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1.
Mol Cell ; 74(2): 347-362.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853401

RESUMO

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Peroxissomos/química , Peroxissomos/genética , Fosforilação , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
2.
J Biosci Bioeng ; 128(1): 33-38, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30711353

RESUMO

In this work, we analyzed several genes participating in the rearrangement pathway for xylulose 5-phosphate (Xu5P) in the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii). P. pastoris has two set of genes for fructose-1,6-bisphosphate aldolase (FBA1 and FBA2) and transaldolase (TAL1 and TAL2), although there are single-copy genes for fructose-1,6-bisphosphatase (FBP1) and transketolase (TKL1), respectively. Expressions of FBP1 and TAL2 were upregulated by non-fermentative carbon sources, especially methanol was the best inducer for them, and FBA2 was induced only by methanol. On the other hand, FBA1, TAL1 and TKL1 showed constitutive expression. Strain fbp1Δ showed severe growth defect on methanol and non-fermentable carbon sources, and growth rate of strain fba2Δ in methanol medium was slightly decreased. Moreover, Fba2p and Tal2p possessed peroxisome targeting signal type 1 (PTS1), and EGFP-Fba2p and EGFP-Tal2p were found to be localized in peroxisomes. From these findings, it was suggested that Fba2p, Fbp1p and Tal2p participate in the rearrangement pathway for Xu5P in peroxisomes, and that the altered Calvin cycle and non-oxidative pentose phosphate pathway involving Tal2p function in a complementary manner.


Assuntos
Frutose-Bifosfato Aldolase/genética , Metanol/metabolismo , Pentosefosfatos/metabolismo , Pichia , Transaldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Redes e Vias Metabólicas/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Pichia/enzimologia , Pichia/genética , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Transaldolase/metabolismo , Transcetolase/genética , Transcetolase/metabolismo
3.
Methods Enzymol ; 617: 83-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784416

RESUMO

Microbial synthesis represents an alternative approach for the sustainable production of chemicals, fuels, and medicines. However, construction of biosynthetic pathways always suffers from side reactions, toxicity of intermediates, or low efficiency of substrate channeling. Subcellular compartmentalization may contribute to a more efficient production of target products by reducing side reactions and toxic effects within a compact insular space. The peroxisome, a type of organelle that is involved in catabolism of fatty acids and reactive oxygen species, has attracted a great deal of attention in the construction of eukaryotic cell factories with little impact on essential cellular function. In this chapter, we will systematically review recent advances in peroxisomal compartmentalization for microbial production of valuable biomolecules. Additionally, detailed experimental designs and protocols are also described. We hope a comprehensive understanding of peroxisomes will promote their application in metabolic engineering and synthetic biology.


Assuntos
Engenharia Metabólica/métodos , Peroxissomos/metabolismo , Leveduras/metabolismo , Vias Biossintéticas , Carotenoides/genética , Carotenoides/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Microbiologia Industrial/métodos , Penicilinas/metabolismo , Peroxissomos/genética , Biologia Sintética/métodos , Leveduras/genética
4.
Redox Biol ; 20: 321-333, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391825

RESUMO

PGC-1α is a key regulator of oxidative metabolism facilitating the expression of genes critical for the function and biogenesis of the two key oxidative organelles, mitochondria and peroxisomes, in skeletal muscle (SKM) and other organs. Our recent studies have found that the transcription factor Bhlhe40 negatively regulates PGC-1α gene expression and its coactivational activity, therefore, this factor should have profound influence on the biogenesis and metabolic activity of mitochondria and peroxisomes. Here we found that both the number and activity of peroxisomes were increased upon knockdown of Bhlhe40 expression but were repressed by its over-expression. Mitochondrial efficiency was significantly reduced by Bhlhe40 knockdown, resulting in the burst of ROS. Over-expression of a constitutively active PGC-1α-interactive domain (named as VBH135) of Bhlhe40 mimicked the effects of its knockdown on peroxisomes but simultaneously reduced ROS level. Furthermore, the efficiency, but not the number, of mitochondria was also increased by VBH135, suggesting differential regulation of peroxisomes and mitochondria by Bhlhe40. Unsaturated fatty acid oxidation, insulin response, and oxidative respiration were highly enhanced in Bhlhe40 knockdown or VBH135 over-expressed cells, suggesting the importance of Bhlhe40 in the regulation of unsaturated fatty acid and glucose oxidative metabolism. Expression profiling of genes important for either organelle also supports differential regulation of peroxisomes and mitochondria by Bhlhe40. These observations have established the important role of Bhlhe40 in SKM oxidative metabolism as the critical regulator of peroxisome and mitochondrion biogenesis and functions, and thus should provide a novel route for developing drugs targeting SKM metabolic diseases.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Catalase/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Oxirredução , Consumo de Oxigênio , RNA Interferente Pequeno/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo
5.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30530632

