Peroxisomal Protein Research Articles

The Aspergillus niger Major Allergen (Asp n 3) DNA-Specific Sequence Is a Reliable Marker to Identify Early Fungal Contamination and Postharvest Damage in Mangifera indica Fruit.

The aim of this work was to study the value of the main allergen Asp n 3 of Aspergillus niger as a molecular marker of allergenicity and pathogenicity with the potential to be used in the identification of A. niger as a contaminant and cause of spoilage of Mangifera indica. Real-time polymerase chain reaction (RT-PCR) was used for the amplification of Asp n 3 gene. Two pairs of primers were designed: one for the amplification of the entire sequence and another one for the amplification of the most conserved region of this peroxisomal protein. The presence of A. niger was demonstrated by the early detection of the allergenic protein Asp n 3 coding gene, which could be considered a species-specific marker. The use of primers designed based on the conserved region of the Asp n 3 encoding gene allowed us to identify the presence of the closely related fungal species Aspergillus fumigatus by detecting Asp n 3 homologous protein, which can be cross-reactive. The use of conserved segments of the Asp n 3 gene or its entire sequence allows us to detect phylogenetically closely related species within the Aspergilaceae family or to identify species-specific contaminating fungi.

In-Pero: Exploiting Deep Learning Embeddings of Protein Sequences to Predict the Localisation of Peroxisomal Proteins.

Peroxisomes are ubiquitous membrane-bound organelles, and aberrant localisation of peroxisomal proteins contributes to the pathogenesis of several disorders. Many computational methods focus on assigning protein sequences to subcellular compartments, but there are no specific tools tailored for the sub-localisation (matrix vs. membrane) of peroxisome proteins. We present here In-Pero, a new method for predicting protein sub-peroxisomal cellular localisation. In-Pero combines standard machine learning approaches with recently proposed multi-dimensional deep-learning representations of the protein amino-acid sequence. It showed a classification accuracy above 0.9 in predicting peroxisomal matrix and membrane proteins. The method is trained and tested using a double cross-validation approach on a curated data set comprising 160 peroxisomal proteins with experimental evidence for sub-peroxisomal localisation. We further show that the proposed approach can be easily adapted (In-Mito) to the prediction of mitochondrial protein localisation obtaining performances for certain classes of proteins (matrix and inner-membrane) superior to existing tools.

Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis.

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

Peroxisomes in the mouse parotid glands: An in-depth morphological and molecular analysis

BackgroundThe parotid gland is a major salivary gland that has important roles in the digestive and immune system. Peroxisomes are ubiquitous, single-membrane-bound organelles that are present in all eukaryotic cells. Peroxisomes help mediate lipid and reactive oxygen species metabolism, as well as polyunsaturated fatty acid, cholesterol and plasmalogen synthesis. Much of the knowledge on peroxisomes has derived from metabolic organs, however no detailed knowledge is available on peroxisomes in the parotid glands. We thus aimed to comprehensively delineate the localization and characterization of peroxisomal proteins in the murine parotid gland. MethodsWe characterized peroxisomes in the acinar and striated duct cells of the murine parotid gland by fluorescence and electron microscopy, as well as protein and mRNA expression analyses for important peroxisomal genes and proteins. ResultsWe found that peroxisomes are present in all cell types of the mouse parotid gland, however, exhibit notable cell-specific differences in their abundance and enzyme content. We also observed that mouse parotid glands contain high levels of peroxisomal β-oxidation enzymes (including Acox1, Mfp2 and Acaa1), catalase and other peroxisomal anti-oxidative enzymes. ConclusionsThis data suggests that peroxisomes are highly abundant in the murine parotid gland and might help to protect against oxidative stress. This comprehensive description of peroxisomes in the parotid gland lays the groundwork for further research concerning their role in the pathogenesis of parotid gland diseases and tumors.

Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

ABSTRACT Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer. Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.

PEX6 Mutations in Peroxisomal Biogenesis Disorders: An Usher Syndrome Mimic.

