Peroxin Proteins Research Articles

Multivariate analysis and model building for classifying patients in the peroxisomal disorders X-linked adrenoleukodystrophy and Zellweger syndrome in Chinese pediatric patients

BackgroundThe peroxisome is a ubiquitous single membrane-enclosed organelle with an important metabolic role. Peroxisomal disorders represent a class of medical conditions caused by deficiencies in peroxisome function and are segmented into enzyme-and-transporter defects (defects in single peroxisomal proteins) and peroxisome biogenesis disorders (defects in the peroxin proteins, critical for normal peroxisome assembly and biogenesis). In this study, we employed multivariate supervised and non-supervised statistical methods and utilized mass spectrometry data of neurological patients, peroxisomal disorder patients (X-linked adrenoleukodystrophy and Zellweger syndrome), and healthy controls to analyze the role of common metabolites in peroxisomal disorders, to develop and refine a classification models of X-linked adrenoleukodystrophy and Zellweger syndrome, and to explore analytes with utility in rapid screening and diagnostics.ResultsT-SNE, PCA, and (sparse) PLS-DA, operated on mass spectrometry data of patients and healthy controls were utilized in this study. The performance of exploratory PLS-DA models was assessed to determine a suitable number of latent components and variables to retain for sparse PLS-DA models. Reduced-features (sparse) PLS-DA models achieved excellent classification performance of X-linked adrenoleukodystrophy and Zellweger syndrome patients.ConclusionsOur study demonstrated metabolic differences between healthy controls, neurological patients, and peroxisomal disorder (X-linked adrenoleukodystrophy and Zellweger syndrome) patients, refined classification models and showed the potential utility of hexacosanoylcarnitine (C26:0-carnitine) as a screening analyte for Chinese patients in the context of a multivariate discriminant model predictive of peroxisomal disorders.

Peroxisome Deficiency Dysregulates Fatty Acid Oxidization and Exacerbates Lipotoxicity in β Cells.

An adverse intrauterine environment impairs the development of pancreatic islets in the fetus and leads to insufficient β cell mass and β cell dysfunction. We previously reported that Pex14, a peroxin protein involved in the biogenesis and degradation of peroxisomes, is markedly reduced in the pancreas of an intrauterine growth restriction fetus and last into adulthood. Peroxisomes function in a wide range of metabolic processes including fatty acid oxidization, ROS detoxification, and anti-inflammatory responses. To elucidate the impact of downregulation of the Pex14 gene on β cell, Pex14 was knocked down by siRNA in INS-1 cells. Pex14 knockdown disturbed peroxisomal biogenesis and dysregulated fatty acid metabolism and lipid storage capability, thereby increased ROS level and blunted insulin secretion. Moreover, Pex14 knockdown upregulated inflammation factors and regulators of endoplasmic reticulum stress. The lipotoxicity of fatty acid (including palmitic acid and linoleic acid) in β cells was exacerbated by knockdown of Pex14, as indicated by H2O2 accumulation and increased programmed cell death. The present results demonstrate the vital role of Pex14 in maintaining normal peroxisome function and β cell viability and highlight the importance of a functional peroxisomal metabolism for the detoxification of excess FAs in β cells.

The Pex4p-Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization.

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.

Colletotrichum orbiculare FAM1 Encodes a Novel Woronin Body-Associated Pex22 Peroxin Required for Appressorium-Mediated Plant Infection.

ABSTRACTThe cucumber anthracnose fungus Colletotrichum orbiculare forms specialized cells called appressoria for host penetration. We identified a gene, FAM1, encoding a novel peroxin protein that is essential for peroxisome biogenesis and that associates with Woronin bodies (WBs), dense-core vesicles found only in filamentous ascomycete fungi which function to maintain cellular integrity. The fam1 disrupted mutants were unable to grow on medium containing oleic acids as the sole carbon source and were nonpathogenic, being defective in both appressorium melanization and host penetration. Fluorescent proteins carrying peroxisomal targeting signals (PTSs) were not imported into the peroxisomes of fam1 mutants, suggesting that FAM1 is a novel peroxisomal biogenesis gene (peroxin). FAM1 did not show significant homology to any Saccharomyces cerevisiae peroxins but resembled conserved filamentous ascomycete-specific Pex22-like proteins which contain a predicted Pex4-binding site and are potentially involved in recycling PTS receptors from peroxisomes to the cytosol. C. orbiculare FAM1 complemented the peroxisomal matrix protein import defect of the S. cerevisiae pex22 mutant. Confocal microscopy of Fam1-GFP (green fluorescent protein) fusion proteins and immunoelectron microscopy with anti-Fam1 antibodies showed that Fam1 localized to nascent WBs budding from peroxisomes and mature WBs. Association of Fam1 with WBs was confirmed by colocalization with WB matrix protein CoHex1 (C. orbiculare Hex1) and WB membrane protein CoWsc (C. orbiculare Wsc) and by subcellular fractionation and Western blotting with antibodies to Fam1 and CoHex1. In WB-deficient cohex1 mutants, Fam1 was redirected to the peroxisome membrane. Our results show that Fam1 is a WB-associated peroxin required for pathogenesis and raise the possibility that localized receptor recycling occurs in WBs.