RESUMO

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.


Assuntos
Retículo Endoplasmático/genética , Proteínas de Membrana/biossíntese , Mitocôndrias/genética , Peroxissomos/genética , Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Homeostase/genética , Humanos , Proteínas de Membrana/genética , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Peroxissomos/metabolismo
6.
Genome Biol Evol ; 11(1): 41-53, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500900

RESUMO

The phylum Apicomplexa is a quintessentially parasitic lineage, whose members infect a broad range of animals. One exception to this may be the apicomplexan genus Nephromyces, which has been described as having a mutualistic relationship with its host. Here we analyze transcriptome data from Nephromyces and its parasitic sister taxon, Cardiosporidium, revealing an ancestral purine degradation pathway thought to have been lost early in apicomplexan evolution. The predicted localization of many of the purine degradation enzymes to peroxisomes, and the in silico identification of a full set of peroxisome proteins, indicates that loss of both features in other apicomplexans occurred multiple times. The degradation of purines is thought to play a key role in the unusual relationship between Nephromyces and its host. Transcriptome data confirm previous biochemical results of a functional pathway for the utilization of uric acid as a primary nitrogen source for this unusual apicomplexan.


Assuntos
Apicomplexa/genética , Peroxissomos/genética , Purinas/metabolismo , Apicomplexa/metabolismo , Ácido Úrico/metabolismo
7.
Subcell Biochem ; 89: 125-138, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30378021

RESUMO

Our knowledge of the proteome of plant peroxisomes is far from being complete, and the functional complexity and plasticity of this cell organelle are amazingly high particularly in plants, as exemplified by the model species Arabidopsis thaliana. Plant-specific peroxisome functions that have been uncovered only recently include, for instance, the participation of peroxisomes in phylloquinone and biotin biosynthesis. Experimental proteome studies have been proved very successful in defining the proteome of Arabidopsis peroxisomes but this approach also faces significant challenges and limitations. Complementary to experimental approaches, computational methods have emerged as important powerful tools to define the proteome of soluble matrix proteins of plant peroxisomes. Compared to other cell organelles such as mitochondria, plastids and the ER, the simultaneous operation of two major import pathways for soluble proteins in peroxisomes is rather atypical. Novel machine learning prediction approaches have been developed for peroxisome targeting signals type 1 (PTS1) and revealed high sensitivity and specificity, as validated by in vivo subcellular targeting analyses in diverse transient plant expression systems. Accordingly, the algorithms allow the correct prediction of many novel peroxisome-targeted proteins from plant genome sequences and the discovery of additional organelle functions. In contrast, the prediction of PTS2 proteins largely remains restricted to genome searches by conserved patterns contrary to more advanced machine learning methods. Here, we summarize and discuss the capabilities and accuracies of available prediction algorithms for PTS1 and PTS2 carrying proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Peroxissomos/química , Peroxissomos/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Genoma de Planta/genética , Peroxissomos/genética , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico , Proteoma/análise , Proteoma/genética
8.
Subcell Biochem ; 89: 157-199, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30378023

