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In peroxisomes, acyl-CoA oxidase (ACOX) oxidizes fatty acids and produces H2O2, and the latter is decomposed by catalase. If ethanol is present, ethanol will be oxidized by catalase coupling with decomposition of H2O2. Peroxisome proliferator-activated receptor α (PPARα) agonist WY-14,643 escalated ethanol clearance, which was not observed in catalase knockout (Cat-/-) mice or partially blocked by an ACOX1 inhibitor. WY-14,643 induced peroxisome proliferation via peroxin 16 (PEX16). PEX16 liver-specific knockout (Pex16Alb-Cre) mice lack intact peroxisomes in liver, but catalase and ACOX1 were upregulated. Due to lacking intact peroxisomes, the upregulated catalase and ACOX1 in the Pex16Alb-Cre mice were mislocated in cytosol and microsomes, and the escalated ethanol clearance was not observed in the Pex16Alb-Cre mice, implicating that the intact functional peroxisomes are essential for ACOX1/catalase to metabolize ethanol. Alcohol-associated liver disease (ALD) is a spectrum of liver disorders ranging from alcoholic steatosis to steatohepatitis. WY-14,643 ameliorated alcoholic steatosis but tended to enhance alcoholic steatohepatitis. In mice lacking nuclear factor erythroid 2-related factor 2 (Nrf2-/-), WY-14,643 still induced PEX16, ACOX1 and catalase to escalate ethanol clearance and blunt alcoholic steatosis, which was not observed in the PPARα-absent Nrf2-/- mice (Pparα-/-/Nrf2-/-) mice, suggesting that WY-14,643 escalates ethanol clearance through PPARα but not through Nrf2.

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Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.

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Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 coreceptor, support a model in which the exportomer tail anchored to the peroxisome membrane represses Atg36 phosphorylation on Pex3 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.

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Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD)-are primarily autosomal recessive disorders caused by mutations in any of 13 PEX genes involved in peroxisome assembly. Compared to other PEX-related disorders, some PEX16 defects are associated with an atypical phenotype consisting of spasticity, cerebellar dysfunction, preserved cognition, and prolonged survival. In this case series, medical records and brain MRIs from 7 patients with this PEX16 presentation were reviewed to further characterize this phenotype. Classic PBD features such as sensory deficits and amelogenesis imperfecta were absent in all 7 patients, while all patients had hypertonia. Five patients were noted to have dystonia and received a treatment trial of levodopa/carbidopa. Four treated patients had partial but significant improvements in their dystonia and tremors, and 1 patient had only minimal response. Brain MRI studies commonly showed T2/FLAIR hyperintensities in the brainstem, superior and middle cerebellar peduncles, corticospinal tracts, and splenium of the corpus callosum. Genetic analysis revealed novel biallelic variants in 3 probands (c.683C > T/372delG; c.692A > G homozygous; c.865C > G/451C > T) and 1 novel variant (c.956_958delCGC) in another proband. We demonstrated residual PEX16 protein amounts by immunoblotting in fibroblasts available from 5 patients with this atypical PEX16 disease (3 from this series, 2 previously reported), in contrast to the absence of PEX16 protein in fibroblasts from a patient with the severe ZSD presentation. This study further characterizes the phenotype of PEX16 defects by highlighting novel and distinctive clinical, neuroradiological, and molecular features of the disease and proposes a potential treatment for the dystonia. ClinicalTrials.gov Identifier: NCT01668186. Date of registration: January 2012.

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Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62-an autophagy adaptor protein-with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.

