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1.
BMJ Case Rep ; 13(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31911407

RESUMO

A 66-year-old man presented with upper back cellulitis and imaging findings consistent with a necrotising soft tissue infection. He was started on broad-spectrum intravenous antibiotics and was taken to the operating room for immediate surgical debridement. On postoperative day 5, the culture was noted to be growing Gemella morbillorum, an exceedingly rare cause of necrotising soft tissue infections in immunocompetent hosts. His condition improved, and he was transitioned to oral antibiotics and discharged home.

2.
Autophagy ; : 1-15, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31964216

RESUMO

Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes in vitro and in vivo. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in Drosophila, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.

3.
Int J Mol Sci ; 21(2)2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31963200

RESUMO

The removal of damaged or superfluous organelles from the cytosol by selective autophagy is required to maintain organelle function, quality control and overall cellular homeostasis. Precisely how substrate selectivity is achieved, and how individual substrates are degraded during selective autophagy in response to both extracellular and intracellular cues is not well understood. The aim of this review is to highlight pexophagy, the autophagic degradation of peroxisomes, as a model for selective autophagy. Peroxisomes are dynamic organelles whose abundance is rapidly modulated in response to metabolic demands. Peroxisomes are routinely turned over by pexophagy for organelle quality control yet can also be degraded by pexophagy in response to external stimuli such as amino acid starvation or hypoxia. This review discusses the molecular machinery and regulatory mechanisms governing substrate selectivity during both quality-control pexophagy and pexophagy in response to external stimuli, in yeast and mammalian systems. We draw lessons from pexophagy to infer how the cell may coordinate the degradation of individual substrates by selective autophagy across different cellular cues.

4.
Int J Surg Case Rep ; 63: 89-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31574456

RESUMO

INTRODUCTION: Midgut malrotation results from abnormalities in the 270-degree counterclockwise rotation of the midgut around the axis of the superior mesenteric artery during embryological development, and classically presents early in life with symptoms of intestinal obstruction. Nevertheless, adult cases have occasionally been reported. PRESENTATION OF CASE: An 80-year-old female with no surgical history was brought to our emergency department for acutely altered mental status. On exam, her abdomen was distended and diffusely tender to palpation. Computed tomography (CT) scan of the abdomen and pelvis showed a dilated loop of jejunum with evidence of mesenteric twist concerning for closed-loop small bowel obstruction. The patient was taken for exploratory laparotomy and was found to have Ladd bands and other findings suggestive of intestinal malrotation. A Ladd procedure was performed and the patient remained under observation. She experienced intermittent abdominal distension and bilious nasogastric tube output, but subsequent CT scans revealed no evidence of obstruction. She was discharged following clinical improvement and ability to tolerate a diet. DISCUSSION: Malrotation of the small bowel exists on a spectrum depending on the embryologic stage during which anomalous rotation occurs. Classic findings on CT imaging (including abnormal mesenteric vasculature, right-sided duodenojejunal junction, whirlpool signs, and left-sided ascending colon) can provide clues to the existence of malrotation. CONCLUSION: Although malrotation is rare in adults, clinical and radiologic findings play an important role in the correct diagnosis of adult malrotation for appropriate and timely intervention.

6.
World J Emerg Surg ; 14: 8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30858872

RESUMO

In the last three decades, Clostridium difficile infection (CDI) has increased in incidence and severity in many countries worldwide. The increase in CDI incidence has been particularly apparent among surgical patients. Therefore, prevention of CDI and optimization of management in the surgical patient are paramount. An international multidisciplinary panel of experts from the World Society of Emergency Surgery (WSES) updated its guidelines for management of CDI in surgical patients according to the most recent available literature. The update includes recent changes introduced in the management of this infection.


Assuntos
Infecções por Clostridium/terapia , Clostridium difficile/patogenicidade , Complicações Pós-Operatórias/terapia , Antibacterianos/uso terapêutico , Gestão de Antimicrobianos , Infecções por Clostridium/diagnóstico , Enterocolite Pseudomembranosa/etiologia , Enterocolite Pseudomembranosa/prevenção & controle , Transplante de Microbiota Fecal/métodos , Transplante de Microbiota Fecal/tendências , Guias como Assunto , Humanos , Incidência , Controle de Infecções/métodos , Controle de Infecções/tendências , Fatores de Risco
7.
Cytoskeleton (Hoboken) ; 76(1): 63-72, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30176126

RESUMO

Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes, including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified Saccharomyces cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a direct stochastic optical reconstruction microscopy and photoactivated localization microscopy study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero-octamers and an approximate 30-35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9.