Contribution of Peroxisomes to Secondary Metabolism and Pathogenicity in the Fungal Plant Pathogen Alternaria alternata

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1, AKT2, AKT3, and AKTR, involved in AK toxin biosynthesis. AKT1, AKT2, and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated AaPEX6, which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made AaPEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The DeltaAaPEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid beta oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the DeltaAaPEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The DeltaAaPEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata. We also observed that mutation of AaPEX6 caused a marked reduction of conidiation.

Drosophila models of peroxisomal biogenesis disorder: peroxins are required for spermatogenesis and very-long-chain fatty acid metabolism

Peroxisomes are vital eukaryotic organelles that participate in lipid metabolism, in particular the metabolism of very-long-chain fatty acids (VLCFA). The biogenesis of peroxisomes is regulated by a set of peroxin proteins (PEX). In humans, mutations affecting peroxin protein production or function result in devastating diseases classified as peroxisome biogenesis disorders (PBD). The way in which peroxisomal dysfunction leads to the pathophysiological consequences of PBD is not well understood. Here we report that Drosophila pex mutants faithfully recapitulate several key features of human PBD, including impaired peroxisomal protein import, elevated VLCFA levels and growth retardation. Moreover, disruption of pex function results in spermatogenesis defects, including spermatocyte cytokinesis failure in Drosophila. Importantly, increased VLCFA levels enhance these spermatogenesis defects whereas reduced VLCFA levels alleviate them. Thus, regulation of proper VLCFA levels by pex genes is crucial for spermatogenesis. Together our study reveals an indispensable function of pex genes during spermatogenesis and provides a causative link between the phenotypic severity of pex mutants and VLCFA levels.

Arabidopsis Peroxin 16 Coexists at Steady State in Peroxisomes and Endoplasmic Reticulum

Homologs of peroxin 16 genes (PEX16) have been identified only in Yarrowia lipolytica, humans (Homo sapiens), and Arabidopsis (Arabidopsis thaliana). The Arabidopsis gene (AtPEX16), previously reported as the SSE1 gene, codes for a predicted 42-kD membrane peroxin protein (AtPex16p). Lin et al. (Y. Lin, J.E. Cluette-Brown, H.M. Goodman [2004] Plant Physiol 135: 814-827) reported that SSE1/AtPEX16 was essential for endoplasmic reticulum (ER)-dependent oil and protein body biogenesis in peroxisome-deficient maturing seeds and likely also was involved in peroxisomal biogenesis based on localization of stably expressed green fluorescent protein::AtPex16p in peroxisomes of Arabidopsis plants. In this study with Arabidopsis suspension-cultured cells, combined in vivo and in vitro experiments revealed a novel dual organelle localization and corresponding membrane association/topology of endogenous AtPex16p. Immunofluorescence microscopy with antigen affinity-purified IgGs showed an unambiguous, steady-state coexistence of AtPex16p in suspension cell peroxisomes and ER. AtPex16p also was observed in peroxisomes and ER of root and leaf cells. Cell fractionation experiments surprisingly revealed two immunorelated polypeptides, 42 kD (expected) and 52 kD (unexpected), in homogenates and microsome membrane pellets derived from roots, inflorescence, and suspension cells. Suc-gradient purifications confirmed the presence of both 42-kD and 52-kD polypeptides in isolated peroxisomes (isopycnic separation) and in rough ER vesicles (Mg2+ shifted). They were found peripherally associated with peroxisome and ER membranes but not as covalently bound subunits of AtPex16p. Both were mostly on the matrix side of peroxisomal membranes and unexpectedly mostly on the cytosolic side of ER membranes. In summary, AtPex16p is the only authentic plant peroxin homolog known to coexist at steady state within peroxisomes and ER; these data provide new insights in support of its ER-related, multifunctional roles in organelle biogenesis.

PEX1 deficiency presenting as Leber congenital amaurosis

Peroxisome biogenesis disorders result from defects in peroxin proteins involved in peroxisomal matrix and membrane protein import. Peroxins are encoded in peroxin protein genes; to date, the PEX genes responsible for all 12 peroxisome biogenesis disorders complementation groups are known. Peroxin protein 1 deficiency associated with complementation group 1 is responsible for disease in approximately two thirds of all patients with a peroxisome biogenesis disorder. Their phenotypes range from severe to mild, and it appears to be a phenotype-genotype relationship. This case report describes a patient with peroxin protein 1 deficiency presenting as Leber congenital amaurosis, in whom the diagnosis was questioned at the age of 2 years when seizures first appeared and mild facial dysmorphia became evident.

Identification of PEX5p-related novel peroxisome-targeting signal 1 (PTS1)-binding proteins in mammals

Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as ‘Pex5p-related proteins’ (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5−/− mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2–3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.

Identification of PEX5p-related novel peroxisome-targeting signal 1 (PTS1)-binding proteins in mammals.

Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as 'Pex5p-related proteins' (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5(-/-) mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2--3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.

C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal.

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivo and in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP-PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP-PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.