PurposePeroxisomal biogenesis disorders (PBDs) represent a spectrum of conditions that result in vision loss, sensorineural hearing loss, neurologic dysfunction, and other abnormalities resulting from aberrant peroxisomal function caused by mutations in PEX genes. With no treatments currently available, we sought to investigate the disease mechanism in a patient with a PBD caused by defects in PEX6 and to probe whether overexpression of PEX6 could restore peroxisome function and potentially offer therapeutic benefit.DesignLaboratory-based study.ParticipantsA 12-year-old boy sought treatment with hearing loss and retinopathy. After negative results in an Usher syndrome panel, targeted genetic testing revealed compound heterozygous mutations in PEX6. These included a 14-nucleotide deletion (c.802_815del: p.(Asp268Cysfs∗8)) and a milder missense variant (c.35T→C:(p.Phe12Ser)).MethodsPatient-derived skin fibroblasts were cultured, and a PEX6 knockout cell line was developed using clustered regularly interspaced short palindromic repeats and Cas9 technology in HEK293T cells to emulate a more severe disease phenotype. Immunoblot analysis of whole cell lysates was performed to assess peroxisome number. Immunofluorescence studies used antibodies against components of the peroxisomal protein import pathway to interrogate the effects of mutations in PEX6 on protein trafficking.Main Outcome MeasuresPrimary outcome measures were peroxisome abundance and matrix protein import.ResultsPeroxisome number was not significantly different between control fibroblasts and patient fibroblasts; however, fewer peroxisomes were observed in PEX6 knockout cells compared with wild-type cells (P = 0.04). Analysis by immunofluorescent microscopy showed significantly impaired peroxisomal targeting signal 1- and peroxisomal targeting signal 2-mediated matrix protein import in both patient fibroblasts and PEX6 knockout cells. Overexpressing PEX6 resulted in improved matrix protein import in PEX6 knockout cells.ConclusionsMutations in PEX6 were responsible for combined hearing loss and retinopathy in our patient. The primary peroxisomal defect in our patient’s skin fibroblasts was impaired peroxisomal protein import as opposed to reduction in the number of peroxisomes. Genetic strategies that introduce wild-type PEX6 into cells deficient in PEX6 protein show promise in restoring peroxisome function. Future studies of patient-specific induced pluripotent stem cell-derived retinal pigment epithelium cells may clarify the role of PEX6 in the retina and the potential for gene therapy in these patients.

Adrenal Insufficiency in an Adolescent Boy With Type 1 Diabetes Mellitus - the Importance of Considering X-Linked Adrenoleukodystrophy

Background: Primary adrenal insufficiency (PAI) is the rare, life-threatening failure of the adrenal glands to produce sufficient glucocorticoids and mineralocorticoids, presenting with fatigue, weakness, weight loss, hyperpigmentation, hypoglycemia, and hypotension. In adults, PAI is primarily autoimmune, occurring independently or in conjunction with thyroiditis and/or Type I diabetes mellitus (T1DM) as a component of Autoimmune Polyglandular Syndrome-2 (APS-2, Schmidt syndrome). APS-2 is rare (prevalence 1 in 20,000) and more common in females with a peak incidence in the third and fourth decades of life. In children, PAI is most often due to congenital adrenal hyperplasia (CAH), followed by autoimmune disease, but as many as 50% of cases of non-CAH pediatric PAI are not autoimmune. PAI is often the first documented clinical finding in boys with X-linked adrenoleukodystrophy (X-ALD), a rare disorder (prevalence 1 in 15,000) in which deficiency of a peroxisomal membrane protein leads to very long chain fatty acid (VLCFA) accumulation and progressive destruction of the adrenal cortex. A subset of boys with X-ALD develop cerebral ALD (cALD), characterized by progressive central demyelination, neurocognitive decline, and death. 70–80% of boys with X-ALD present with PAI prior to demonstrating neurologic symptoms. Diagnostic workup for X-ALD in pediatric patients presenting with PAI is crucial as timely intervention with hematopoietic stem cell transplant (HSCT) can stop progression of cerebral disease. Clinical Case: An eleven-year-old male was diagnosed with PAI after presenting with poor school performance, growth delay and skin hyperpigmentation. Medical history was significant for well controlled T1DM diagnosed at eight years old. Given his history of T1DM, his PAI was presumed to be autoimmune and further diagnostic testing was not performed. Eleven months later, a brain MRI performed for complaints of visual disturbance and chronic headaches revealed extensive demyelination with gadolinium enhancement consistent with cALD. Elevated VLCFAs and a mutation in the ABCD1 gene confirmed the diagnosis of X-ALD. Unfortunately, the extent of cerebral involvement was so severe that HSCT would not be of significant clinical benefit. In these situations, progressive neurological decline that leads to disability and ultimately death would be expected. Clinical Lessons: This case illustrates that VLCFA testing should be performed in all boys with PAI to rule out X-ALD. In boys, PAI should not be assumed to be autoimmune, even with co-existing autoimmune diseases since APS-2 (Schmidt syndrome) is rare and more likely to occur in adult women. PAI presents early and precedes neurologic symptoms in a majority of boys with cALD. Early identification of X-ALD through VLCFA testing prior to development of severe cALD is critical to allow early intervention with lifesaving HSCT.

