Peroxisomal Import Research Articles

Structure and function of the peroxisomal ubiquitin ligase complex.

Peroxisomes are membrane-bounded organelles that exist in most eukaryotic cells and are involved in the oxidation of fatty acids and the destruction of reactive oxygen species. Depending on the organism, they house additional metabolic reactions that range from glycolysis in parasitic protozoa to the production of ether lipids in animals and antibiotics in fungi. The importance of peroxisomes for human health is revealed by various disorders - notably the Zellweger spectrum - that are caused by defects in peroxisome biogenesis and are often fatal. Most peroxisomal metabolic enzymes reside in the lumen, but are synthesized in the cytosol and imported into the organelle by mobile receptors. The receptors accompany cargo all the way into the lumen and must return to the cytosol to start a new import cycle. Recycling requires receptor monoubiquitination by a membrane-embedded ubiquitin ligase complex composed of three RING finger (RF) domain-containing proteins: PEX2, PEX10, and PEX12. A recent cryo-electron microscopy (cryo-EM) structure of the complex reveals its function as a retro-translocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that assemble into an open channel. The N terminus of a receptor likely inserts into the pore from the lumenal side, and is then monoubiquitinated by one of the RFs to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitinated by the concerted action of the other two RFs and ultimately degraded. The new data provide mechanistic insight into a crucial step of peroxisomal protein import.

Gene expression analysis of the ATP-binding cassette transporter ABCD1 in petunia (Petunia hybrida) and tobacco (Nicotiana spp.)

Most plant metabolites are biosynthesized via organelles, including peroxisome. From the mutant analyses, Arabidopsis ATP-binding cassette (ABC) transporter ABCD1 is reported to be implicated in the peroxisomal import of β-oxidation substrates, which are biosynthetic intermediates for central and specialized metabolites. However, little was explored about ABCD1 in plants except for AtABCD1 and two barley homologs, HvABCD1 and 2. The present study investigated the expression profiles of ABCD1 genes of petunia and tobacco that produce various metabolites, including volatile benzenoid/phenylpropanoids (VBPs) and ubiquinone. PhABCD1 expression was ubiquitous and at particularly high levels (2.2- to 2.8-fold compared to roots) in leaves, limbs, and tubes. NsABCD1 was expressed in roots, stems, leaves, sepals, and flowers; specifically, a relatively high expression level (3.2-fold compared to roots) was observed in sepals. Yeast extract and methyl jasmonate induced the expression of the NtABCD1 gene significantly (1.3- and 2.5-fold, respectively) in N. tabacum BY-2 cells. These results suggest that the expression of ABCD1 would be related to the biosynthesis of metabolites beneficial for plant environmental adaptation in petunia and tobacco. The current study provides new insights into the mechanism of metabolite biosynthesis and ABC transporter function in Solanaceae plants.

Antioxidant Response in Human X-Linked Adrenoleukodystrophy Fibroblasts.

Redox imbalance, mitochondrial dysfunction, and inflammation play a major role in the pathophysiology of X-linked adrenoleukodystrophy (X-ALD), an inherited neurodegenerative disease caused by mutations in the ABCD1 gene, encoding the protein responsible for peroxisomal import and degradation of very long chain fatty acids (VLCFAs). Therefore, VLCFAs accumulate in tissues and plasma, constituting a pathognomonic biomarker for diagnosis. However, the precise role of VLCFA accumulation on the diverse clinical phenotypes of X-ALD and the pathogenic link between VLCFAs and oxidative stress remain currently unclear. This study proposes ferroptosis as a crucial contributor to the disease development and progression. The expression profiles of "GPX4-glutathione" and "NQO1-CoQ10" ferroptosis pathways have been analyzed in fibroblasts of one patient with AMN, the late onset and slowly progressive form of X-ALD, and in two patients with cALD, the cerebral inflammatory demyelinating form of early childhood. Furthermore, as no effective treatments are currently available, especially for the rapidly progressing form of X-ALD (cALD), the efficacy of NAC treatment has also been evaluated to open the way toward novel combined therapies. Our findings demonstrate that lipid peroxides accumulate in X-ALD fibroblasts and ferroptosis-counteracting enzymes are dysregulated, highlighting a different antioxidant response in patients with AMN and cALD.

Dynamics of the translocation pore of the human peroxisomal protein import machinery.

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

A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1–4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1–4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

Therapeutic potential of deuterium-stabilized (R)-pioglitazone-PXL065-for X-linked adrenoleukodystrophy.

X‐linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and β‐oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C‐ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to‐date for ALD. Pioglitazone, an anti‐diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and nongenomic (mitochondrial pyruvate carrier—MPC, long‐chain acyl‐CoA synthetase 4—ACSL4, inhibition). However, its use is limited by PPARγ‐driven side effects (e.g. weight gain, edema). PXL065 is a clinical‐stage deuterium‐stabilized (R)‐enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient‐derived cells (both AMN and C‐ALD) and glial cells from Abcd1‐null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1‐null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted.

Two Pex5 Proteins With Different Cargo Specificity Are Critical for Peroxisome Function in Ustilago maydis.

Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.

Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves

Main conclusionThe oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by ≈5%.Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 °C, 500 µmol photons m−2 s−1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin–Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin–Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if ≤ 21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of ≥ 62%. Last, we discuss interactions between the OPPP and the cytosolic oxidation–reduction cycle in supplying leaf cytosolic NADPH.

