Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.
- MeSH
- acyltransferasy metabolismus MeSH
- Barthův syndrom * metabolismus MeSH
- fenotyp MeSH
- fosfatidylglyceroly * antagonisté a inhibitory metabolismus MeSH
- kardiolipiny * genetika metabolismus MeSH
- lidé MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transkripční faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Idiopathic inflammatory myopathies/IIM are associated with changes in muscle-specific microRNA/miR. Exercise improves muscle function and metabolism in parallel with changes in miR expression. We investigated the effects of disease and exercise on miRs in differentiated muscle cells/myotubes from IIM patients and controls. METHODS: Samples of m. vastus lateralis were obtained by needle biopsy from IIM patients before/after 6-month training and from matched sedentary healthy controls. Muscle cell cultures were established and exposed to saturated fatty acid during differentiation. MiR-133a,-133b,-206,-1 and their target genes (qPCR), fat oxidation (FOx), lipids (chromatography) and mitochondrial oxidative phosphorylation (OxPHOS) complexes (immunoblotting) were measured. Interrelations between in vitro miRs and metabolism of myotubes as well as clinical parameters and disease activity/MITAX were explored. RESULTS: Levels of miRs were higher in myotubes derived from IIM patients compared to healthy controls (up to 3.5-fold, p<0.05). Neither 6-month training (IIM patients) nor in vitro palmitate treatment modulated myomiRs in myotubes. However, miR-133a,-133b, and miR-1 correlated negatively with FOx (p<0.01), triacylglycerols (p<0.05) and OxPHOS complex-V (p<0.05) and positively with OxPHOS complex-I (p<0.05) in myotubes. MiR-133a and miR-133b in myotubes were related to disease activity and fasting glycaemia in vivo (both p<0.05). CONCLUSIONS: Upregulation of microRNAs involved in myogenesis and regeneration in muscle cells derived from IIM patients indicates activation of compensatory epigenetic mechanisms, potentially aimed to counteract disease progression. Relationships of microRNAs with in vitro metabolic profile of muscle cells as well as with clinical parameters support the role of muscle-specific microRNAs in modulating muscle metabolism and clinical state of patients.
- MeSH
- cvičení fyziologie MeSH
- kosterní svalová vlákna metabolismus patologie MeSH
- kosterní svaly fyziologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- myozitida * patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.
The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- fosfatidylglyceroly metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- homeostáza MeSH
- lipidová tělíska chemie MeSH
- metabolismus lipidů MeSH
- mitochondrie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We describe a novel mechanism of mRNA decay regulation, which takes place under the conditions of glucose deprivation in the yeast Saccharomyces cerevisiae. The regulation is based on temporally stable sequestration of the main 5'-3' mRNA exoribonuclease Xrn1 at the eisosome, a plasma membrane-associated protein complex organizing a specialized membrane microdomain. As documented by monitoring the decay of a specific mRNA substrate in time, Xrn1-mediated mRNA degradation ceases during the accumulation of Xrn1 at eisosome, but the eisosome-associated Xrn1 retains its functionality and can be re-activated when released to cytoplasm following the addition of glucose. In cells lacking the eisosome organizer Pil1, Xrn1 does not associate with the plasma membrane and its activity is preserved till the stationary phase. Thus, properly assembled eisosome is necessary for this kind of Xrn1 regulation, which occurs in a liquid culture as well as in a differentiated colony.
- MeSH
- buněčná membrána genetika metabolismus MeSH
- cytoplazma genetika metabolismus MeSH
- exoribonukleasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- membránové mikrodomény genetika metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- messenger RNA metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- stabilita RNA genetika MeSH
- Publikační typ
- časopisecké články MeSH
In yeast, phosphatidylglycerol (PG) is a minor phospholipid under standard conditions; it can be utilized for cardiolipin (CL) biosynthesis by CL synthase, Crd1p, or alternatively degraded by the phospholipase Pgc1p. The Saccharomyces cerevisiae deletion mutants crd1Δ and pgc1Δ both accumulate PG. Based on analyses of the phospholipid content of pgc1Δ and crd1Δ yeast, we revealed that in yeast mitochondria, two separate pools of PG are present, which differ in their fatty acid composition and accessibility for Pgc1p-catalyzed degradation. In contrast to CL-deficient crd1Δ yeast, the pgc1Δ mutant contains normal levels of CL. This makes the pgc1Δ strain a suitable model to study the effect of accumulation of PG per se. Using fluorescence microscopy, we show that accumulation of PG with normal levels of CL resulted in increased fragmentation of mitochondria, while in the absence of CL, accumulation of PG led to the formation of large mitochondrial sheets. We also show that pgc1Δ mitochondria exhibited increased respiration rates due to increased activity of cytochrome c oxidase. Taken together, our results indicate that not only a lack of anionic phospholipids, but also excess PG, or unbalanced ratios of anionic phospholipids in mitochondrial membranes, have harmful consequences on mitochondrial morphology and function.
