5-dependent signature as a promising prognostic tool for gliomas.

Tubular extensions emerging from plastids, termed stromules, have received renewed attention due to advancements in imaging techniques. Stromules are widespread in plant and algal species; however, their role in organelle communication and physiology is yet to be elucidated. Initially, stromules were thought to facilitate interplastid communication; however, this proposition is still debated. Stromules with diameters of 0.3-0.8 µm enable protein movement via diffusion and Adenosine Triphosphate (ATP)-dependent transport. Stromule formation is more evident in non-photosynthetic plastids and is induced by various biotic and abiotic stresses, suggesting the involvement of stress-triggered signal transduction via phytohormones and redox changes. Recent studies have emphasized the significance of stromules in plant immunity, especially in response to viral and bacterial effectors, where they serve as conduits for the transport of retrograde signaling molecules from the plastids to the nucleus. Peroxules and matrixules, extending from peroxisomes and mitochondria, respectively, are parallel tubular extensions that were originally found in plant cells, while similar structures also exist in mammalian cells. The response of these extensions to stress may contribute to the management of Reactive Oxygen Species (ROS) and organelle proliferation. This short review discusses the potential roles of the organelle extensions in retrograde signaling pathways.

Peroxisomes are metabolically active organelles that are known for exerting oxidative metabolism, but the precise mechanism remains unclear in diabetic nephropathy (DN). Here, we used proteomics to uncover a correlation between the antioxidant protein disulfide-bond A oxidoreductase-like protein (DsbA-L) and peroxisomal function. In vivo, renal tubular injury, oxidative stress, and cell apoptosis in high-fat diet plus streptozotocin (STZ)-induced diabetic mice were significantly increased, and these changes were accompanied by a ''ghost'' peroxisomal phenotype, which was further aggravated in DsbA-L-deficient diabetic mice. In vitro, the overexpression of DsbA-L in peroxisomes could improve peroxisomal phenotype and function, reduce oxidative stress and cell apoptosis induced by high glucose (HG, 30 mM) and palmitic acid (PA, 250 μM), but this effect was reversed by 3-Amino-1,2,4-triazole (3-AT, a catalase inhibitor). Mechanistically, DsbA-L regulated the activity of catalase by binding to it, thereby reducing peroxisomal leakage and proteasomal degradation of peroxisomal matrix proteins induced by HG and PA. Additionally, the expression of DsbA-L in renal tubules of patients with DN significantly decreased and was positively correlated with peroxisomal function. Taken together, these results highlight an important role of DsbA-L in ameliorating tubular injury in DN by improving peroxisomal function.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

SUMMARY: Photorespiration is an essential metabolic mechanism associated with photosynthesis; however, little is known about the photorespiratory pathway of conifer gymnosperms. Metabolite analyses of the leaves of 27 tree species showed that the mean glycerate content in conifer leaves was lower than that in angiosperm leaves. We performed experiments where [13C]‐serine was fed to detached shoots of a conifer (Cryptomeria japonica), via the transpiration stream, and compared the labeling patterns of photorespiratory metabolites with those of an angiosperm tree (Populus nigra), because glycerate is produced from serine via hydroxypyruvate in peroxisomes. In P. nigra, hydroxypyruvate, glycerate and glycine were labeled with 13C, whereas in C. japonica, glycolate and a non‐canonical photorespiratory metabolite, formate, were also labeled, suggesting that an H2O2‐mediated non‐enzymatic decarboxylation (NED) reaction occurs in C. japonica. We analyzed changes in the metabolite contents of leaves kept in the dark and leaves exposed to illuminated photorespiration‐promoting conditions: a positive relationship between formate and serine levels in C. japonica implied that the active C1‐metabolism pathway synthesizes serine from formate. Leaf gas exchange analyses revealed that CO2 produced through NED was recaptured by chloroplasts. Database analysis of the peroxisomal targeting signal motifs of an H2O2‐scavenging enzyme, catalase, derived from various species, including nine coniferous species, as well as analyses of peroxisomal fractions isolated from C. japonica and P. nigra leaves indicated that conifer peroxisomes had less catalase activity. These results suggest that NED and the subsequent C1 metabolism are involved in the photorespiratory pathway of conifer leaves, where peroxisomes have intrinsically low catalase activity. Significance Statement: Photorespiration is a crucial metabolism that has a significant impact on the net CO2 fixation rates and energy‐use efficiencies of plants. It is thought that the photorespiratory pathway is identical among tree species; however, this study demonstrates that conifer gymnosperms have a different photorespiratory mechanism than that of angiosperm trees. [ABSTRACT FROM AUTHOR]