RESUMO

Peroxisomes harbor a plethora of proteins, but the peroxisomal proteome as the entirety of all peroxisomal proteins is still unknown for mammalian species. Computational algorithms can be used to predict the subcellular localization of proteins based on their amino acid sequence and this method has been amply used to forecast the intracellular fate of individual proteins. However, when applying such algorithms systematically to all proteins of an organism the prediction of its peroxisomal proteome in silico should be possible. Therefore, a reliable detection of peroxisomal targeting signals (PTS ) acting as postal codes for the intracellular distribution of the encoding protein is crucial. Peroxisomal proteins can utilize different routes to reach their destination depending on the type of PTS. Accordingly, independent prediction algorithms have been developed for each type of PTS, but only those for type-1 motifs (PTS1) have so far reached a satisfying predictive performance. This is partially due to the low number of peroxisomal proteins limiting the power of statistical analyses and partially due to specific properties of peroxisomal protein import, which render functional PTS motifs inactive in specific contexts. Moreover, the prediction of the peroxisomal proteome is limited by the high number of proteins encoded in mammalian genomes, which causes numerous false positive predictions even when using reliable algorithms and buries the few yet unidentified peroxisomal proteins. Thus, the application of prediction algorithms to identify all peroxisomal proteins is currently ineffective as stand-alone method, but can display its full potential when combined with other methods.


Assuntos
Mamíferos/metabolismo , Sinais de Orientação para Peroxissomos/fisiologia , Peroxissomos/metabolismo , Proteoma/química , Proteoma/metabolismo , Animais , Mamíferos/genética , Sinais de Orientação para Peroxissomos/genética , Peroxissomos/genética , Proteoma/genética
9.
Subcell Biochem ; 89: 201-219, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30378024

RESUMO

In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as a starting point, proteins are imported into organelles by specific targeting signals. Many proteins, however, act in more than one cellular compartment. In this chapter, we discuss mechanisms by which proteins can be targeted to multiple organelles with a focus on a novel gene regulatory mechanism, functional translational readthrough, that permits multiple targeting of proteins to the peroxisome and other organelles. In mammals, lactate and malate dehydrogenase are the best-characterized enzymes whose targeting is controlled by functional translational readthrough.


Assuntos
Peroxissomos/metabolismo , Biossíntese de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Animais , Citoplasma/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Peroxissomos/genética , Transporte Proteico/genética
10.
Subcell Biochem ; 89: 367-382, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30378032

RESUMO

Peroxisome proliferation involves signal recognition and computation by molecular networks that direct molecular events of gene expression, metabolism, membrane biogenesis, organelle proliferation, protein import, and organelle inheritance. Peroxisome biogenesis in yeast has served as a model system for exploring the regulatory networks controlling this process. Yeast is an outstanding model system to develop tools and approaches to study molecular networks and cellular responses and because the mechanisms of peroxisome biogenesis and key aspects of the transcriptional regulatory networks are remarkably conserved from yeast to humans. In this chapter, we focus on the complex regulatory networks that respond to environmental cues leading to peroxisome assembly and the molecular events of organelle assembly. Ultimately, understanding the mechanisms of the entire peroxisome biogenesis program holds promise for predictive modeling approaches and for guiding rational intervention strategies that could treat human conditions associated with peroxisome function.


Assuntos
Redes e Vias Metabólicas , Peroxissomos/metabolismo , Humanos , Modelos Biológicos , Peroxissomos/química , Peroxissomos/genética , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
11.
Int J Biochem Cell Biol ; 105: 24-34, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30268746

RESUMO

Peroxisomes are single membrane enclosed cell organelles, which are present in almost all eukaryotic cells. In addition to the common peroxisomal pathways such as ß-oxidation of fatty acids and decomposition of H2O2, these organelles fulfil a range of metabolic and non-metabolic functions. Peroxisomes are very important since various human disorders exist that are caused by a defect in peroxisome function. Here we describe our current knowledge on the molecular mechanisms of peroxisome biogenesis in yeast, including peroxisomal protein sorting, organelle dynamics and peroxisomal membrane contact sites.