A Novel Graph Based Semi-Supervised Learning Approach to Identify Pathways Contributing to the Development of Diabetes and Obesity

Abstract Background: Gestational diabetes (GDM) has profound effects on the intrauterine metabolic milieu, induces marked abnormalities in fetal glucose and insulin secretion and is linked to obesity and diabetes in the offspring, but the mechanisms remain largely unknown. Epigenetic modifications in stems cells may be one mechanism by which an in utero exposure can lead to the development of diabetes and obesity later in life. Objective: To identify novel pathways contributing to the development of diabetes and obesity in offspring exposed to GDM in utero by integrating data generated from transcriptome and methylome analysis from second trimester human amniocytes exposed to GDM in utero. Methods: We analyzed RNAseq and genome wide DNA methylation data (ERRBS) generated from second trimester amniocytes obtained from women with GDM (n=14). Amniocytes have stem cells-like characteristics and are derived from the fetus. Expression data of 22,271 genes were retrieved from RNAseq data. CpGs with significant changes in DNA methylation were mapped into 20,028 genes by collapsing methylation probes into promoter and gene regions. To better understand the associations among diverse gene sets or among gene sets and GDM,we first constructed two weighted co-expression networks from RNAseq and DNA methylation data, respectively. Then, two co-expression networks were integrated using a linear combination. With the integrated co-expression network, graph-based label propagation algorithm was employed to prioritize GDM-associated genes. Results: From the differential expression analysis between GDM and control, the top 20 query genes, including 11 genes and 9 methylated genes, were selected for label propagation. Finally, the top 100 genes were picked up for the pathway enrichment testusing an over-representation analysis approach. Significantly enriched pathways included: Interferon Signaling, N-glycan Antennae Elongation, Sphingolipid Pathway and Metabolism, Classical Complement Pathway, Complement and Coagulation Cascades, Tryptophan Metabolism, Peroxisomal Protein Import, Unsaturated Fatty Acid Metabolism, Complement Activation, Human Innate Immune Response, Ceramide Metabolism, Fertilization Pathway, Keratan Sulfate Biosynthesis Pathway, Transport to the Golgi and Modification Pathway (FDR q<0.05 for all pathways). Conclusion: Using a novel bioinformatic approach to synthesize transcriptome and methylome data derived from human amniocytes exposed to GDM in utero, we identified potential pathways involved in programming of diabetes and obesity in offspring including pathways involving the immune response, complex lipid metabolism, the complement pathway, and protein transport and processing. Further investigation of these pathways may yield new mechanisms contributing to diabetes and obesity.

The effect of intracerebroventricular amyloid beta 1–42 application on cognitive functions in aged rats supplemented with taurine and the change of peroxisomal proteins in this process