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INTRODUCTION: X-linked adrenoleukodystrophy (ALD) and Zellweger spectrum disorder (ZSD) are peroxisomal diseases characterized by accumulation of very long chain fatty acids (VLCFA) in plasma and tissues. Considering the wide variability of manifestation, patients of ALD and atypical ZSD are easily misdiagnosed as hereditary spastic paraplegia (HSP) on their clinical grounds. Here, we aimed to determine the frequency of peroxisome diseases and compare their phenotypic spectra with HSP. METHODS: We first applied targeted sequencing in 120 pedigrees with spastic paraplegia, and subsequently confirmed 74 HSP families. We then performed whole exome sequencing for the probands of the 46 remaining pedigrees lacking known HSP-causal genes. Detailed clinical, radiological features, and VLCFA analyses are presented. RESULTS: Seven ALD pedigrees with ABCD1 mutations and one ZSD family harboring bi-allelic mutations of PEX16 were identified. Clinically, in addition to spastic paraplegia, four ALD probands presented adrenocortical insufficiency, and the ZSD proband and her affected sister both developed thyroid problems. VLCFA analysis showed that ratios of C24/C22 and C26/C22 were specifically increased in ALD probands. Moreover, three ALD probands and the ZSD proband had abnormalities in brain or spinal imaging. CONCLUSIONS: Our study reports the first ZSD case in China that manifested spastic paraplegia, and emphasized the finding that peroxisomal diseases comprise a significant proportion (8/120) of spastic paraplegia entities. These findings extend our current understanding of the ALD and ZSD diseases.

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BACKGROUND: Mutations in PEX16 cause peroxisome biogenesis disorder (PBD). Zellweger syndrome characterized by neurological dysfunction, dysmorphic features, liver disease and early death represents the severe end of this clinical spectrum. Here we discuss the diagnostic challenge of atypical PEX16 related PBD in 3 patients from highly inbred kindred and describe the role of specific metabolites analyses, fibroblasts studies, whole-exome sequencing (WES) and metabolomics profiling to establish the diagnosis. METHODS AND PATIENTS: The proband is a 12-year-old male born to consanguineous parents. Despite normal development in the first year, regression and progressive spastic diplegia, poor coordination and dysarthria occurred thereafter. Patient 2 (3-year old female) and Patient 3 (19-month old female) shared similar clinical course with the proband. Biochemical studies on plasma and fibroblasts, WES and global metabolomics analyses were performed. RESULTS: Very-long-chain fatty acids analysis showed subtle elevations in C26 and C26/C22. Global Metabolomics-Assisted Pathway profiling was not remarkable. Immunocytochemical investigations on fibroblasts revealed fewer catalase and PMP70-containing particles indicating aberrant peroxisomal assembly. Complementation studies were inconclusive. WES revealed a novel homozygous variant in PEX16 (c.859C>T). The biochemical profiles of Patient 2 and Patient 3 were similar to the proband and the same genotype was confirmed. CONCLUSION: This paper highlights the diagnostic challenge of PEX16 patients due to the widely variable clinical and biochemical phenotypes. It also emphasizes the important roles of combined biochemical assays with next generation sequencing techniques in reaching diagnosis in the context of atypical clinical presentations, subtle biomarker abnormalities and consanguinity.

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We characterized Pex16 in Aspergillus luchuensis mut. kawachii to examine the role of peroxisomes on citric acid production during the shochu-fermentation process. Rice koji made using a Δpex16 strain exhibited no significant change in citric acid accumulation but a 1.4-fold increase in formic acid production. Microscopic observation of mRFP-SKL (a peroxisome protein marker) showed that pex16 disruption decreased the number of dot-like structures per hyphal cell to 5% of the control. Pex16-GFP exclusively co-localized with mRFP-SKL throughout the hyphae including the very close position to the septal pore. Moreover, the Δpex16 strain was hypersensitive to calcofluor white, which appeared to induce bursting of the hyphal tip and translocation of mRFP-SKL signals to the septal pore. These results indicate that Pex16 does not play a role in citric acid accumulation but is significantly involved in peroxisome and Woronin body formation in Aspergillus kawachii.

