PEX5 Research Articles

Peroxisomal disorders

Peroxisomes are multifunctional microorganelles that play a key role in numerous biochemical processes adapting dynamically to the current physiological requirements of the cell. The disturbance of the peroxisome structure due to mutations in different PEX and non-PEX genes coding functional peroxisomal proteins is the pathogenic basis of the peroxisomal disorders. The β-oxidation process of very long-chain fatty acids (VLCFA) is a unique metabolic pathway located exclusively in the peroxisome. This determines that VLCFA is the main biomarker for the diagnosis of peroxisomal diseases. Peroxisomal disorders present a broad spectrum of clinical symptoms from the neonatal, severe Zellweger syndrome with dysmorphia, multi-organ dysfunction to the late symptomatic adult form of X-linked adrenoleukodystrophy. Relatively common the use of highly specialized analytical techniques causes it is a still growing group of rare metabolic diseases.

Zellweger spectrum disorder patient–derived fibroblasts with the PEX1‐Gly843Asp allele recover peroxisome functions in response to flavonoids

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5',7'-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D-associated ZSD.

Clinical and Neuroimaging Spectrum of Peroxisomal Disorders.

Peroxisomes play vital roles in a broad spectrum of cellular metabolic pathways. Defects in genes encoding peroxisomal proteins can result in a wide array of disorders, depending upon the metabolic pathways affected. These disorders can be broadly classified into 2 main groups; peroxisome biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies. Peroxisomal enzyme deficiencies are result of dysfunction of a specific metabolic pathway, while PBDs are due to generalized peroxisomal dysfunction. Mutations in PEX1 gene are the most common cause of PBDs, accounting for two-thirds of cases. Peroxisomal fission defects is a recently recognized entity, included under the subgroup of PBDs. The aim of this article is to provide a comprehensive review on the clinical and neuroimaging spectrum of peroxisomal disorders.

Expanding the spectrum of PEX16 mutations and novel insights into disease mechanisms

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.

Super-resolution imaging reveals the sub-diffraction phenotype of Zellweger Syndrome ghosts and wild-type peroxisomes

Peroxisomes are ubiquitous cell organelles involved in many metabolic and signaling functions. Their assembly requires peroxins, encoded by PEX genes. Mutations in PEX genes are the cause of Zellweger Syndrome spectrum (ZSS), a heterogeneous group of peroxisomal biogenesis disorders (PBD). The size and morphological features of peroxisomes are below the diffraction limit of light, which makes them attractive for super-resolution imaging. We applied Stimulated Emission Depletion (STED) microscopy to study the morphology of human peroxisomes and peroxisomal protein localization in human controls and ZSS patients. We defined the peroxisome morphology in healthy skin fibroblasts and the sub-diffraction phenotype of residual peroxisomal structures (‘ghosts’) in ZSS patients that revealed a relation between mutation severity and clinical phenotype. Further, we investigated the 70 kDa peroxisomal membrane protein (PMP70) abundance in relationship to the ZSS sub-diffraction phenotype. This work improves the morphological definition of peroxisomes. It expands current knowledge about peroxisome biogenesis and ZSS pathoethiology to the sub-diffraction phenotype including key peroxins and the characteristics of ghost peroxisomes.

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y.lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y.lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

PEX13 Enters the RING, Lives Fast, Dies Young

PEX13 Enters the RING, Lives Fast, Dies Young

PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

Defects in the PEX5 gene impair the import of peroxisomal matrix proteins, leading to nonfunctional peroxisomes and other associated pathological defects such as Zellweger syndrome. Although PEX5 regulates autophagy process in a stress condition, the mechanisms controlling autophagy by PEX5 under nutrient deprivation are largely unknown. Herein, we show a novel function of PEX5 in the regulation of autophagy via Transcription Factor EB (TFEB). Under serum-starved conditions, when PEX5 is depleted, the mammalian target of rapamycin (mTORC1) inhibitor TSC2 is downregulated, which results in increased phosphorylation of the mTORC1 substrates, including 70S6K, S6K, and 4E-BP-1. mTORC1 activation further suppresses the nuclear localization of TFEB, as indicated by decreased mRNA levels of TFEB, LIPA, and LAMP1. Interestingly, peroxisomal mRNA and protein levels are also reduced by TFEB inactivation, indicating that TFEB might control peroxisome biogenesis at a transcriptional level. Conversely, pharmacological inhibition of mTOR resulting from PEX5 depletion during nutrient starvation activates TFEB by promoting nuclear localization of the protein. In addition, mTORC1 inhibition recovers the damaged-peroxisome biogenesis. These data suggest that PEX5 may be a critical regulator of lysosomal gene expression and autophagy through the mTOR-TFEB-autophagy axis under nutrient deprivation.

Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica.

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the β-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.

