RESUMO
Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.
Assuntos
Peroxissomos , Receptores Citoplasmáticos e Nucleares , Proteínas de Transporte/metabolismo , Humanos , Ligases/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/química , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina/metabolismoRESUMO
Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
Assuntos
Leishmania donovani/metabolismo , Microcorpos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Fosfatidilgliceróis/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Fracionamento Celular , Colesterol/química , Colesterol/metabolismo , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Leishmania donovani/genética , Fluidez de Membrana , Microcorpos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Different pull-down strategies were successfully applied to gain novel insight into the interactome of human membrane-associated proteins. Here, we compare the outcome, efficiency and potential of pull-down strategies applied to human peroxisomal membrane proteins. Stable membrane-bound protein complexes can be affinity-purified from genetically engineered human cells or subfractions thereof after detergent solubilization, followed by size exclusion chromatography and analysis by mass spectrometry (MS). As exemplified for Protein A-tagged human PEX14, one of the central constituents of the peroxisomal matrix protein import machinery, MS analyses of the affinity-purified complexes revealed an unexpected association of PEX14 with other protein assemblies like the microtubular network or the insertion apparatus for peroxisomal membrane proteins comprising PEX3, PEX16 and PEX19. The latter association was recently supported by using a different pull-down strategy following in vivo proximity labeling with biotin, named BioID, which enabled the identification of various membrane proteins in close proximity of PEX16 in living cells.
Assuntos
Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Linhagem Celular , Humanos , Membranas Intracelulares/metabolismo , Proteínas Repressoras/metabolismoRESUMO
A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5-cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1-197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1-125)) still interacted correctly with the DTM; however, this species was largely accessible to exogenously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1-125)-DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138-639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5-DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Transporte Biológico , Endopeptidase K/metabolismo , Deleção de Genes , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , SolubilidadeRESUMO
Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Biotina/química , Biotinilação , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Helianthus , Malato Desidrogenase/metabolismo , Peptídeo Hidrolases/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
The peroxisomal proteins (peroxins) that mediate the import of peroxisomal matrix proteins have been identified. Recently, the purification of a functional peroxisomal translocon has been reported. However, the molecular details of the import pathways and the mechanisms by which the cargo is translocated into the lumen of the organelle are still poorly understood. Structural studies have begun to provide insight into molecular mechanisms of peroxisomal import pathways for cargo proteins that harbor peroxisomal targeting signals, PTS1 and PTS2, at their C- and N-termini, respectively. So far structures have been reported for binary or tertiary protein-protein interfaces, and highlight the role of intrinsically disordered regions for these interactions. Here, we provide an overview of the currently available structural biology of peroxisomal import pathways. Current challenges and future perspectives of the structural biology of peroxisomal protein translocation are discussed.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017.
Assuntos
Carcinoma Hepatocelular/patologia , Catalase/metabolismo , Poluentes Ambientais/toxicidade , Neoplasias Hepáticas/patologia , Peroxissomos/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Trinitrotolueno/toxicidade , Animais , Antioxidantes/metabolismo , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Substâncias Explosivas/toxicidade , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxissomos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Regulação para Cima/efeitos dos fármacosRESUMO