Assuntos
Peroxissomos/metabolismo , Leveduras/genética , Leveduras/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Biogênese de Organelas , Peroxinas/genética , Peroxinas/metabolismo , Sinais de Orientação para Peroxissomos , Peroxissomos/genética , Peroxissomos/ultraestrutura , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/ultraestrutura
12.
Sci Rep ; 8(1): 16014, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375424

RESUMO

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.


Assuntos
Receptor 1 de Sinal de Orientação para Peroxissomos/química , Peroxissomos/química , Mapas de Interação de Proteínas/genética , Transporte Proteico/genética , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Sequência de Aminoácidos/genética , Animais , Química Click , Citosol/química , Citosol/metabolismo , Fibroblastos/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação , Receptor 2 de Sinal de Orientação para Peroxissomos/química , Receptor 2 de Sinal de Orientação para Peroxissomos/genética , Sinais de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/genética , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação/genética
13.
Rev. neurol. (Ed. impr.) ; 67(8): 298-302, 16 oct., 2018. ilus, tab
Artigo em Espanhol | IBECS | ID: ibc-175226

RESUMO

Introducción. Los trastornos de la biogénesis de los peroxisomas se deben a mutaciones en los genes PEX, que codifican peroxinas requeridas para la biogénesis peroxisómica. Clínicamente se expresan como un espectro del síndrome de Zellweger, y hay una amplia variedad fenotípica. Su diagnóstico se realiza bioquímicamente y la confirmación es molecular. El objetivo de este caso ilustrativo es resaltar la importancia de la clínica y de las pruebas bioquímicas en el abordaje de una enfermedad peroxisómica. Caso clínico. Niño de 3 años con hipotonía neonatal, retraso global del desarrollo y fallo de medro, con un patrón en resonancia cerebral de leucodistrofia hipomielinizante, en quien se había sospechado un trastorno de la biogénesis de los peroxisomas por encontrarse una variante de significado incierto en PEX5, pero su clínica, los estudios bioquímicos y el análisis crítico de las pruebas moleculares hacían improbable este diagnóstico. Se hace énfasis en el abordaje que debería tenerse cuando se sospecha un trastorno del espectro del síndrome de Zellweger. Conclusión. En el caso descrito se sospechó un trastorno de la biogénesis de los peroxisomas por una secuenciación exómica que, al analizarse críticamente junto con la clínica y los hallazgos bioquímicos, hacía muy poco probable una enfermedad peroxisómica. Cuando se tiene sospecha clínica y por neuroimágenes, el abordaje diagnóstico principal debe partir del análisis bioquímico. Aunque la confirmación es molecular, estas pruebas deben interpretarse con precaución


Introduction. 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. Case report. 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. Conclusion. 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


Assuntos
Humanos , Masculino , Pré-Escolar , Peroxissomos/genética , Bioquímica , Hipotonia Muscular/genética , Síndrome de Zellweger/diagnóstico , Transtornos Peroxissômicos/sangue , Transtornos Peroxissômicos/urina , Espectroscopia de Ressonância Magnética/métodos , Polimicrogiria/diagnóstico por imagem , Neuroimagem , Transtornos Peroxissômicos/diagnóstico
14.
PLoS One ; 13(9): e0203466, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30212482

RESUMO

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, ß-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells.


Assuntos
Brônquios/metabolismo , Regulação da Expressão Gênica , PPAR alfa/biossíntese , PPAR gama/deficiência , Peroxissomos/metabolismo , Enfisema Pulmonar/metabolismo , Anilidas/farmacologia , Animais , Brônquios/patologia , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , PPAR alfa/antagonistas & inibidores , PPAR alfa/genética , Peroxissomos/genética , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia
15.
FEMS Yeast Res ; 18(8)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30124827