Mitochondria (MITO) and peroxisomes (PEXO) are the major organelles involved in the oxidative metabolism of cells, but detailed examination of their dynamics and functional adaptations during skeletal muscle (SKM) development (myogenesis) is still lacking. In this study, we found that during myogenesis, MITO DNA, ROS level, and redox ratio increased in myotubes, but the membrane potential (Δψm) and ATP content reduced, implying that the MITO efficiency might reduce during myogenesis. The PEXO number and density both increased during myogenesis, which probably resulted from the accumulation and increased biogenesis of PEXO. The expression of PEXO biogenesis factors was induced during myogenesis in vitro and in utero, and their promoters were also activated by MyoD. Knockdown of the biogenesis factors Pex3 repressed not only the PEXO density and functions but also the levels of MITO genes and functions, suggesting a close coupling between PEXO biogenesis and MITO functions. Surprisingly, Pex3 knockdown by the CRISPRi system repressed myogenic differentiation, indicating critical involvement of PEXO biogenesis in myogenesis. Taken together, these observations suggest that the dynamics and functions of both MITO and PEXO are coupled with each other and with the metabolic changes that occur during myogenesis, and these metabolic couplings are critical to myogenesis. [ABSTRACT FROM AUTHOR]

Peroxisomal disorders (PDs) are a heterogenous group of diseases caused by defects in peroxisome biogenesis or functions. X-linked adrenoleukodystrophy is the most prevalent form of PDs and results from mutations in the ABCD1 gene, which encodes a transporter mediating the uptake of very long-chain fatty acids (VLCFAs). The curative approaches for PDs are very limited. Here, we investigated whether cholesterol accumulation in the lysosomes is a biochemical feature shared by a broad spectrum of PDs. We individually knocked down fifteen PD-associated genes in cultured cells and found ten induced cholesterol accumulation in the lysosome. 2-Hydroxypropyl-β-cyclodextrin (HPCD) effectively alleviated the cholesterol accumulation phenotype in PD-mimicking cells through reducing intracellular cholesterol content as well as promoting cholesterol redistribution to other cellular membranes. In ABCD1 knockdown cells, HPCD treatment lowered reactive oxygen species and VLCFA to normal levels. In Abcd1 knockout mice, HPCD injections reduced cholesterol and VLCFA sequestration in the brain and adrenal cortex. The plasma levels of adrenocortical hormones were increased and the behavioral abnormalities were greatly ameliorated upon HPCD administration. Together, our results suggest that defective cholesterol transport underlies most, if not all, PDs, and that HPCD can serve as a novel and effective strategy for the treatment of PDs. [ABSTRACT FROM AUTHOR]

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 Arabidopsis 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 miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al., Nat. Commun. 4, 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7, 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 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 et al., Cells 11, 1515 (2022)]. When expressed in Arabidopsis, human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome—a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions. [ABSTRACT FROM AUTHOR]

Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells. [ABSTRACT FROM AUTHOR]

Tubular extensions emerging from plastids, termed stromules, have received renewed attention due to advancements in imaging techniques. Stromules are widespread in plant and algal species; however, their role in organelle communication and physiology is yet to be elucidated. Initially, stromules were thought to facilitate interplastid communication; however, this proposition is still debated. Stromules with diameters of 0.3-0.8 µm enable protein movement via diffusion and Adenosine Triphosphate (ATP)-dependent transport. Stromule formation is more evident in nonphotosynthetic plastids and is induced by various biotic and abiotic stresses, suggesting the involvement of stress-triggered signal transduction via phytohormones and redox changes. Recent studies have emphasized the significance of stromules in plant immunity, especially in response to viral and bacterial effectors, where they serve as conduits for the transport of retrograde signaling molecules from the plastids to the nucleus. Peroxules and matrixules, extending from peroxisomes and mitochondria, respectively, are parallel tubular extensions that were originally found in plant cells, while similar structures also exist in mammalian cells. The response of these extensions to stress may contribute to the management of Reactive Oxygen Species (ROS) and organelle proliferation. This short review discusses the potential roles of the organelle extensions in retrograde signaling pathways. [ABSTRACT FROM AUTHOR]

