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1.
Nat Commun ; 11(1): 5267, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077711

RESUMO

Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Estresse Oxidativo , Peroxissomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ataxia/genética , Ataxia/metabolismo , Ataxia/fisiopatologia , Transporte Biológico , Linhagem Celular , Proteínas de Choque Térmico HSC70/genética , Humanos , Macroautofagia , Peroxissomos/genética , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Nature ; 585(7826): 603-608, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939090

RESUMO

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.


Assuntos
Éteres/metabolismo , Ferroptose , Peroxissomos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Éteres/química , Feminino , Edição de Genes , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Peroxidação de Lipídeos , Masculino , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Peroxissomos/genética
3.
Proc Natl Acad Sci U S A ; 117(26): 14970-14977, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541053

RESUMO

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Transporte Proteico
4.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118709, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32224193

RESUMO

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/metabolismo
5.
Sci Rep ; 10(1): 3735, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111926

RESUMO

Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.


Assuntos
Autofagia , Células da Medula Óssea/metabolismo , Senescência Celular , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/citologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , MicroRNAs/genética , Peroxissomos/genética , Peroxissomos/metabolismo
6.
mSphere ; 5(1)2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075879

RESUMO

Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous.IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.


Assuntos
Microcorpos/metabolismo , Peroxinas/metabolismo , Peroxissomos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Citosol/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peroxinas/genética , Peroxissomos/genética , Transporte Proteico , Proteínas de Protozoários/genética
7.
J Agric Food Chem ; 68(5): 1382-1389, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31944688

RESUMO

Metabolic engineering of Saccharomyces cerevisiae focusing on the cytoplasm for sustainable terpenoid production is commonly practiced. However, engineering organelles for terpenoid production is rarely reported. Herein, peroxisomes, together with the cytoplasm, were engineered to boost sesquiterpene α-humulene synthesis in S. cerevisiae. The farnesyl diphosphate synthetic pathway and α-humulene synthase were successfully expressed inside yeast peroxisomes to enable high-level α-humulene production with glucose as the sole carbon source. With the combination of peroxisomal and cytoplasmic engineering, α-humulene production was increased by 2.5-fold compared to that in cytoplasm-engineered recombinant strains. Finally, the α-humulene titer of 1726.78 mg/L was achieved by fed-batch fermentation in a 5 L bioreactor. The strategy presented here offers an efficient method for terpenoid production in S. cerevisiae.


Assuntos
Acetilcoenzima A/metabolismo , Citosol/metabolismo , Sesquiterpenos Monocíclicos/metabolismo , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica , Peroxissomos/genética , Sesquiterpenos/metabolismo
8.
Nat Commun ; 11(1): 578, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996685

RESUMO

Lipid droplets (LDs) are key subcellular organelles for regulating lipid metabolism. Although several subcellular organelles participate in lipid metabolism, it remains elusive whether physical contacts between subcellular organelles and LDs might be involved in lipolysis upon nutritional deprivation. Here, we demonstrate that peroxisomes and peroxisomal protein PEX5 mediate fasting-induced lipolysis by stimulating adipose triglyceride lipase (ATGL) translocation onto LDs. During fasting, physical contacts between peroxisomes and LDs are increased by KIFC3-dependent movement of peroxisomes toward LDs, which facilitates spatial translocations of ATGL onto LDs. In addition, PEX5 could escort ATGL to contact points between peroxisomes and LDs in the presence of fasting cues. Moreover, in adipocyte-specific PEX5-knockout mice, the recruitment of ATGL onto LDs was defective and fasting-induced lipolysis is attenuated. Collectively, these data suggest that physical contacts between peroxisomes and LDs are required for spatiotemporal translocation of ATGL, which is escorted by PEX5 upon fasting, to maintain energy homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Jejum/efeitos adversos , Gotículas Lipídicas/metabolismo , Lipólise/fisiologia , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Análise Espaço-Temporal , Células 3T3-L1/metabolismo , Adipócitos/metabolismo , Animais , Caenorhabditis elegans , Sinais (Psicologia) , Citoesqueleto , Cinesina/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nutrientes , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/genética , Transdução de Sinais
9.
Proc Natl Acad Sci U S A ; 117(4): 2065-2075, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932444

RESUMO

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either ß-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.