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Different pull-down strategies were successfully applied to gain novel insight into the interactome of human membrane-associated proteins. Here, we compare the outcome, efficiency and potential of pull-down strategies applied to human peroxisomal membrane proteins. Stable membrane-bound protein complexes can be affinity-purified from genetically engineered human cells or subfractions thereof after detergent solubilization, followed by size exclusion chromatography and analysis by mass spectrometry (MS). As exemplified for Protein A-tagged human PEX14, one of the central constituents of the peroxisomal matrix protein import machinery, MS analyses of the affinity-purified complexes revealed an unexpected association of PEX14 with other protein assemblies like the microtubular network or the insertion apparatus for peroxisomal membrane proteins comprising PEX3, PEX16 and PEX19. The latter association was recently supported by using a different pull-down strategy following in vivo proximity labeling with biotin, named BioID, which enabled the identification of various membrane proteins in close proximity of PEX16 in living cells.

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Zellweger syndrome spectrum disorders are caused by mutations in any of at least 12 different PEX genes. This includes PEX16, an important regulator of peroxisome biogenesis. Using whole genome sequencing, we detected previously unreported, biallelic variants in PEX16 [NM_004813.2:c.658G>A, p.(Ala220Thr) and NM_004813.2:c.830G>A, p.(Arg277Gln)] in an individual with leukodystrophy, spastic paraplegia, cerebellar ataxia, and craniocervical dystonia with normal plasma very long chain fatty acids. Using olfactory-neurosphere derived cells, a population of neural stem cells, we showed patient cells had reduced peroxisome density and increased peroxisome size, replicating previously reported findings in PEX16 cell lines. Along with alterations in peroxisome morphology, patient cells also had impaired peroxisome function with reduced catalase activity. Furthermore, patient cells had reduced oxidative stress levels after exposure to hydrogen-peroxide (H2O2), which may be a result of compensation by H2O2 metabolising enzymes other than catalase to preserve peroxisome-related cell functions. Our findings of impaired catalase activity and altered oxidative stress response are novel. Our study expands the phenotype of PEX16 mutations by including dystonia and provides further insights into the pathological mechanisms underlying PEX16-associated disorders. Additional studies of the full spectrum of peroxisomal dysfunction could improve our understanding of the mechanism underlying PEX16-associated disorders.

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The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.

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We present a patient with a unique neurological phenotype with a progressive neurodegenerative phenotype. An 18-year diagnostic odyssey for the patient ended when exome sequencing identified a homozygous PEX16 mutation suggesting an atypical peroxisomal biogenesis disorder (PBD). Interestingly, the patient's peroxisomal biochemical abnormalities were subtle, such that plasma very-long-chain fatty acids initially failed to provide a diagnosis. This case suggests next-generation sequencing may be diagnostic in some atypical peroxisomal biogenesis disorders.

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Protozoan parasites of the family Trypanosomatidae infect humans as well as livestock causing devastating diseases like sleeping sickness, Chagas disease, and Leishmaniasis. These parasites compartmentalize glycolytic enzymes within unique organelles, the glycosomes. Glycosomes represent a subclass of peroxisomes and they are essential for the parasite survival. Hence, disruption of glycosome biogenesis is an attractive drug target for these Neglected Tropical Diseases (NTDs). Peroxin 16 (PEX16) plays an essential role in peroxisomal membrane protein targeting and de novo biogenesis of peroxisomes from endoplasmic reticulum (ER). We identified trypanosomal PEX16 based on specific sequence characteristics and demonstrate that it is an integral glycosomal membrane protein of procyclic and bloodstream form trypanosomes. RNAi mediated partial knockdown of Trypanosoma brucei PEX16 in bloodstream form trypanosomes led to severe ATP depletion, motility defects and cell death. Microscopic and biochemical analysis revealed drastic reduction in glycosome number and mislocalization of the glycosomal matrix enzymes to the cytosol. Asymmetry of the localization of the remaining glycosomes was observed with a severe depletion in the posterior part. The results demonstrate that trypanosomal PEX16 is essential for glycosome biogenesis and thereby, provides a potential drug target for sleeping sickness and related diseases.