Pex1p and Pex6p are required for the relocation of the import receptor Pex5p from the peroxisomal membrane to the cytosol. We herein show that mammalian Pex26p directly binds to Pex14p, the initial docking receptor of Pex5p, and interacts with Pex5p via Pex14p. The binding affinity of Pex26p to Pex14p is altered by Pex5p. Further evidence suggests that the N-terminal region in Pex26p acts as a scaffold protein to recruit Pex14p·Pex5p complex together with Pex1p·Pex6p complexes on peroxisomes. Pex26p binding to Pex14p was suppressed by overexpression of Pex1p and Pex6p in an ATP-dependent manner, whereas Pex14p was not competed out by Pex1p and Pex6p from Pex26p mutant defective in peroxisomal matrix protein import. These results suggested that peroxisome biogenesis requires Pex1p- and Pex6p-regulated dissociation of Pex14p from Pex26p. Pex1p homo-oligomer directly binds to Pex5p as assessed by a surface plasmon resonance-based assay. Moreover, cytosolic Pex1p is likely to maintain the functional oligomer of Pex5p. Taken together, in the peroxisomal protein import, AAA peroxins modulate the interaction between Pex26p and Pex14p on peroxisome membrane as well as Pex5p oligomer in the cytosol.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Membrana/fisiologia , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Primers do DNA , Células HEK293 , Humanos , Eletroforese em Gel de Poliacrilamida Nativa , Reação em Cadeia da Polimerase , Ligação ProteicaRESUMO
Zellweger syndrome, one of the peroxisome biogenesis disorders, is an autosomal recessive disease caused by mutations in PEX genes. It is characterized by severe hypotonia, failure to thrive, psychomotor retardation, liver dysfunction, and sensorineural hearing impairment. Most of the patients with this disease die before the age of 1 year. PEX14 is the 13th PEX gene responsible for peroxisome biogenesis disorders. Thus far, only two patients with PEX14 deficiency have been reported. Here, we report the first case of a Japanese patient with a PEX14 mutation who showed severe hypotonia, psychomotor retardation, demyelination, and developed rickets at the age of 5 months. An increased excretion of 3,6-epoxydicarboxylic acids leads to the diagnosis of Zellweger syndrome and a mutation analysis of PEX14 revealed a homozygous mutation of c.538C>T (p.Q180X). The patient survived for a prolonged period of time but died of liver failure at the age of 46 months.
Assuntos
DNA/genética , Proteínas de Membrana/genética , Mutação , Proteínas Repressoras/genética , Síndrome de Zellweger/genética , Análise Mutacional de DNA , Evolução Fatal , Humanos , Recém-Nascido , Japão , Masculino , Proteínas de Membrana/metabolismo , Proteínas Repressoras/metabolismo , Síndrome de Zellweger/metabolismoRESUMO
Peroxisomes are ubiquitous cellular organelles participating in a variety of critical metabolic reactions. PEX14 is an essential peroxin responsible for peroxisome biogenesis. In this study, we identified the human PEX14 homolog in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). N. lugens PEX14 (NlPEX14) showed significant topological similarity to its human counterpart. It is expressed throughout all developmental stages, with the highest expression observed in adult insects. Down-regulation of NlPEX14 through injection of NlPEX14-specific double-strand RNA impaired nymphal development. Moreover, females subjected to dsNlPEX14 treatment exhibited a significantly reduced lifespan. Additionally, we found abnormal ovarian development and a significant decrease in the number of eggs laid in NlPEX14-downregulated females. Further experiments support that the shortening of lifespan and the decrease in female fecundity can be attributed, at least partially, to the accumulation of fatty acids and reduced expression of vitellogenin. Together, our study reveals an indispensable function of NlPEX14 for insect reproduction and establishes a causal connection between the phenotypes and peroxisome biogenesis, shedding light on the importance of peroxisomes in female fecundity.
Assuntos
Fertilidade , Hemípteros , Proteínas de Insetos , Animais , Hemípteros/genética , Hemípteros/metabolismo , Hemípteros/fisiologia , Hemípteros/crescimento & desenvolvimento , Feminino , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Peroxissomos/metabolismo , Longevidade , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Ninfa/genética , Peroxinas/metabolismo , Peroxinas/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Vitelogeninas/metabolismo , Vitelogeninas/genéticaRESUMO
Peroxisome biogenesis disorders are caused by pathogenic variants in genes involved in biogenesis and maintenance of peroxisomes. However, mitochondria are also often affected in these diseases. Peroxisomal membrane proteins, including PEX14, have been found to mislocalise to mitochondria in cells lacking peroxisomes. Recent studies indicated that this mislocalisation contributes to mitochondrial abnormalities in PEX3-deficient patient fibroblasts cells. Here, we studied whether mitochondrial morphology is also affected in PEX3-deficient HEK293 cells and whether PEX14 mislocalises to mitochondria in these cells. Using high-resolution imaging techniques, we show that although endogenous PEX14 mislocalises to mitochondria, mitochondrial morphology was normal in PEX3-KO HEK293 cells. However, we discovered that overexpression of tagged PEX14 in wild-type HEK293 cells resulted in its mitochondrial localisation, accompanied by altered mitochondrial morphology. Our data indicate that overexpression of tagged PEX14 alone directly or indirectly cause mitochondrial abnormalities in cells containing peroxisomes.