Assuntos
Archamoebae/metabolismo , Peroxissomos/metabolismo , Anaerobiose , Archamoebae/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Peroxinas/genética , Peroxinas/metabolismo , Peroxissomos/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
G3 (Bethesda) ; 10(1): 69-77, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31767637

RESUMO

Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, we conducted a genetic screen on a collection of lethal mutations on the X chromosome in Drosophila Using the number, size and morphology of GFP tagged peroxisomes as a readout, we screened for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, we identified eighteen genes that cause increases in peroxisome number or altered morphology when mutated. We examined the human homologs of these genes and found that they are involved in a diverse array of cellular processes. Interestingly, the human homologs from the X-chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This in vivo screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in vivo in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes.


Assuntos
Genes de Insetos , Peroxissomos/genética , Homologia de Sequência do Ácido Nucleico , Síndrome de Zellweger/genética , Animais , Drosophila melanogaster , Humanos , Mutação , Peroxissomos/patologia , Cromossomo X/genética
11.
Folia Microbiol (Praha) ; 65(2): 423-429, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31273644

RESUMO

Baker's yeast is a valuable model system for the study of biological aging as it can be utilized for the measurement of replicative and chronological life spans in response to interventions. Whereas replicative aging in Saccharomyces cerevisiae mirrors dividing mammalian cells, chronological aging is seen in non-dividing cells. Aging is strongly influenced by the cellular organelles, especially by mitochondria which house essential functions like oxidative phosphorylation. Additionally, peroxisomes were shown to modulate the aging process, mainly by their turnover of reactive oxygen species. There is a fundamental interest in understanding how mitochondria and peroxisomes contribute to cellular aging. This work analyzes chronological aging in yeast mutants that are affected in peroxisomal proliferation and inheritance. Deletion of INP1 (retention of peroxisomes in the mother cell) or PEX11 (division of peroxisomes) leads to clearly reduced life spans compared to the wild-type control under conditions which depend on peroxisomal metabolism. Δinp1 cells are long-lived in contrast to the wild type and Δpex11 when assayed under conditions that not necessitate peroxisome function. Neither treatment affects the index of respiratory capacity, indicating fully functional mitochondria. Evaluation of stress resistances reveals that Δinp1 has significantly higher resistance to the apoptosis elicitor acetic acid. Old Δpex11 cells from an oleate culture are more susceptible to hydrogen peroxide treatment compared to Δinp1 and the wild type. Finally, aged cells are hyper-sensitive to heat shock treatment in contrast to young cells.


Assuntos
Proteínas de Membrana/genética , Peroxinas/genética , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proliferação de Células , Deleção de Genes , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Peroxinas/metabolismo , Peroxissomos/genética , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Biochem J ; 476(22): 3521-3532, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31688904

RESUMO

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the ß-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the ß-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate ß-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the ß-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m-2 s-1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Coenzima A Ligases/metabolismo , Peroxissomos/metabolismo , Ubiquinona/análogos & derivados , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Coenzima A Ligases/genética , Regulação da Expressão Gênica de Plantas , Estrutura Molecular , Oxirredução , Peroxissomos/química , Peroxissomos/genética , Ubiquinona/biossíntese , Ubiquinona/química
13.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31614978