AB037. rAAV mediated PEX1 gene augmentation improves visual function in a mouse model for Zellweger spectrum disorder

Background: Zellweger spectrum disorder (ZSD) is an autosomal recessive disease caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly. Retinopathy leading to blindness is one of the major handicaps faced by affected individuals, but treatment for this is supportive only. To test whether we could improve visual function in ZSD, we performed a proof-of-concept trial for PEX1 gene augmentation therapy using the Pex1-G844D mouse model, which bears the equivalent to a common human mutation. This model exhibits a gradual decline in scotopic ffERG response, an always residual photopic ffERG response, diminished visual acuity, and cone and bipolar cell anomalies.

A Missense Mutation of G257A at Exon 3 in PEX7 CDS Was Responsible for the Incidence of Rhizomelic Chondrodysplasia Punctata Type 1

Background Rhizomelic chondrodysplasia punctata (RCDP) type 1 is among of the rare autosomal recessive peroxisome biogenesis disorders caused by mutations in the PEX7 gene. RCDP patients with the classic form of RCDP1 do not live more than 10- year. Materials and Methods In the present study, a two-year-old girl with skeletal abnormalities and dysmorphic facial appearance is reported to be suffered from RCDP. The patient's parents were second cousins and healthy and there was no similar case in the parents’ family. PEX7 gene was sequenced in the patient and her parents. Results A homozygous mutation, G257A, was identified PEX7 in the genome of patient while the parents were compound heterozygous. Conclusion Taken together, clinical presentation and peroxisome profile of the patient suggested a missense mutation led to formation of a pathogenic PEX7, responsible for incidence of RCDP.

Importancia de la semiología y la bioquímica en el abordaje diagnóstico de un trastorno de la biogénesis peroxisómica

Peroxisomal biogenesis disorders are due to mutations in the PEX genes, which code for peroxins that are required for peroxisomal biogenesis. Clinically, they are expressed as a Zellweger syndrome spectrum, and there is a wide phenotypic variety. They are diagnosed biochemically, and confirmation is molecular. The aim of this illustrative case is to highlight the importance of the clinical features and biochemical testing in the management of a peroxisomal disease. A 3-year-old boy with neonatal hypotonia, overall developmental delay and failure to thrive and a pattern of hypomyelinating leukodystrophy in brain resonance. The suspected diagnosis was a disorder affecting the biogenesis of the peroxisomes due to having found a variant with an uncertain meaning in PEX5. The clinical features, the biochemical studies and critical analysis, however, made this diagnosis unlikely. Emphasis is placed on the management that must be applied when a Zellweger syndrome spectrum is suspected. In the case reported here, a peroxisomal biogenesis disorder was suspected owing to an exome sequencing which, on being critically analysed together with the clinical features and the biochemical findings, made a peroxisomal disease very unlikely. In cases of clinical suspicion, backed up by neuroimaging, the main diagnostic management must be based on the biochemistry analysis. Although confirmation is molecular, these tests must be interpreted with caution.

Unbalanced lipolysis results in lipotoxicity and mitochondrial damage in peroxisome-deficient Pex19 mutants.

Inherited peroxisomal biogenesis disorders (PBDs) are characterized by the absence of functional peroxisomes. They are caused by mutations of peroxisomal biogenesis factors encoded by Pex genes, and result in childhood lethality. Owing to the many metabolic functions fulfilled by peroxisomes, PBD pathology is complex and incompletely understood. Besides accumulation of peroxisomal educts (like very-long-chain fatty acids [VLCFAs] or branched-chain fatty acids) and lack of products (like bile acids or plasmalogens), many peroxisomal defects lead to detrimental mitochondrial abnormalities for unknown reasons. We generated Pex19 Drosophila mutants, which recapitulate the hallmarks of PBDs, like absence of peroxisomes, reduced viability, neurodegeneration, mitochondrial abnormalities, and accumulation of VLCFAs. We present a model of hepatocyte nuclear factor 4 (Hnf4)-induced lipotoxicity and accumulation of free fatty acids as the cause for mitochondrial damage in consequence of peroxisome loss in Pex19 mutants. Hyperactive Hnf4 signaling leads to up-regulation of lipase 3 and enzymes for mitochondrial β-oxidation. This results in enhanced lipolysis, elevated concentrations of free fatty acids, maximal β-oxidation, and mitochondrial abnormalities. Increased acid lipase expression and accumulation of free fatty acids are also present in a Pex19-deficient patient skin fibroblast line, suggesting the conservation of key aspects of our findings.

Allelic Expression Imbalance Promoting a Mutant PEX6 Allele Causes Zellweger Spectrum Disorder

Zellweger spectrum disorders (ZSDs) are autosomal-recessive disorders that are caused by defects in peroxisome biogenesis due to bi-allelic mutations in any of 13 different PEX genes. Here, we identified seven unrelated individuals affected with an apparent dominant ZSD in whom a heterozygous mutant PEX6 allele (c.2578C>T [p.Arg860Trp]) was overrepresented due to allelic expression imbalance (AEI). We demonstrated that AEI of PEX6 is a common phenomenon and is correlated with heterozygosity for a frequent variant in the 3' untranslated region (UTR) of the mutant allele, which disrupts the most distal of two polyadenylation sites. Asymptomatic parents, who were heterozygous for PEX c.2578C>T, did not show AEI and were homozygous for the 3' UTR variant. Overexpression models confirmed that the overrepresentation of the pathogenic PEX6 c.2578T variant compared to wild-type PEX6 c.2578C results in a peroxisome biogenesis defect and thus constitutes the cause of disease in the affected individuals. AEI promoting the overrepresentation of a mutant allele might also play a role in other autosomal-recessive disorders, in which only one heterozygous pathogenic variant is identified.