ObjectiveThe aim of our study is to investigate the change of peroxisomal proteins in the neurodegenerative and oxidative process caused by the neurotoxicity of Aβ 1–42 in aged rats supplemented with taurine and to show the possible positive effects of taurine in this process. Methods30 Wistar albino rats were randomly divided into 5 groups as control, sham, Aβ 1–42, taurine, and Aβ 1–42+taurine. Taurine administration continued for 6 weeks (1000 mg/kg/day with drinking water). Stereotaxic surgery was applied to all groups (intracerebroventricular per lateral ventricle needle only or 5 μl, PBS, or Aβ 1–42). Spatial learning and memory performances of the animals were evaluated with Morris water maze and elevated plus maze. The levels of MDA and GSH were measured as oxidative stress parameters in the cerebral cortex and hippocampus. Expressions of CAT, PEX14, PMP70 of peroxisomal membrane proteins were indicated by Western blot analysis. ResultsOur results showed that injection of Aβ 1–42 decreased the spatial learning and memory performance, cortex CAT and hippocampus PEX14, PMP70 and GSH levels, and increased cortex and hippocampus MDA levels (p < 0.05). Although the administration of taurine partially ameliorated the adverse effects of Aβ 1–42 injection, a significant difference was found only at the hippocampus GSH levels (p < 0.05). Also, taurine caused anxiety at this dose (p < 0.05). DiscussionIn conclusion, decreased peroxisomal proteins and antioxidant capacity in neurodegenerative and oxidative processes induced by intracerebroventricular Aβ 1–42 injection showed that peroxisomes may play a role in this process and taurine supplementation may have positive effects especially in increasing antioxidant capacity.

Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14.

Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine.

Phosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.

Autophagy Inhibitors Do Not Restore Peroxisomal Functions in Cells With the Most Common Peroxisome Biogenesis Defect.

Peroxisome biogenesis disorders within the Zellweger spectrum (PBD-ZSDs) are most frequently associated with the c.2528G>A (p.G843D) mutation in the PEX1 gene (PEX1-G843D), which results in impaired import of peroxisomal matrix proteins and, consequently, defective peroxisomal functions. A recent study suggested that treatment with autophagy inhibitors, in particular hydroxychloroquine, would be a potential therapeutic option for PBD-ZSD patients carrying the PEX1-G843D mutation. Here, we studied whether autophagy inhibition by chloroquine, hydroxychloroquine and 3-methyladenine indeed can improve peroxisomal functions in four different cell types with the PEX1-G843D mutation, including primary patient cells. Furthermore, we studied whether autophagy inhibition may be the mechanism underlying the previously reported improvement of peroxisomal functions by L-arginine in PEX1-G843D cells. In contrast to L-arginine, we observed no improvement but a worsening of peroxisomal metabolic functions and peroxisomal matrix protein import by the autophagy inhibitors, while genetic knock-down of ATG5 and NBR1 in primary patient cells resulted in only a minimal improvement. Our results do not support the use of autophagy inhibitors as potential treatment for PBD-ZSD patients, whereas L-arginine remains a therapeutically promising compound.

The peroxisomal enzyme, FAR1, is induced during ER stress in an ATF6-dependent manner in cardiac myocytes.

Although peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by pharmacological and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia-reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.NEW & NOTEWORTHY The peroxisomal enzyme, FAR1, was shown to be an ER stress- and ATF6-inducible protein that localizes to peroxisomes in cardiac myocytes. FAR1 decreases myocyte viability during oxidative stress.

Nitric Oxide (NO) Scaffolds the Peroxisomal Protein-Protein Interaction Network in Higher Plants.

The peroxisome is a single-membrane subcellular compartment present in almost all eukaryotic cells from simple protists and fungi to complex organisms such as higher plants and animals. Historically, the name of the peroxisome came from a subcellular structure that contained high levels of hydrogen peroxide (H2O2) and the antioxidant enzyme catalase, which indicated that this organelle had basically an oxidative metabolism. During the last 20 years, it has been shown that plant peroxisomes also contain nitric oxide (NO), a radical molecule than leads to a family of derived molecules designated as reactive nitrogen species (RNS). These reactive species can mediate post-translational modifications (PTMs) of proteins, such as S-nitrosation and tyrosine nitration, thus affecting their function. This review aims to provide a comprehensive overview of how NO could affect peroxisomal metabolism and its internal protein-protein interactions (PPIs). Remarkably, many of the identified NO-target proteins in plant peroxisomes are involved in the metabolism of reactive oxygen species (ROS), either in its generation or its scavenging. Therefore, it is proposed that NO is a molecule with signaling properties with the capacity to modulate the peroxisomal protein-protein network and consequently the peroxisomal functions, especially under adverse environmental conditions.