Assuntos
Proteínas de Membrana , Mitocôndrias , Peroxissomos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células HEK293 , Peroxissomos/metabolismo , Peroxissomos/genética , Peroxinas/metabolismo , Peroxinas/genética , Transporte Proteico , Lipoproteínas , Proteínas RepressorasRESUMO
Peroxisomes are eukaryotic organelles that rapidly change in number depending on the metabolic requirement of distinct cell types and tissues. In the brain, these organelles are essential for neuronal migration and myelination during development and their dysfunction is associated with age-related neurodegenerative diseases. Except for one study analysing ABCD3-positive peroxisomes in neurons of the frontal neocortex of Alzheimer disease (AD) patients, no data on other brain regions or peroxisomal proteins are available. In the present morphometric study, we quantified peroxisomes labelled with PEX14, a metabolism-independent peroxisome marker, in 13 different brain areas of 8 patients each either with low, intermediate or high AD neuropathological changes compared to 10 control patients. Classification of patient samples was based on the official ABC score. During AD-stage progression, the peroxisome density decreased in the area entorhinalis, parietal/occipital neocortex and cerebellum, it increased and in later AD-stage patients decreased in the subiculum and hippocampal CA3 region, frontal neocortex and pontine gray and it remained unchanged in the gyrus dentatus, temporal neocortex, striatum and inferior olive. Moreover, we investigated the density of catalase-positive peroxisomes in a subset of patients (> 80 years), focussing on regions with significant alterations of PEX14-positive peroxisomes. In hippocampal neurons, only one third of all peroxisomes contained detectable levels of catalase exhibiting constant density at all AD stages. Whereas the density of all peroxisomes in neocortical neurons was only half of the one of the hippocampus, two thirds of them were catalase-positive exhibiting increased levels at higher ABC scores. In conclusion, we observed spatiotemporal differences in the response of peroxisomes to different stages of AD-associated pathologies.
Assuntos
Doença de Alzheimer , Neocórtex , Humanos , Doença de Alzheimer/patologia , Peroxissomos/metabolismo , Peroxissomos/patologia , Catalase/metabolismo , Projetos Piloto , Neocórtex/patologiaRESUMO
The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region. Various PTS1-PEX5 interactions have been studied by biophysical methods using isolated proteins or in heterologous systems such as two-hybrid assays, but a recently established approach based on Försters resonance energy transfer (FRET) allows a quantifying investigation in living cells. FRET is the radiation-free energy transfer between two fluorophores in close proximity and can be used to estimate the fraction of acceptor molecules bound to a donor molecule. For PTS1-PEX5 this method relies on the measurement of FRET-efficiency between the PTS1-binding TPR-domain of PEX5 tagged with mCherry and EGFP fused to a PTS1 peptide. However, this method is less suitable for binding partners with low affinity and protein complexes involving large proteins such as the interaction between full-length PTS1-carrying cargo proteins and PEX5. To overcome this limitation, we introduce a life-cell competition assay based on the same FRET approach but including a fusion protein of Cerulean with the protein of interest as a competitor. After implementing the mathematical description of competitive binding experiments into a fitting algorithm, we demonstrate the functionality of this approach using known interaction partners, its ability to circumvent previous limitations of FRET-measurements and its ability to study the interaction between PEX5 and its full-length cargo proteins. We find that some proteins (SCP2 and AGXT) bind PEX5 with higher affinity than their PTS1-peptides alone, but other proteins (ACOX3, DAO, PerCR-SRL) bind with lower but reasonable affinity, whereas GSTK1 binds with very low affinity. This binding strength was not increased upon elongating the PEX5 TPR-domain at its N-terminus, PEX5(N-TPR), although it interacts specifically with the N-terminal domain of PEX14. Finally, we demonstrate that the latter reduces the interaction strength between PEX5(N-TPR) and PTS1 by a dose-dependent but apparently non-competitive mechanism. Altogether, this demonstrates the power of this novel FRET-based competition approach for studying cargo recognition by PEX5 and protein complexes including large proteins in general.