Assuntos
Septinas/metabolismo , Animais , Mamíferos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Nat Microbiol ; 3(12): 1472-1485, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30478389

RESUMO

Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Animais , Autofagia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Colesterol/metabolismo , Modelos Animais de Doenças , Exocitose , Células HeLa , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Humanos , Listeria monocytogenes/metabolismo , Lisossomos , Masculino , Camundongos
9.
J Biol Chem ; 293(42): 16464-16478, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30201609

RESUMO

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , Alvo Mecanístico do Complexo 2 de Rapamicina/fisiologia , Inanição/metabolismo , Acetilglucosamina/biossíntese , Animais , Vias Biossintéticas , Humanos , Fosforilação , Serina/metabolismo , Uridina Difosfato N-Acetilglicosamina/biossíntese
10.
J Biol Chem ; 293(38): 14723-14739, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30061153

RESUMO

Glycogen synthase kinase 3ß (GSK3ß) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3ß substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3ß function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3ß from the cytosol to the nucleus and to a GSK3ß-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3ß nuclear localization in an mTORC1-dependent manner. GSK3ß was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3ß from lysosomes and in enhanced GSK3ß nuclear localization as well as GSK3ß-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3ß is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3ß-dependent regulation of transcriptional networks controlling cellular biomass production.


Assuntos
Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Endossomos/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina/metabolismo
11.
Mol Cell Proteomics ; 17(11): 2242-2255, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30037810

RESUMO

Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.


Assuntos
Organelas/virologia , Mapas de Interação de Proteínas , Zika virus/fisiologia , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Peroxissomos/metabolismo , Proteínas Virais/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia
12.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(4): 447-457, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29343430

RESUMO

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes.


Assuntos
Cardiolipinas/biossíntese , Cardiolipinas/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Detergentes/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Microscopia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos
13.
J Cell Sci ; 130(15): 2579-2590, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600323

RESUMO

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.


Assuntos
Endossomos/metabolismo , Lipoilação , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endossomos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/genética
14.
Autophagy ; 13(5): 868-884, 2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28521612

RESUMO

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Autofagia/fisiologia , Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Mutação/genética , Transtornos Peroxissômicos/genética , Transporte Proteico/fisiologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo
15.
J Cell Biol ; 216(2): 367-377, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28108526

RESUMO

Lipid exchange between the endoplasmic reticulum (ER) and peroxisomes is necessary for the synthesis and catabolism of lipids, the trafficking of cholesterol, and peroxisome biogenesis in mammalian cells. However, how lipids are exchanged between these two organelles is not understood. In this study, we report that the ER-resident VAMP-associated proteins A and B (VAPA and VAPB) interact with the peroxisomal membrane protein acyl-CoA binding domain containing 5 (ACBD5) and that this interaction is required to tether the two organelles together, thereby facilitating the lipid exchange between them. Depletion of either ACBD5 or VAP expression results in increased peroxisome mobility, suggesting that VAP-ACBD5 complex acts as the primary ER-peroxisome tether. We also demonstrate that tethering of peroxisomes to the ER is necessary for peroxisome growth, the synthesis of plasmalogen phospholipids, and the maintenance of cellular cholesterol levels. Collectively, our data highlight the importance of VAP-ACBD5-mediated contact between the ER and peroxisomes for organelle maintenance and lipid homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Homeostase , Humanos , Proteínas de Membrana/genética , Microscopia Confocal , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas de Transporte Vesicular/genética
16.
Mol Cell ; 63(5): 811-26, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27570073

RESUMO

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis.


Assuntos
Fibroblastos/metabolismo , Hexosaminas/biossíntese , Complexos Multiproteicos/metabolismo , Transferases de Grupos Nitrogenados/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Fibroblastos/citologia , Regulação da Expressão Gênica , Glucose/metabolismo , Glutamina/metabolismo , Células HeLa , Homeostase , Humanos , Ácidos Cetoglutáricos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Metaboloma/genética , Metabolômica , Camundongos , Complexos Multiproteicos/genética , Transferases de Grupos Nitrogenados/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Proteína 1 de Ligação a X-Box/genética
17.
J Hepatol ; 65(6): 1198-1208, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27312946