RESUMO

TGF-ß/Smad signaling is a major pathway in progressive fibrotic processes, and further studies on the molecular mechanisms of TGF-ß/Smad signaling are still needed for their therapeutic targeting. Recently, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) was shown to improve renal fibrosis, making it an attractive target for chronic kidney diseases (CKDs). Here, we show the mechanism by which PGC-1α regulates the TGF-ß/Smad signaling pathway using HK-2 cell lines stably overexpressing empty vector (mock cells) or human PGC1α (PGC1α cells). Stable PGC-1α overexpression negatively regulated the expression of TGF-ß-induced epithelial-mesenchymal transition (EMT) markers (fibronectin, E-cadherin, vimentin, and α-SMA) and EMT-related transcription factors (Snail and Slug) compared to mock cells, inhibiting fibrotic progression. Interestingly, among molecules upstream of Smad2/3 activation, the gene expression of only TGFßRI, but not TGFßRII, was downregulated in PGC-1α cells. In addition, the downregulation of TGFßRI by PGC-1α was associated with the upregulation of let-7b/c, miRNA for which the 3' untranslated region (UTR) of TGFßRI contains a binding site. In conclusion, PGC-1α suppresses TGF-ß/Smad signaling activation via targeting TGFßRI downregulation by let-7b/c upregulation.


Assuntos
MicroRNAs/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Insuficiência Renal Crônica/genética , Sítios de Ligação/genética , Linhagem Celular , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/genética , Humanos , Peroxissomos/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Insuficiência Renal Crônica/patologia , Transdução de Sinais , Proteínas Smad/genética , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta/genética
14.
Sci Rep ; 9(1): 14485, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597940

RESUMO

The endoplasmic reticulum (ER) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. The SEC59 gene encodes dolichol kinase, required for protein glycosylation in the ER. The mutation of sec59-1 caused a protein N-glycosylation defect mediated ER stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced. In the sec59-1∆ cell, the N-glycosylation of vacuolar carboxy peptidase-Y (CPY) was significantly reduced; whereas the ER stress marker Kar2p and unfolded protein response (UPR) were significantly increased. Increased levels of Triacylglycerol (TAG), sterol ester (SE), and lipid droplets (LD) could be attributed to up-regulation of DPP1, LRO1, and ARE2 in the sec 59-1∆ cell. Also, the diacylglycerol (DAG), sterol (STE), and free fatty acids (FFA) levels were significantly increased, whereas the genes involved in peroxisome biogenesis and Pex3-EGFP levels were reduced when compared to the wild-type. The microarray data also revealed increased expression of genes involved in phospholipid, TAG, fatty acid, sterol synthesis, and phospholipid transport resulting in dysregulation of lipid homeostasis in the sec59-1∆ cell. We conclude that SEC59 dependent N-glycosylation is required for lipid homeostasis, peroxisome biogenesis, and ER protein quality control.


Assuntos
Metabolismo dos Lipídeos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Glicosilação , Metabolismo dos Lipídeos/genética , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Mutação , Peroxissomos/genética , Peroxissomos/metabolismo , Fosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Esteróis/metabolismo , Resposta a Proteínas não Dobradas/genética
15.
Antioxid Redox Signal ; 31(18): 1339-1351, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31530170

RESUMO

Aims: Peroxisome is a critical organelle for fatty acid oxidation (FAO) and metabolism of reactive oxygen species (ROS). Increased oxidative stress in adipose tissue contributes to the development of insulin resistance and metabolic syndrome in obesity. This study aimed to investigate the role of peroxisomal fitness in maintaining adipocyte function, which has been under-rated in the obesity research area. Results: Reduced peroxisomal gene expressions in white adipose tissue (WAT) of obese mice suggested a close correlation between peroxisomes and obesity. Peroxisomal biogenesis factor 5 siRNA increased cellular ROS and inflammatory mediators in 3T3-L1 adipocytes. On the contrary, hydrogen peroxide or tumor necrosis factor-α treatment significantly decreased biogenesis- and function-related peroxisomal proteins, suggesting a positive feedback loop of ROS/inflammation and peroxisomal dysfunction. Correspondingly, catalase (a major peroxisomal antioxidant)-knockout mice fed with high-fat diet (HFD) exhibited suppressed peroxisomal proteins along with increased oxidative stress and accelerated obesity. In response to fenofibrate (a peroxisomal proliferator) treatment, WAT of HFD-fed wild-type mice showed not only increases in peroxisomal biogenesis and FAO but also attenuated features of adipocyte dysfunction and obesity. However, these results were not observed in peroxisome proliferator-activated receptor-alpha null obese mice. Innovation: Impaired peroxisomal fitness enhanced oxidative stress and inflammation in adipocytes, which exacerbates obesity. Conclusion: Adipose tissue peroxisomal homeostasis plays an important role in attenuating the features of obesity, and it can be a potential therapeutic target of obesity.