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Peroxisomes fulfil many anabolic and catabolic functions, thereby importing all required proteins post-translationally from the cytosol. Dysfunction of the peroxisomal import process leads to severe diseases making this process a study of utmost importance. Peroxisomal proteins are recognized in the cytosol by the import receptor PEX5 and transported towards the peroxisomal membrane. Here we study the diffusion of the cargo loaded receptor in the cytosol by combining advanced live-cell microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and superresolution STED and Minflux microscopy to present a detailed characterization of the diffusion and thus interaction dynamics as well as spatial organization of the peroxisomal import receptor PEX5.

Peroxisomal Stress Response and Inter-Organelle Communication in Cellular Homeostasis and Aging.

Peroxisomes are key regulators of cellular and metabolic homeostasis. These organelles play important roles in redox metabolism, the oxidation of very-long-chain fatty acids (VLCFAs), and the biosynthesis of ether phospholipids. Given the essential role of peroxisomes in cellular homeostasis, peroxisomal dysfunction has been linked to various pathological conditions, tissue functional decline, and aging. In the past few decades, a variety of cellular signaling and metabolic changes have been reported to be associated with defective peroxisomes, suggesting that many cellular processes and functions depend on peroxisomes. Peroxisomes communicate with other subcellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum (ER), and lysosomes. These inter-organelle communications are highly linked to the key mechanisms by which cells surveil defective peroxisomes and mount adaptive responses to protect them from damages. In this review, we highlight the major cellular changes that accompany peroxisomal dysfunction and peroxisomal inter-organelle communication through membrane contact sites, metabolic signaling, and retrograde signaling. We also discuss the age-related decline of peroxisomal protein import and its role in animal aging and age-related diseases. Unlike other organelle stress response pathways, such as the unfolded protein response (UPR) in the ER and mitochondria, the cellular signaling pathways that mediate stress responses to malfunctioning peroxisomes have not been systematically studied and investigated. Here, we coin these signaling pathways as “peroxisomal stress response pathways”. Understanding peroxisomal stress response pathways and how peroxisomes communicate with other organelles are important and emerging areas of peroxisome research.

A Floppy Infant with Facial Dysmorphism.

A Floppy Infant with Facial Dysmorphism.

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris

Synthetic biology offers several routes for CO2 conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a μmax of 0.008 h−1, which was further improved to 0.018 h−1 by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO2 for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.

Computer-Aided Design and Synthesis of a New Class of PEX14 Inhibitors: Substituted 2,3,4,5-Tetrahydrobenzo[F][1,4]oxazepines as Potential New Trypanocidal Agents.

African and American trypanosomiases are estimated to affect several million people across the world, with effective treatments distinctly lacking. New, ideally oral, treatments with higher efficacy against these diseases are desperately needed. Peroxisomal import matrix (PEX) proteins represent a very interesting target for structure- and ligand-based drug design. The PEX5-PEX14 protein-protein interface in particular has been highlighted as a target, with inhibitors shown to disrupt essential cell processes in trypanosomes, leading to cell death. In this work, we present a drug development campaign that utilizes the synergy between structural biology, computer-aided drug design, and medicinal chemistry in the quest to discover and develop new potential compounds to treat trypanosomiasis by targeting the PEX14-PEX5 interaction. Using the structure of the known lead compounds discovered by Dawidowski et al. as the template for a chemically advanced template search (CATS) algorithm, we performed scaffold-hopping to obtain a new class of compounds with trypanocidal activity, based on 2,3,4,5-tetrahydrobenzo[f][1,4]oxazepines chemistry. The initial compounds obtained were taken forward to a first round of hit-to-lead optimization by synthesis of derivatives, which show activities in the range of low- to high-digit micromolar IC50 in the in vitro tests. The NMR measurements confirm binding to PEX14 in solution, while immunofluorescent microscopy indicates disruption of protein import into the glycosomes, indicating that the PEX14-PEX5 protein-protein interface was successfully disrupted. These studies result in development of a novel scaffold for future lead optimization, while ADME testing gives an indication of further areas of improvement in the path from lead molecules toward a new drug active against trypanosomes.

Different contributions of the peroxisomal import protein Pex5 and Pex7 to development, stress response and virulence of insect fungal pathogen Beauveria bassiana.

Peroxins Pex5 and Pex7 belong to the peroxisomal import machinery and recognize proteins containing peroxisomal targeting signal (PTS) type 1 and type 2, respectively. This study seeks to characterize these two peroxins in the entomopathogenic fungus Beauveria bassiana. The orthologs of Pex5 and Pex7 in B. bassiana (BbPex5 and BbPex7) were functionally analyzed via protein localization and gene disruption. BbPex5 and BbPex7 were associated with peroxisome and specifically required for PTS1 and PTS2 pathways, respectively, which were demonstrated to be involved in development, tolerance to oxidative stress and virulence. ΔBbPex5mutant displayed additionally defectives that were undetected in ΔBbPex7 in vegetative growth and resistance to osmotic and cell wall-perturbing stresses. Notably, Woronin body major protein Hex1 with PTS1linked this organelle to the development and virulence of B. bassiana, which indicates that Woronin body is associated with the roles of PTS1 pathway. Both PTS1 and PTS2 pathways are involved in broad physiological process, and the PTS1 pathway acts as a main peroxisomal import pathway. This study shows the functional divergence of different peroxins and improves our understanding of organellar physiology involved in biocontrol potential of the entomopathogenic fungi.

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.

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.

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.

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.