Peroxisomes are ubiquitous eukaryotic organelles housing not only many important oxidative metabolic reactions, but also some reductive reactions that are less known. Members of the short-chain dehydrogenase/reductase (SDR) superfamily, which are NAD(P)(H)-dependent oxidoreductases, play important roles in plant peroxisomes, including the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), auxiliary b-oxidation of fatty acids, and benzaldehyde production. To further explore the function of this family of proteins in the plant peroxisome, we performed an in silico search for peroxisomal SDR proteins from Arabidopsis based on the presence of peroxisome targeting signal peptides. A total of 11 proteins were discovered, among which four were experimentally confirmed to be peroxisomal in this study. Phylogenetic analyses showed the presence of peroxisomal SDR proteins in diverse plant species, indicating the functional conservation of this protein family in peroxisomal metabolism. Knowledge about the known peroxisomal SDRs from other species also allowed us to predict the function of plant SDR proteins within the same subgroup. Furthermore, in silico gene expression profiling revealed strong expression of most SDR genes in floral tissues and during seed germination, suggesting their involvement in reproduction and seed development. Finally, we explored the function of SDRj, a member of a novel subgroup of peroxisomal SDR proteins, by generating and analyzing CRISPR/Cas mutant lines. This work provides a foundation for future research on the biological activities of peroxisomal SDRs to fully understand the redox control of peroxisome functions. [ABSTRACT FROM AUTHOR]

X-linked adrenoleukodystrophy (X-ALD), the most frequent, inherited peroxisomal disease, is caused by mutations in the ABCD1 gene encoding a peroxisomal lipid transporter importing very long-chain fatty acids (VLCFAs) from the cytosol into peroxisomes for degradation via β-oxidation. ABCD1 deficiency results in accumulation of VLCFAs in tissues and body fluids of X-ALD patients with a wide range of phenotypic manifestations. The most severe variant, cerebral X-ALD (CALD) is characterized by progressive inflammation, loss of the myelin-producing oligodendrocytes and demyelination of the cerebral white matter. Whether the oligodendrocyte loss and demyelination in CALD are caused by a primary cell autonomous defect or injury to oligodendrocytes or by a secondary effect of the inflammatory reaction remains unresolved. To address the role of X-ALD oligodendrocytes in demyelinating pathophysiology, we combined the Abcd1 deficient X-ALD mouse model, in which VLCFAs accumulate without spontaneous demyelination, with the cuprizone model of toxic demyelination. In mice, the copper chelator cuprizone induces reproducible demyelination in the corpus callosum, followed by remyelination upon cuprizone removal. By immunohistochemical analyses of oligodendrocytes, myelin, axonal damage and microglia activation during de-and remyelination, we found that the mature oligodendrocytes of Abcd1 KO mice are more susceptible to cuprizone-induced cell death compared to WT mice in the early demyelinating phase. Furthermore, this effect was mirrored by a greater extent of acute axonal damage during demyelination in the KO mice. Abcd1 deficiency did not affect the function of microglia in either phase of the treatment. Also, the proliferation and differentiation of oligodendrocyte precursor cells and remyelination progressed at similar rates in both genotypes. Taken together, our findings point to an effect of Abcd1 deficiency on mature oligodendrocytes and the oligodendrocyte-axon unit, leading to increased vulnerability in the context of a demyelinating insult. [ABSTRACT FROM AUTHOR]

Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position. [ABSTRACT FROM AUTHOR]

SUMMARY: CCT is required to regulate interorganelle contact sites through kinesin‐1 dynamics, modulating peroxisome and mitochondria activity, and ultimately controlling lipid cell content and metabolism. 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. [ABSTRACT FROM AUTHOR]