Assuntos
Adipócitos/metabolismo , Estresse Oxidativo , Peroxissomos/metabolismo , Células 3T3-L1 , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/análise , Diferenciação Celular , Células Cultivadas , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Peroxissomos/genética , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo
16.
Fungal Genet Biol ; 132: 103259, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31394175

RESUMO

In an in silico search for correlated gene loss with fungal peroxisomal uric acid oxidase (UOX), we identified PMP22-like proteins, some of which function as promiscuous channels in organellar membranes. To investigate whether PMP22 channels have a role in peroxisomal uric acid transport and catabolism, we functionally analyzed the closest homologue in Aspergillus nidulans, named SspA. We confirmed that SspA is a peroxisomal membrane protein that co-localizes significantly with PTS1-tagged mRFP, UOX or HexA, the latter considered a protein of Woronin bodies (WB), organelles originating from peroxisomes that dynamically plug septal pores in ascomycetes. Our results suggest that in A. nidulans, unlike some other ascomycetes, there is no strict protein segregation of peroxisomal and WB-specific proteins. Importantly, genetic deletion of sspA, but not of hexA, led to lack of peroxisomal localization at septal pores, suggesting that SspA is a key factor for septal pore functioning. Additionally, ΔsspA resulted in increased sensitivity to oxidative stress, apparently as a consequence of not only the inability to plug septal pores, but also a recorded reduction in peroxisome biogenesis. However, deleting sspA had no effect on uric acid or purine utilization, as we hypothesized, a result also in line with the observation that expression of SspA was not affected by regulatory mutants and conditions known to control purine catabolic enzymes. Our results are discussed within the framework of previous studies of SspA homologues in other fungi, as well as, the observed gene losses of PMP22 and peroxisomal uric acid oxidase.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Peroxissomos/metabolismo , Purinas/metabolismo , Deleção de Genes , Peroxissomos/genética , Ácido Úrico/metabolismo
17.
J Inherit Metab Dis ; 42(5): 955-965, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31150129

RESUMO

Patients with a Zellweger spectrum disorder (ZSD) have a defect in the assembly or maintenance of peroxisomes, leading to a multisystem disease with variable outcome. Liver disease is an important feature in patients with severe and milder phenotypes and a frequent cause of death. However, the course and histology of liver disease in ZSD patients are ill-defined. We reviewed the hepatic symptoms and histological findings of 13 patients with a ZSD in which one or several liver biopsies have been performed (patient age 0.2-39 years). All patients had at least some histological liver abnormalities, ranging from minor fibrosis to cirrhosis. Five patients demonstrated significant disease progression with liver failure and early death. In others, liver-related symptoms were absent, although some still silently developed cirrhosis. Patients with peroxisomal mosaicism had a better prognosis. In addition, we show that patients are at risk to develop a hepatocellular carcinoma (HCC), as one patient developed a HCC at the age of 36 years and one patient a precancerous lesion at the age of 18 years. Thus, regular examination to detect fibrosis or cirrhosis should be included in the standard care of ZSD patients. In case of advanced fibrosis/cirrhosis expert consultation and HCC screening should be initiated. This study further delineates the spectrum and significance of liver involvement in ZSDs.


Assuntos
Carcinoma Hepatocelular/etiologia , Cirrose Hepática/etiologia , Neoplasias Hepáticas/etiologia , Fígado/patologia , Síndrome de Zellweger/complicações , Adolescente , Adulto , Carcinoma Hepatocelular/patologia , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Lactente , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Países Baixos , Peroxissomos/genética , Síndrome de Zellweger/genética
18.
Metabolism ; 98: 53-61, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31226353

RESUMO

Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM). AIMS: We hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply. MATERIALS AND METHODS: HSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene (PEX) expression were assessed using western blotting and realtime qRT-PCR respectively. 1-14C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively. RESULTS: 1) Under fasting conditions, HSkM tissue demonstrated a significant correlation (P ≪ 0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes (Pex19, PEX11A, and PEX5) were significantly (P ≪ 0.05) upregulated. 3) Increments in PMP70, carnitine octanoyl transferase (CrOT), PGC-1α, and ERRα mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly (P ≪ 0.05) elevated 48-h post lipid treatment. CONCLUSIONS: These results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue.