RESUMO

BACKGROUND & AIMS: Severe malnutrition in young children is associated with signs of hepatic dysfunction such as steatosis and hypoalbuminemia, but its etiology is unknown. Peroxisomes and mitochondria play key roles in various hepatic metabolic functions including lipid metabolism and energy production. To investigate the involvement of these organelles in the mechanisms underlying malnutrition-induced hepatic dysfunction we developed a rat model of malnutrition. METHODS: Weanling rats were placed on a low protein or control diet (5% or 20% of calories from protein, respectively) for four weeks. Peroxisomal and mitochondrial structural features were characterized using immunofluorescence and electron microscopy. Mitochondrial function was assessed using high-resolution respirometry. A novel targeted quantitative proteomics method was applied to analyze 47 mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle and fatty acid ß-oxidation pathways. RESULTS: Low protein diet-fed rats developed hypoalbuminemia and hepatic steatosis, consistent with the human phenotype. Hepatic peroxisome content was decreased and metabolomic analysis indicated peroxisomal dysfunction. This was followed by changes in mitochondrial ultrastructure and increased mitochondrial content. Mitochondrial function was impaired due to multiple defects affecting respiratory chain complex I and IV, pyruvate uptake and several ß-oxidation enzymes, leading to strongly reduced hepatic ATP levels. Fenofibrate supplementation restored hepatic peroxisome abundance and increased mitochondrial ß-oxidation capacity, resulting in reduced steatosis and normalization of ATP and plasma albumin levels. CONCLUSIONS: Malnutrition leads to severe impairments in hepatic peroxisomal and mitochondrial function, and hepatic metabolic dysfunction. We discuss the potential future implications of our findings for the clinical management of malnourished children. LAY SUMMARY: Severe malnutrition in children is associated with metabolic disturbances that are poorly understood. In order to study this further, we developed a malnutrition animal model and found that severe malnutrition leads to an impaired function of liver mitochondria which are essential for energy production and a loss of peroxisomes, which are important for normal liver metabolic function.


Assuntos
Desnutrição , Trifosfato de Adenosina , Animais , Criança , Fígado Gorduroso , Humanos , Fígado , Mitocôndrias , Oxirredução , Ratos
18.
Biochem J ; 473(4): 509-23, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26635352

RESUMO

Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Células Cultivadas , Insulina/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fosforilação , Conformação Proteica , Proteína Quinase C/química , Serina-Treonina Quinases TOR/metabolismo
19.
Biochim Biophys Acta ; 1863(5): 881-91, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26408931

RESUMO

Peroxisomes are dynamic organelles that can adjust their size and number in response to cellular demand and environmental stimuli. They can propagate from pre-existing peroxisomes through growth and division, as well as de novo from the endoplasmic reticulum (ER). However, to what extend that these two distinct peroxisome biogenesis pathways are involved in maintaining peroxisome numbers in cycling cells is unclear. Recent studies in yeast suggest that the ER plays a direct role in the maintenance of peroxisomes. However, the role of the ER in mammalian system is under debate. In this review, we outline the recent progress in understanding the biogenesis of mammalian peroxisomes. We herein discuss some of the discrepancies in the literature and the outstanding questions in the field.


Assuntos
Retículo Endoplasmático/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Animais , Retículo Endoplasmático/química , Regulação da Expressão Gênica , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/química , Mitocôndrias/metabolismo , Biogênese de Organelas , Peroxinas , Peroxissomos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais
20.
Autophagy ; 11(4): 595-606, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25915564

RESUMO

The selective degradation of mitochondria by the process of autophagy, termed mitophagy, is one of the major mechanisms of mitochondrial quality control. The best-studied mitophagy pathway is the one mediated by PINK1 and PARK2/Parkin. From recent studies it has become clear that ubiquitin-ligation plays a pivotal role and most of the focus has been on the role of ubiquitination of mitochondrial proteins in mitophagy. Even though ubiquitination is a reversible process, very little is known about the role of deubiquitinating enzymes (DUBs) in mitophagy. Here, we report that 2 mitochondrial DUBs, USP30 and USP35, regulate PARK2-mediated mitophagy. We show that USP30 and USP35 can delay PARK2-mediated mitophagy using a quantitative mitophagy assay. Furthermore, we show that USP30 delays mitophagy by delaying PARK2 recruitment to the mitochondria during mitophagy. USP35 does not delay PARK2 recruitment, suggesting that it regulates mitophagy through an alternative mechanism. Interestingly, USP35 only associates with polarized mitochondria, and rapidly translocates to the cytosol during CCCP-induced mitophagy. It is clear that PARK2-mediated mitophagy is regulated at many steps in this important quality control pathway. Taken together, these findings demonstrate an important role of mitochondrial-associated DUBs in mitophagy. Because defects in mitochondria quality control are implicated in many neurodegenerative disorders, our study provides clear rationales for the design and development of drugs for the therapeutic treatment of neurodegenerative diseases such as Parkinson and Alzheimer diseases.


Assuntos
Autofagia/fisiologia , Endopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Citosol/metabolismo , Humanos , Ubiquitina/metabolismo
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