Versatile allosteric properties in Pex5-like tetratricopeptide repeat proteins to induce diverse downstream function.

Proteins composed of tetratricopeptide repeat (TPR) arrays belong to the α-solenoid tandem-repeat family that have unique properties in terms of their overall conformational flexibility and ability to bind to multiple protein ligands. The peroxisomal matrix protein import receptor Pex5 comprises two TPR triplets that recognize protein cargos with a specific C-terminal Peroxisomal Targeting Signal (PTS) 1 motif. Import of PTS1-containing protein cargos into peroxisomes through a transient pore is mainly driven by allosteric binding, coupling and release mechanisms, without a need for external energy. A very similar TPR architecture is found in the functionally unrelated TRIP8b, a regulator of the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. TRIP8b binds to the HCN ion channel via a C-terminal sequence motif that is nearly identical to the PTS1 motif of Pex5 receptor cargos. Pex5, Pex5-related Pex9, and TRIP8b also share a less conserved N-terminal domain. This domain provides a second protein cargo-binding site and plays a distinct role in allosteric coupling of initial cargo loading by PTS1 motif-mediated interactions and different downstream functional readouts. The data reviewed here highlight the overarching role of molecular allostery in driving the diverse functions of TPR array proteins, which could form a model for other α-solenoid tandem-repeat proteins involved in translocation processes across membranes.

Lipid Inhibitory Effect of (-)-loliolide Isolated from Sargassum horneri in 3T3-L1 Adipocytes: Inhibitory Mechanism of Adipose-Specific Proteins.

Sargassum horneri (S. horneri) is a well-known brown seaweed widely distributed worldwide. Several biological activities of S. horneri have been reported. However, its effects on lipid metabolism and the underlying mechanisms remain elusive. In the present study, we examined the inhibitory effect of the active compound “(−)-loliolide ((6S,7aR)-6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one (HTT))” from S. horneri extract on lipid accumulation in differentiated adipocytes. MTT assays demonstrated that (−)-loliolide is not toxic to 3T3-L1 adipocytes in a range of concentrations. (−)-loliolide significantly reduced intracellular lipid accumulation in the differentiated phase of 3T3-L1 adipocytes as shown by Oil Red O staining. Western blot analysis revealed that (−)-loliolide increased the expression of lipolytic protein phospho-hormone-sensitive lipase (p-HSL) and thermogenic protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1). Additionally, (−)-loliolide decreased expression of adipogenic and lipogenic proteins, including sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), and fatty acid-binding protein 4 (FABP4) in 3T3-L1 adipocytes. These results indicate that (−)-loliolide from S. horneri could suppress lipid accumulation via regulation of antiadipogenic and prolipolytic mechanisms in 3T3-L1 cells. Considering the multifunctional effect of (−)-loliolide, it can be useful as a lipid-lowering agent in the management of patients who suffer from obesity.

Depletion of HNRNPA1 induces peroxisomal autophagy by regulating PEX1 expression

Peroxisomes play an essential role in cellular homeostasis by regulating lipid metabolism and the conversion of reactive oxygen species (ROS). Several peroxisomal proteins, known as peroxins (PEXs), control peroxisome biogenesis and degradation. Various mutations in the PEX genes are genetic causes for the development of inheritable peroxisomal-biogenesis disorders, such as Zellweger syndrome. Among the peroxins, PEX1 defects are the most common mutations in Zellweger syndrome. PEX1 is an AAA-ATPase that regulates the recycling of PEX5, which is essential for importing peroxisome matrix proteins. However, the post-transcriptional regulation of PEX1 is largely unknown. Here, we showed that heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) controls PEX1 expression. In addition, we found that depletion of HNRNPA1 induces autophagic degradation of peroxisome, which is blocked in ATG5-knockout cells. In addition, depletion of HNRNPA1 increased peroxisomal ROS levels. Inhibition of the generation of peroxisomal ROS by treatment with NAC significantly suppressed pexophagy in HNRNPA1-deficient cells. Taken together, our results suggest that depletion of HNRNPA1 increases peroxisomal ROS and pexophagy by downregulating PEX1 expression.

Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal β-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p-chloromercuribenzoic acid (pCMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2–1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.