Assuntos
Dieta Hiperlipídica , Expressão Gênica/fisiologia , Músculo Esquelético/metabolismo , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Biópsia , Ácidos Graxos/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Peroxissomos/genética , Cultura Primária de Células , Adulto Jovem
19.
Plant J ; 99(6): 1144-1158, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31108001

RESUMO

Although peroxisomes play a key role in plant metabolism under both normal and stressful growth conditions, the impact of drought and heat stress on the peroxisomes remains unknown. Quinoa represents an informative system for dissecting the impact of abiotic stress on peroxisome proliferation because it is adapted to marginal environments. Here we determined the correlation of peroxisome abundance with physiological responses and yield under heat, drought and heat plus drought stresses in eight genotypes of quinoa. We found that all stresses caused a reduction in stomatal conductance and yield. Furthermore, H2 O2 content increased under drought and heat plus drought. Principal component analysis demonstrated that peroxisome abundance correlated positively with H2 O2 content in leaves and correlated negatively with yield. Pearson correlation coefficient for yield and peroxisome abundance (r = -0.59) was higher than for commonly used photosynthetic efficiency (r = 0.23), but comparable to those for classical stress indicators such as soil moisture content (r = 0.51) or stomatal conductance (r = 0.62). Our work established peroxisome abundance as a cellular sensor for measuring responses to heat and drought stress in the genetically diverse populations. As heat waves threaten agricultural productivity in arid climates, our findings will facilitate identification of genetic markers for improving yield of crops under extreme weather patterns.


Assuntos
Chenopodium quinoa/genética , Chenopodium quinoa/metabolismo , Resposta ao Choque Térmico/fisiologia , Peroxissomos/metabolismo , Produtos Agrícolas/metabolismo , Secas , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Peroxissomos/genética , Fotossíntese/genética , Fotossíntese/fisiologia , Filogenia , Estômatos de Plantas/metabolismo
20.
Curr Genet ; 65(6): 1383-1396, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31111312

RESUMO

Peroxisomes are ubiquitous single-membrane-bound organelles that perform a variety of biochemical functions in eukaryotic cells. Proteins involved in peroxisomal biogenesis are collectively called peroxins. Currently, functions of most peroxins in phytopathogenic fungi are poorly understood. Here, we report identification of PEX1 and PEX10 in the phytopathogenic fungus, Fusarium graminearum, namely FgPEX1 and FgPEX10, the orthologs of yeast ScPEX1 and ScPEX10. To functionally characterize FgPEX1 and FgPEX10, we constructed deletion mutants of FgPEX1 and FgPEX10 (ΔPEX1 and ΔPEX10) by targeting gene-replacement strategies. Our data demonstrate that both mutants displayed reduced mycelial growth, conidiation, and production of perithecia. Deletion of FgPEX1 and FgPEX10 resulted in a shortage of acetyl-CoA, which is an important reason for the reduced deoxynivalenol production and inhibited virulence of F. graminearum. Moreover, ΔPEX1 and ΔPEX10 showed an increased accumulation of lipid droplets and endogenous reactive oxygen species. In addition, FgPEX1 and FgPEX10 were found to be involved in the maintenance of cell wall integrity and Woronin bodies.


Assuntos
Proteínas Fúngicas/fisiologia , Fusarium/genética , Fusarium/patogenicidade , Peroxinas/fisiologia , Peroxissomos/ultraestrutura , ATPases Associadas a Diversas Atividades Celulares/genética , Acetilcoenzima A/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Fusarium/citologia , Fusarium/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Peroxinas/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/fisiologia , Tricotecenos/metabolismo , Virulência/genética
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