RESUMO

We report the permanent introduction of the human peroxisomal ß-oxidation enzymatic machinery required for straight chain degradation of fatty acids into the yeast, Saccharomyces cerevisiae. Peroxisomal ß-oxidation encompasses four sequential reactions that are confined to three enzymes. The genes encoding human acyl-CoA oxidase 1, peroxisomal multifunctional enzyme type 2 and 3-ketoacyl-CoA thiolase were introduced into the genomic loci of their yeast gene equivalents. The human ß-oxidation genes were individually tagged with sequence coding for GFP and expression of the protein chimeras as well as their targeting to peroxisomes was confirmed. Functional complementation of the ß-oxidation pathway was assessed by growth on media containing fatty acids of different chain lengths. Yeast cells exhibited distinctive substrate specificities depending on whether they expressed the human or their endogenous ß-oxidation machinery. The genetic engineering of yeast to contain a 'humanized' organelle is a first step for the in vivo study of human peroxisome disorders in a model organism.


Assuntos
Ácidos Graxos/metabolismo , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Teste de Complementação Genética , Humanos , Organismos Geneticamente Modificados/enzimologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Oxirredução , Peroxissomos/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética
16.
J Cell Sci ; 131(17)2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30131444

RESUMO

The import of most of peroxisomal proteins into the lumen of their target organelle is driven by C-terminal (PTS1) or N-terminal (PTS2) signals recognized by the Pex5p or Pex7p receptors, respectively. However, some proteins in budding yeast, such as acyl-CoA oxidase (AOx) and carnitine acetyltransferase (Cat2p), are imported into peroxisomes via an alternative route that does not rely on known PTS signals and involves the Pex5p receptor N-terminal region. Here, we show that two other budding yeast peroxisomal proteins, a multifunctional enzyme from the ß-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal. The I264K amino acid substitution in Pex5p adjacent to its FxxxW diaromatic motif, previously shown to abolish the import of AOx and Cat2p into peroxisomes, also affects Fox2p and Cta1p import. Moreover, we demonstrate that Pex9p, a newly discovered paralog of Pex5p that was recently implicated in the import of malate synthases in budding yeast, also exhibits weak receptor activity towards Fox2p and Cta1p. These findings indicate the need to re-evaluate the peroxisomal import paradigm.This article has an associated First Person interview with the first author of the paper.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Enoil-CoA Hidratase/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , Motivos de Aminoácidos , Enoil-CoA Hidratase/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/genética , Domínios Proteicos , Transporte Proteico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
BMC Microbiol ; 18(1): 90, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30134836

RESUMO

BACKGROUND: Apple canker is a devastating branch disease caused by Valsa mali (Vm). The endophytic actinomycete Saccharothrix yanglingensis Hhs.015 (Sy Hhs.015) can effectively inhibit the growth of Vm. To reveal the mechanism, by which Vm respond to Sy Hhs.015, the transcriptome of Vm was analyzed using RNA-seq technology. RESULTS: Compared with normal growing Vm in the control group, 1476 genes were significantly differentially expressed in the Sy Hhs.015's treatment group, of which 851 genes were up-regulated and 625 genes were down-regulated. Combined gene function and pathway analysis of differentially expressed genes (DEGs) revealed that Sy Hhs.015 affected the carbohydrate metabolic pathway, which is utilized by Vm for energy production. Approximately 82% of the glycoside hydrolase genes were down-regulated, including three pectinase genes (PGs), which are key pathogenic factors. The cell wall structure of Vm was disrupted by Sy Hhs.015 and cell wall-related genes were found to be down-regulated. Of the peroxisome associated genes, those encoding catalase (CAT) and superoxide dismutase (SOD) which scavenge reactive oxygen species (ROS), as well as those encoding AMACR and ACAA1 which are related to the ß-oxidation of fatty acids, were down-regulated. MS and ICL, key genes in glyoxylate cycle, were also down-regulated. In response to the stress of Sy Hhs.015 exposure, Vm increased amino acid metabolism to synthesize the required nitrogenous compounds, while alpha-keto acids, which involved in the TCA cycle, could be used to produce energy by deamination or transamination. Retinol dehydrogenase, associated with cell wall dextran synthesis, and sterol 24-C-methyltransferase, related to cell membrane ergosterol synthesis, were up-regulated. The genes encoding glutathione S-transferase, (GST), which has antioxidant activity and ABC transporters which have an efflux function, were also up-regulated. CONCLUSION: These results show that the response of Vm to Sy Hhs.015 exposure is a complicated and highly regulated process, and provide a theoretical basis for both clarifying the biocontrol mechanism of Sy Hhs.015 and the response of Vm to stress.