In the N-degron pathway, N-recognins recognize cognate substrates for degradation via the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). We have recently shown that the autophagy receptor SQSTM1/p62 (sequestosome 1) is an N-recognin that binds the N-terminal arginine (Nt-Arg) as an N-degron to modulate autophagic proteolysis. Here, we show that the N-degron pathway mediates pexophagy, in which damaged peroxisomal fragments are degraded by autophagy under normal and oxidative stress conditions. This degradative process initiates when the Nt-Cys of ACAD10 (acyl-CoA dehydrogenase family, member 10), a receptor in pexophagy, is oxidized into Cys sulfinic (CysO2) or sulfonic acid (CysO3) by ADO (2-aminoethanethiol (cysteamine) dioxygenase). Under oxidative stress, the Nt-Cys of ACAD10 is chemically oxidized by reactive oxygen species (ROS). The oxidized Nt-Cys2 is arginylated by ATE1-encoded R-transferases, generating the RCOX N-degron. RCOX-ACAD10 marks the site of pexophagy via the interaction with PEX5 and binds the ZZ domain of SQSTM1/p62, recruiting LC3+-autophagic membranes. In mice, knockout of either Ate1 responsible for Nt-arginylation or Sqstm1/p62 leads to increased levels of peroxisomes. In the cells from patients with peroxisome biogenesis disorders (PBDs), characterized by peroxisomal loss due to uncontrolled pexophagy, inhibition of either ATE1 or SQSTM1/p62 was sufficient to recover the level of peroxisomes. Our results demonstrate that the Cys-N-degron pathway generates an N-degron that regulates the removal of damaged peroxisomal membranes along with their contents. We suggest that tannic acid, a commercially available drug on the market, has a potential to treat PBDs through its activity to inhibit ATE1 R-transferases. Abbreviations: ACAA1, acetyl-Coenzyme A acyltransferase 1; ACAD, acyl-Coenzyme A dehydrogenase; ADO, 2-aminoethanethiol (cysteamine) dioxygenase; ATE1, arginyltransferase 1; CDO1, cysteine dioxygenase type 1; ER, endoplasmic reticulum; LIR, LC3-interacting region; MOXD1, monooxygenase, DBH-like 1; NAC, N-acetyl-cysteine; Nt-Arg, N-terminal arginine; Nt-Cys, N-terminal cysteine; PB1, Phox and Bem1p; PBD, peroxisome biogenesis disorder; PCO, plant cysteine oxidase; PDI, protein disulfide isomerase; PTS, peroxisomal targeting signal; R-COX, Nt-Arg-CysOX; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNP, sodium nitroprusside; UBA, ubiquitin-associated; UPS, ubiquitinproteasome system. [ABSTRACT FROM AUTHOR]

Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity. [ABSTRACT FROM AUTHOR]

Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products.Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation.

We present the OrganelX e-Science Web Server that provides a user-friendly implementation of the In-Pero and In-Mito classifiers for sub-peroxisomal and sub-mitochondrial localization of peroxisomal and mitochondrial proteins and the Is-PTS1 algorithm for detecting and validating potential peroxisomal proteins carrying a PTS1 signal sequence. The OrganelX e-Science Web Server is available at https://organelx.hpc.rug.nl/fasta/.

Abstract Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi‐ome, remains undefined. Uncovering the peroxi‐ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high‐content microscopy to uncover peroxisomal proteins in the model eukaryote – Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi‐ome by ~40% and paved the way for performing systematic, whole‐organellar proteome assays. By characterizing the sub‐organellar localization and protein targeting dependencies into the organelle, we unveiled non‐canonical targeting routes. Metabolomic analysis of the peroxi‐ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) has been extensively engineered for protein production, and is attracting attention as a chassis cell for methanol biotransformation toward production of small molecules. However, the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic efficiency. We here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose, respectively. There were 243, 158 up-regulated proteins and 244, 304 down-regulated proteins in log and stationary phase, respectively, when cultivated in methanol medium compared with that of glucose medium. Peroxisome enrichment further improved the characterization of more differentially expressed proteins (481 proteins in log phase and 524 proteins in stationary phase). We demonstrated the transaldolase isoenzyme (Tal2, Protein ID: C4R244) was highly up-regulated in methanol medium cultivation, which plays an important role in methanol utilization. Our work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in P. pastoris.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Catalase (CAT) is an extensively studied if somewhat enigmatic enzyme that is at the heart of eukaryotic antioxidant systems with a canonical role in peroxisomal function. The CAT family of proteins exert control over a wide range of plant growth and defence processes. CAT proteins are subject to many types of post-translational modification (PTM), which modify activity, ligand binding, stability, compartmentation and function. The CAT interactome involves many cytosolic and nuclear proteins that appear to be essential for protein functions. Hence, the CAT network of roles extends far beyond those associated with peroxisomal metabolism. Some pathogen effector proteins are able to redirect CAT to the nucleus and recent evidence indicates CAT can traffic to the nucleus in the absence of exogenous proteins. While the mechanisms that target CAT to the nucleus are not understood, CAT activity in the cytosol and nucleus is promoted by interactions with nucleoredoxin. Here we discuss recent findings that have been pivotal in generating a step change in our understanding of CAT functions in plant cells. [Display omitted] • Catalase is a central player in the cellular anti-oxidant network (67). • How catalase folds, assembles and imports into peroxisomes is not fully understood (84). • Catalase undergoes many posttranslational modifications (58). • Catalase has a large, dynamic interactome and is targeted by pathogen effectors.(83). • Catalase's cellular location is not fixed and may it exhibit moonlighting functions (85). [ABSTRACT FROM AUTHOR]

Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity. [ABSTRACT FROM AUTHOR]

Dynamin-related proteins (Drps) mediate a variety of membrane remodelling processes. The Saccharomyces cerevisiae Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion, and peroxisome fission and breakdown. How Drps, and in particular Vps1, can function at so many different subcellular locations is of interest to our understanding of cellular organisation. We found that the peroxisomal membrane protein Pex27 is specifically required for Vps1-dependent peroxisome fission in proliferating cells but is not required for Dnm1-dependent peroxisome fission. Pex27 accumulates in constricted regions of peroxisomes and affects peroxisome geometry upon overexpression. Moreover, Pex27 physically interacts with Vps1 in vivo and is required for the accumulation of a GTPase-defective Vps1 mutant (K42A) on peroxisomes. During nitrogen starvation, a condition that halts cell division and induces peroxisome breakdown, Vps1 associates with the pexophagophore. Pex27 is neither required for Vps1 recruitment to the pexophagophore nor for pexophagy. Our study identifies Pex27 as a Vps1-specific partner for the maintenance of peroxisome number in proliferating yeast cells. [ABSTRACT FROM AUTHOR]

Plant peroxisomes are highly dynamic organelles with regard to metabolic pathways, number and morphology and participate in different metabolic processes and cell responses to their environment. Peroxisomes from animal and plant cells house a complex system of reactive oxygen species (ROS) production associated to different metabolic pathways which are under control of an important set of enzymatic and non enzymatic antioxidative defenses. Nitric oxide (NO) and its derivate reactive nitrogen species (RNS) are also produced in these organelles. Peroxisomes can regulate ROS and NO/RNS levels to allow their role as signalling molecules. The metabolism of other reactive species such as carbonyl reactive species (CRS) and sulfur reactive species (SRS) in peroxisomes and their relationship with ROS and NO have not been explored in depth. In this review, we define a peroxisomal reactive species interactome (PRSI), including all reactive species ROS, RNS, CRS and SRS, their interaction and effect on target molecules contributing to the dynamic redox/ROS homeostasis and plasticity of peroxisomes, enabling fine-tuned regulation of signalling networks associated with peroxisome-dependent H 2 O 2. Particular attention will be paid to update the information available on H 2 O 2 -dependent peroxisomal retrograde signalling and to discuss a specific peroxisomal footprint. [Display omitted] • Peroxisomes are a multipurpose organelles necessary for both animal and plant cell functionality. • Peroxisomes participate in the perception of and cell responses to changes in their environment • Peroxisomes can be considered as a platform of signal molecules in the cell. • Peroxisomal reactive species interactome contribute to the redox/ROS/NO homeostasis and plasticity of peroxisomes. • ROS perturbations in peroxisomes trigger specific retrograde signalling. [ABSTRACT FROM AUTHOR]

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. Synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of patients with deficiency in peroxisome biogenesis suggests that de novo synthesis of plasmalogens contributes significantly to plasmalogen homeostasis in humans. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. Dysregulation of plasmalogen synthesis impairs cholesterol synthesis in cells and brain, resulting in the reduced expression of genes such as mRNA encoding myelin basic protein, a phenotype found in the cerebellum of plasmalogen-deficient mice. In this review, we summarize the current knowledge of molecular mechanisms underlying the regulation of plasmalogen biosynthesis and the link between plasmalogen homeostasis and cholesterol biosynthesis, and address the pathogenesis of impaired plasmalogen homeostasis in rodent and humans. [ABSTRACT FROM AUTHOR]

ABSTRACT Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.