RESUMO
Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.
Assuntos
Proteínas de Membrana/química , Microtúbulos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Peroxissomos/metabolismo , Proteínas Repressoras/química , Tubulina (Proteína)/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Transporte Biológico , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Proibitinas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98â Å resolution were obtained from a condition consisting of 1.4â M sodium citrate tribasic dihydrate, 0.1â M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62â Å, ß = 94.68°. Structure determination and characterization are in progress.
Assuntos
Cristalografia por Raios X/métodos , Leishmania donovani/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Clonagem Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.
Assuntos
Proteínas de Membrana/genética , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Peroxissomos/genética , Autofagia/genética , GTP Fosfo-Hidrolases/genética , Humanos , Metabolismo dos Lipídeos/genética , Proteínas Associadas aos Microtúbulos/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Most of peroxisomal matrix proteins including a hydrogen peroxide (H2O2)-decomposing enzyme, catalase, are imported in a peroxisome-targeting signal type-1 (PTS1)-dependent manner. However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H2O2 in mammalian cells. The H2O2-induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively impairs in vitro the interaction of catalase with the Pex14-Pex5 complex. A phosphomimetic mutant Pex14-S232D elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H2O2. We thus suggest that the H2O2-induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.
Assuntos
Catalase/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo/fisiologia , Peroxissomos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Citosol/química , Citosol/enzimologia , Citosol/metabolismo , Peróxido de Hidrogênio , Fígado/química , Masculino , Proteínas de Membrana/química , Camundongos , Fosforilação , Ratos , Proteínas Repressoras/química , Testículo/químicaRESUMO
The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.
RESUMO
Peroxisomes are single membrane cell organelles with a diversity of metabolic functions. Here we studied the peroxisomal dysfunction and oxidative stress after 3-nitropropionic acid (3-NP) induced neurotoxicity and the possible protective effects of oxytocin. Adult male and female rats were subjected to OXT and/or 3-NP treatment. The antioxidant enzymes, Superoxide dismutase (SOD) and Catalase (CAT) activities as well as expression level of Peroxin 14 (Pex14), a marker for peroxisomal number and Peroxisomal membrane protein of 70 kDa (PMP70), a metabolic transporter in peroxisome in different brain regions of both sexes were studied. The results indicated that 3-NP significantly decreased the expression level of Pex14 and PMP70 in various studied areas in male and female rats. In addition, 3-NP reduced the SOD and CAT activity in different brain regions in both sexes. OXT treatment increased the expression level of peroxisomal proteins Pex14 and PMP70 which are representative of peroxisome performance improvement. Besides, it ameliorated the antioxidant system capability through increasing the activity of the SOD and CAT in all studied brain regions including Striatum, Hippocampus, Prefrontal Cortex and Amygdala with no differences in male and female rats. This study demonstrated that toxin 3-NP, could ultimately cause peroxisomal malfunction and so determines the contribution of peroxisomal dysfunction in the etiology of HD pathology. OXT significantly increased peroxisomal function and antioxidant system defense capability, therefore illustrates that OXT might be an alternate treatment approach for the neurodegenerative diseases like HD.
RESUMO
The interaction of Trypanosoma brucei (Tb) Pex5p and its receptor TbPex14p is essential for the translocation of newly synthesized matrix proteins into the glycosome. Here, we reveal that only the third WXXXF/Y motif of TbPex5p is involved in the interaction and that negative charge of the fourth amino acid is important. We suggest that Phe35 and Phe52 of TbPex14p interact with Trp318 and Phe322 in the third motif and that the Lys56 adjacent to Phe35/Phe52 associates with the fourth Glu in the motif to make the complex. This information is expected to be useful for developing anti-trypanosomal drugs.