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Peroxisomes rely on a diverse array of mechanisms to ensure the specific targeting of their protein constituents. Peroxisomal membrane proteins (PMPs), for instance, are targeted by at least two distinct pathways: directly to peroxisomes from their sites of synthesis in the cytosol or indirectly via the endoplasmic reticulum (ER). However, the extent to which each PMP targeting pathway is involved in the maintenance of pre-existing peroxisomes is unclear. Recently, we showed that human PEX16 plays a critical role in the ER-dependent targeting of PMPs by mediating the recruitment of two other PMPs, PEX3 and PMP34, to the ER. Here, we extend these results by carrying out a comprehensive mutational analysis of PEX16 aimed at gaining insights into the molecular targeting signals responsible for its ER-to-peroxisome trafficking and the domain(s) involved in PMP recruitment function at the ER. We also show that the recruitment of PMPs to the ER by PEX16 is conserved in plants. The implications of these results in terms of the function of PEX16 and the role of the ER in peroxisome maintenance in general are discussed.

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Recent increasing evidences showing the interconnection between mitochondria and peroxisome in performing metabolic functions imply that peroxisome dysfunction could lead to a wide variety of human diseases including cancer and osteoarthritis (OA) as mitochondria dysfunction. Even though there is a higher incidence and development of OA in diabetes mellitus (DM) patients, there is not much evidential mechanism study in this inter-regulation between OA and OA with DM in a new view of peroxisome. In this study, we analyzed the alteration of peroxisomal gene expression that could responsible for pathological difference between OA chondrocytes and OA/DM chondrocytes. To discriminate responsible genes in the OA/DM pathogenesis, the expressions of three hundred sixty-two genes reported to differentially relate to peroxisome were analyzed with OA chondrocytes in OA cartilage and OA/DM chondrocytes in the cartilage of OA with DM patient. Among them, PEX-16, a component of peroxisome, was significantly down-regulated in OA/DM chondrocytes and this down-regulation of PEX-16 increased the miR-223 induction. Knockdown studies using PEX-16 null cell line and PEX-16 specific siRNA showed the significant increase in apoptotic cell death. Moreover, over-expression of miR-223 stimulates apoptotic cell death in human articular chondrocytes and induced severe cartilage destruction in db/db mice. In conclusion, our study showed the differential peroxisomal gene expression profiles for OA/DM chondrocytes from OA chondrocytes and suggests the possibility that peroxisomal dysfunction in OA/DM could be responsible for early incidence and development of OA in DM patients.

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PEROXISOMES ARE FORMED BY TWO DISTINCT PATHWAYS: the growth and fission of mature peroxisomes and de novo synthesis at the endoplasmic reticulum (ER). While many of the molecular mechanisms underlying these two pathways remain to be elucidated, it is generally accepted that their relative contribution to peroxisome formation may vary depending on the species, cell type and/or physiological status of the organism. One pertinent example of the apparent differences in the regulation of peroxisome biogenesis among evolutionarily diverse species is the involvement of the peroxin PEX16. In Yarrowia lipolytica, for instance, PEX16 is an intraperoxisomal peripheral membrane protein that participates in peroxisomal fission. By contrast, Human PEX16 is an integral membrane protein that is thought to function at the ER during the early stages of de novo peroxisome formation and also recruits peroxisomal membrane proteins directly to mature peroxisomes. Similarly, PEX16 in the plant Arabidopsis thaliana is speculated to be a PMP receptor at the ER and peroxisomes, and is also required for the formation of other ER-derived organelles, such as oil and protein bodies. Here we briefly review the current knowledge of Y. lipolytica, human and A. thaliana PEX16 in the context of our overall understanding of peroxisome biogenesis and the role of the ER in this process in these three divergent species.