Assuntos
Actinomycetales/fisiologia , Ascomicetos/genética , Ascomicetos/metabolismo , Agentes de Controle Biológico , Perfilação da Expressão Gênica/métodos , Transcriptoma , Aminoácidos/metabolismo , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/patogenicidade , Catalase/genética , Parede Celular/genética , Dextranos/metabolismo , Regulação para Baixo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Malus , Redes e Vias Metabólicas/genética , Oxirredutases/metabolismo , Peroxissomos/genética , Doenças das Plantas/microbiologia , Poligalacturonase/genética , Espécies Reativas de Oxigênio , Superóxido Dismutase/genética , Regulação para Cima
18.
Methods Mol Biol ; 1829: 87-109, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29987716

RESUMO

Plastids are organelles delineated by two envelopes that play important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by signaling molecules and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, the mitochondria, the plasma membrane, the peroxisomes and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still enigmatic. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.


Assuntos
Plastídeos/fisiologia , Transdução de Sinais , Transporte Biológico , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/fisiologia , Metabolismo Energético , Mitocôndrias/genética , Mitocôndrias/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Estresse Fisiológico
19.
Food Funct ; 9(8): 4340-4351, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30043014

RESUMO

Obesity is a medical condition with increasing prevalence, characterized by an accumulation of excess fat that could be improved using some bioactive compounds. However, many of these compounds with in vitro activity fail to respond in vivo, probably due to the sophistication of the physiological energy regulatory networks. In this context, C. elegans has emerged as a plausible model for the identification and characterization of the effect of such compounds on fat storage in a complete organism. However, the results obtained in such a simple model are not easily extrapolated to more complex organisms such as mammals, which hinders its application in the short term. Therefore, it is necessary to obtain new experimental data about the evolutionary conservation of the mechanisms of fat loss between worms and mammals. Previously, we found that some omega-6 fatty acids promote fat loss in C. elegans by up-regulation of peroxisomal fatty acid ß-oxidation in an omega-3 independent manner. In this work, we prove that the omega-6 fatty acids' effects on worms are also seen when they are supplemented with a natural omega-6 source (borage seed oil, BSO). Additionally, we explore the anti-obesity effects of two doses of BSO in a diet-induced obesity rat model, validating the up-regulation of peroxisomal fatty acid ß-oxidation. The supplementation with BSO significantly reduces body weight gain and energy efficiency and prevents white adipose tissue accumulation without affecting food intake. Moreover, BSO also increases serum HDL-cholesterol levels, improves insulin resistance and promotes the down-regulation of Cebpa, an adipogenesis-related gene. Therefore, we conclude that the effects of omega-6 fatty acids are highly conserved between worms and obesity-induced mammals, so these compounds could be considered to treat or prevent obesity-related disorders.


Assuntos
Borago/química , Caenorhabditis elegans/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Obesidade/dietoterapia , Peroxissomos/metabolismo , Óleos Vegetais/metabolismo , Ácido gama-Linolênico/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Borago/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Caenorhabditis elegans/genética , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Ômega-6/análise , Humanos , Masculino , Obesidade/genética , Obesidade/metabolismo , Oxirredução , Peroxissomos/genética , Óleos Vegetais/química , Ratos , Ratos Wistar , Ácido gama-Linolênico/química
20.
EMBO Rep ; 19(7)2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29895712

RESUMO

USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin-dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1-dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1-substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1- and Parkin-independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1-dependent and a PINK1-independent selective autophagy pathway.


Assuntos
Proteínas Mitocondriais/genética , Proteínas Quinases/genética , Tioléster Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Autofagia/genética , Linhagem Celular , Humanos , Mitocôndrias/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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