Genetic testing for Refsum disease

Abstract We reviewed the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for Refsum disease. The disease has autosomal recessive inheritance, unknown prevalence, and is caused by variations in PEX7 and PHYH genes. Clinical diagnosis is based on clinical findings, ophthalmological examination, electroretinography, optical coherence tomography and phytanic acid assay. The genetic test is useful for confirming diagnosis, for differential diagnosis, couple risk assessment and access to clinical trials.

The functions of PEX genes in peroxisome biogenesis and pathogenicity in phytopathogenic fungi.

Peroxisomes are cellular organelles present ubiquitously in eukaryotic cells and are involved in β-oxidation, glyoxylate cycle and a variety of biochemical metabolisms. Recently peroxisomes have been demonstrated to play vital roles in the host infection processes by plant fungal pathogens. The biogenesis of peroxisomes requires a category of proteins named peroxins, which are encoded by the PEX genes. So far, more than 10 PEX genes were isolated in phytopathogenic fungi, and significant research efforts are focused on the mechanism of peroxisome formation and the roles of peroxisome in the development and pathogenicity of fungal pathogens. In this review, we summarize the latest advances in peroxisome biogenesis and functions in pathogenic fungi, including the roles of PEXs in life cycle of peroxisome, peroxisome related metabolisms, and fungal development, infection and pathogenicity, in order to provide references for future studies in plant pathogenic fungi and the control of disease.

A clinical case of Zellweger syndrome in a patient with a previous history of ocular medulloepithelioma

Peroxisomal biogenesis disorders (PBDs) are autosomal recessive diseases caused by mutations in one of the 14 PEX genes described in the scientific literature.All of these syndromes may be associated with different mutations in the PEX genes, the most frequent being PEX1 for patients with Zellweger syndrome (ZS).In this paper, we present the case of a patient with a peculiar clinical history: evisceration of the left eye (LE) at 4years of age because of a benign ocular teratoid medulloepithelioma and a progressive loss of visual acuity (VA) in the right eye (RE) beginning at 9years of age, leading to the diagnosis of ZS. In addition, the patient presented a mutation in the PEX14 gene that has not been previously described in the literature.This case broadens the spectrum of clinical expression in ZS patients because of not only the presence of a benign ocular teratoid medulloepithelioma at 4years of age but also the late clinical expression of ZS (at 9years of age).

Identification of a novel mutation in PEX10 in a patient with attenuated Zellweger spectrum disorder: a case report

BackgroundThe peroxisome biogenesis disorders, which are caused by mutations in any of 13 different PEX genes, include the Zellweger spectrum disorders. Severe defects in one of these PEX genes result in the absence of functional peroxisomes which is seen in classical Zellweger syndrome. These patients present with hypotonia and seizures shortly after birth. Other typical symptoms are dysmorphic features, liver disease, retinal degeneration, sensorineural deafness, polycystic kidneys, and the patient does not reach any developmental milestones.Case presentationWe report a case of a patient with Zellweger spectrum disorder due to a novel mutation in the PEX10 gene, presenting with a mild late-onset neurological phenotype. The patient, an Assyrian girl originating from Iraq, presented with sensorineural hearing impairment at the age of 5 followed by sensorimotor polyneuropathy, cognitive delay, impaired gross and fine motor skills, and tremor and muscle weakness in her teens. Analyses of biochemical markers for peroxisomal disease suggested a mild peroxisomal defect and functional studies in fibroblasts confirmed the existence of a peroxisome biogenesis disorder. Diagnosis was confirmed by next generation sequencing analysis, which showed a novel homozygous mutation (c.530 T > G (p.Leu177Arg) (NM_153818.1)) in the PEX10 gene predicted to be pathogenic.ConclusionsThis case highlights the importance of performing biochemical, functional, and genetic peroxisomal screening in patients with clinical presentations milder than those usually observed in Zellweger spectrum disorders.

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse.

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.

PEX10 mutation; Autosomal recessive cerebellar ataxia

Objective: Peroxisomal biogenesis disorders typically cause multisystem disease which is characterized clinically by severe global neurological involvement and a variable severity of dysmorphism, retinitis pigmentosa, sensorineural deafness, liver disease, and other systemic features, including death in infancy or early childhood. They are also known as the Zellweger spectrum disorders. Methods: Results: We report twosibling patients of normal intelligence with cerebellar atrophy, slowly progressive ataxia, neuropathy and, hearing loss. In both patients, mutations in PEX10 gene were found. Conclusion: Peroxisomal biogenesis disorders should be considered in the differential diagnosis of autosomal recessive ataxia.