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1.
Cell Death Dis ; 13(11): 947, 2022 11 10.
Article in English | MEDLINE | ID: mdl-36357363

ABSTRACT

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.


Subject(s)
Mitophagy , Parkinson Disease , Humans , Mitophagy/physiology , Ubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Parkinson Disease/metabolism , Mitochondria/metabolism , Ubiquitin-Protein Ligases/metabolism , Autophagy/physiology
2.
PLoS One ; 11(10): e0164864, 2016.
Article in English | MEDLINE | ID: mdl-27768726

ABSTRACT

The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies.


Subject(s)
Adenosine Triphosphatases/physiology , Autophagy , Cell Cycle Proteins/physiology , Frontotemporal Dementia/genetics , Lysosomes/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Mutation , Myositis, Inclusion Body/genetics , Osteitis Deformans/genetics , Animals , Cell Line, Tumor , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Valosin Containing Protein
3.
N Engl J Med ; 370(2): 129-38, 2014 Jan 09.
Article in English | MEDLINE | ID: mdl-24401050

ABSTRACT

BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).


Subject(s)
Fanconi Syndrome/genetics , Kidney Tubules, Proximal/metabolism , Mitochondria/metabolism , Mutation, Missense , Peroxisomal Bifunctional Enzyme/genetics , Amino Acid Sequence , Animals , Black People , Chromosomes, Human, Pair 3 , Disease Models, Animal , Fanconi Syndrome/ethnology , Female , Genetic Linkage , Humans , Male , Mice , Mice, Knockout , Molecular Sequence Data , Pedigree , Peroxisomal Bifunctional Enzyme/chemistry , Peroxisomal Bifunctional Enzyme/metabolism , Phenotype , Sequence Analysis, DNA
4.
Biochim Biophys Acta ; 1807(6): 719-25, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21237131

ABSTRACT

Mitochondria are signal-integrating organelles involved in cell death induction. Mitochondrial alterations and reduction in energy metabolism have been previously reported in the context of glucocorticoid (GC)-triggered apoptosis, although the mechanism is not yet clarified. We analyzed mitochondrial function in a GC-sensitive precursor B-cell acute lymphoblastic leukemia (ALL) model as well as in GC-sensitive and GC-resistant T-ALL model systems. Respiratory activity was preserved in intact GC-sensitive cells up to 24h under treatment with 100 nM dexamethasone before depression of mitochondrial respiration occurred. Severe repression of mitochondrial respiratory function was observed after permeabilization of the cell membrane and provision of exogenous substrates. Several mitochondrial metabolite and protein transporters and two subunits of the ATP synthase were downregulated in the T-ALL and in the precursor B-ALL model at the gene expression level under dexamethasone treatment. These data could partly be confirmed in ALL lymphoblasts from patients, dependent on the molecular abnormality in the ALL cells. GC-resistant cell lines did not show any of these defects after dexamethasone treatment. In conclusion, in GC-sensitive ALL cells, dexamethasone induces changes in membrane properties that together with the reduced expression of mitochondrial transporters of substrates and proteins may lead to repressed mitochondrial respiratory activity and lower ATP levels that contribute to GC-induced apoptosis.


Subject(s)
Glucocorticoids/adverse effects , Mitochondrial Membranes/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Antineoplastic Agents, Hormonal/adverse effects , Antineoplastic Agents, Hormonal/pharmacology , Cell Line, Tumor , Cell Respiration/drug effects , Cell Respiration/genetics , Dexamethasone/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Leukemic/drug effects , Glucocorticoids/pharmacology , Humans , Microarray Analysis , Mitochondria/drug effects , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/physiology , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/pathology , Mitochondrial Membranes/physiology , Oxygen Consumption/drug effects , Oxygen Consumption/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
5.
J Immunol ; 184(3): 1200-9, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20026743

ABSTRACT

High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (< or = 10 mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying the suppression. Uptake of [3-(13)C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-(13)C(2)]glucose to the culture medium and measured glucose uptake and conversion into [2,3-(13)C(2)]lactate. Activation of monocytes increased the glycolytic flux and the secretion of lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-(13)C(2)]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased intracellular [2,3-(13)C(2)]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the tumor environment.


Subject(s)
Acidosis, Lactic/metabolism , Lactic Acid/metabolism , Monocytes/immunology , Monocytes/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Acidosis, Lactic/immunology , Cell Line, Tumor , Cell Survival/immunology , Cells, Cultured , Coculture Techniques , Energy Metabolism/immunology , Glycolysis/immunology , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Humans , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/toxicity , Lactic Acid/toxicity , Melanoma/immunology , Melanoma/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
6.
J Chromatogr A ; 1117(1): 67-73, 2006 Jun 02.
Article in English | MEDLINE | ID: mdl-16600254

ABSTRACT

A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid-liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum-ethyl acetate-methanol-water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%.


Subject(s)
Ascomycota/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Countercurrent Distribution/methods
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