- MeSH
- fosfatidylglyceroly metabolismus MeSH
- fosfolipasy fyziologie MeSH
- kardiolipiny biosyntéza MeSH
- mitochondrie metabolismus ultrastruktura MeSH
- respirační komplex IV metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Východiská: Hráčske problémy je možné posudzovať na určitom kontinuu od rekreačného hráčstva až po hráčstvo patologické. Na posúdenie miery závažnosti patologického hráčstva bolo vo svete vytvorených niekoľko škál, na Slovensku však nemáme dostupné ich štandardizované verzie, čo viedlo aj k vytvoreniu Banskobystrického dotazníka patologického hráčstva (BBDPH). Cieľ: V rámci pilotnej štúdie bola overovaná reliabilita a validita BBDPH, ktorý bol vytvorený na určovanie miery závažnosti patologického hrania. Metodika: Posúdenie vnútornej konzistencie BBDPH s určením koeficientu split-half reliability a určením Cronbachovho alfa, posúdenie korelácie závažnosti hrania vyjadrenej prostredníctvom skóre BBDPH s počtom splnených DSM-IV a DSM-5 kritérií patologického hráčstva. Sledovaný súbor tvorilo 50 patologických hráčov liečených na Psychiatrickom oddelení v Banskej Bystrici. Výsledky: V rámci pilotnej štúdie sa preukázala adekvátna reliabilita a validita BBDPH. Závery: Banskobystrický dotazník patologického hráčstva možno na základe výsledkov pilotnej štúdie predbežne považovať za dostatočne spoľahlivý a užitočný nástroj na určovanie závažnosti patologického hrania, ktorý môže nájsť svoje uplatnenie v klinickej praxi na Slovensku. Na komplexnejšie posúdenie reliability a validity dotazníka, ako aj na zistenie relevantných deskriptívnych parametrov BBDPH vrátane tzv. cut-off skóre bude potrebný ďalší výskum nielen na klinickej vzorke.
Backround: One of the possible ways of rating gambling problems is the continuum from recreational gambling to pathological gambling. Although several scales for measurement of severity of pathological gambling were created abroad, there are no standardized versions of them available in Slovakia, which lead to construction of „Banská Bystrica Pathological Gambling Questionnaire“ (BBDPH). BBDPH was created for measurement of pathological gambling severity. Goal: In a pilot study, reliability and validity of BBDPH were evaluated. Methods: Measurement of the internal consistency of BBDPH with defining split-half reliability coefficient and Cronbach’s alpha, a correlation of severity of pathological gambling considered with BBDPH score with number of fulfilled DSM-IV and DSM-5 criterion of pathological gambling. The research sample consisted of 50 pathological gamblers who were treated at Department of Psychiatry in Banská Bystrica. Results: Within the frame of the pilot study adequate reliability and validity of BBDPH were demonstrated. Conclusion: Based on the results of the pilot study the „Banská Bystrica Pathological Gambling Questionnaire“ is considered as useful and reliable diagnostic tool for the measurement of severity of pathological gambling which may find its position in the clinical practice in Slovakia. For more complex justification of the questionnaire reliability and validity, as well as for ascertaining descriptive BBDPH parameters including cut-off score, another survey, which is not focused only on a clinical sample, will be needed.
- MeSH
- dospělí MeSH
- hráčství diagnóza epidemiologie klasifikace MeSH
- komorbidita MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- poruchy spojené s užíváním psychoaktivních látek diagnóza epidemiologie MeSH
- průzkumy a dotazníky * MeSH
- senzitivita a specificita MeSH
- socioekonomické faktory MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
