Volume 399, Issue 5

Mitochondrial 2-oxoacid dehydrogenase complexes oxidize 2-oxoglutarate, pyruvate, branched-chain 2-oxoacids and 2-oxoadipate to the corresponding acyl-CoAs and reduce NAD + to NADH. The isolated enzyme complexes generate superoxide anion radical or hydrogen peroxide in defined reactions by leaking electrons to oxygen. Studies using isolated mitochondria in media mimicking cytosol suggest that the 2-oxoacid dehydrogenase complexes contribute little to the production of superoxide or hydrogen peroxide relative to other mitochondrial sites at physiological steady states. However, the contributions may increase under pathological conditions, in accordance with the high maximum capacities of superoxide or hydrogen peroxide-generating reactions of the complexes, established in isolated mitochondria. We assess available data on the use of modulations of enzyme activity to infer superoxide or hydrogen peroxide production from particular 2-oxoacid dehydrogenase complexes in cells, and limitations of such methods to discriminate specific superoxide or hydrogen peroxide sources in vivo .

Sarcopenia is a well-known geriatric syndrome that has been endorsed over the years as a biomarker allowing for the discrimination, at a clinical level, of biological from chronological age. Multiple candidate mechanisms have been linked to muscle degeneration during sarcopenia. Among them, there is wide consensus on the central role played by the loss of mitochondrial integrity in myocytes, secondary to dysfunctional quality control mechanisms. Indeed, mitochondria establish direct or indirect contacts with other cellular components (e.g. endoplasmic reticulum, peroxisomes, lysosomes/vacuoles) as well as the extracellular environment through the release of several biomolecules. The functional implications of these interactions in the context of muscle physiology and sarcopenia are not yet fully appreciated and represent a promising area of investigation. Here, we present an overview of recent findings concerning the interrelation between mitochondrial quality control processes, inflammation and the metabolic regulation of muscle mass in the pathogenesis of sarcopenia highlighting those pathways that may be exploited for developing preventive and therapeutic interventions against muscle aging.

Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H 2 O 2 and reactive oxygen species. We and others have shown that Duox2 and H 2 O 2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H 2 O 2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H 2 O 2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H 2 O 2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases.

Gaucher disease (GD) is a lysosomal storage disorder, caused by an impaired function of β-glucocerebrosidase, which results in accumulation of glucocerebroside in cells, and altered membrane ordering. Using electron paramagnetic resonance spin labeling, a statistically significant difference in the order parameter between the peripheral blood mononuclear cell membranes of GD patients and healthy controls was observed. Moreover, the results show that the introduction of the enzyme replacement therapy leads to the restoration of the physiological membrane fluidity. Accordingly, this simple method could serve as a preliminary test for GD diagnosis and therapy efficiency.

Employing hippocampal synaptosomes from amyloid precursor protein (APP)-deleted mice we analyzed the immediate effects of amyloid beta peptide 42 (Aβ 42 ) peptide in its oligomeric or fibrillar assembly or of soluble amyloid precursor protein alpha (sAPPα) protein on their bioenergetic activity. Upon administration of oligomeric Aβ 42 peptide for 30 min we observed a robust decrease both in mitochondrial activity and in mitochondrial membrane potential (MMP). In contrast the respective fibrillary or scrambled peptides showed no effect, indicating that inhibition strictly depends on the oligomerization status of the peptide. Hippocampal synaptosomes from old APP-KO mice revealed a further reduction of their already impaired bioenergetic activity upon incubation with 10 μ m Aβ 42 peptide. In addition we evaluated the influence of the sAPPα protein on mitochondrial activity of hippocampal synaptosomes derived from young or old APP-KO animals. In neither case 20 n m nor 200 n m sAPPα protein had an effect on mitochondrial metabolic activity. Our findings demonstrate that hippocampal synaptosomes derived from APP-KO mice are a most suitable model system to evaluate the impact of Aβ 42 peptide on its bioenergetic activity and to further elucidate the molecular mechanisms underlying the impairments by oligomeric Aβ 42 on mitochondrial function. Our data demonstrate that extracellular Aβ 42 peptide is taken up into synaptosomes where it immediately attenuates mitochondrial activity.

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo . The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro .

This study aimed at investigating the biological functions of long non-coding RNAs (lncRNAs) hox transcript antisense intergenic RNA (HOTAIR) in resistant ovarian cancer cells, exploring the regulation effect of HOTAIR on HOXA7 , and investigating their influence on the chemosensitivity of ovarian cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied for the verification of HOTAIR expression in resistant and sensitive groups. How HOTAIR downregulation affected cell proliferation, migration and invasion, and apoptosis were determined using the MTT assay and the colony formation assay, the Transwell assay and flow cytometry analysis, respectively. Immunohistochemistry was used to inspect the protein expression of HOXA7 in resistant and sensitive ovarian cancer tissues. The regulation relationship between HOTAIR and HOXA7 was investigated by qRT-PCR and Western blot. The effect of HOTAIR and HOXA7 on tumor growth was confirmed by the tumor xenograft model of nude mice. By knocking down HOXA7 , HOTAIR downregulation restrained the ovarian cancer deterioration in functional experiments. Silencing of HOTAIR and HOXA7 could effectively inhibit tumor growth and increase chemosensitivity of ovarian tumors in nude mice. Downregulation of HOTAIR negatively affected the survival and activity of resistant ovarian cancer cells, and suppressed the expression of HOXA7 . Silencing of HOTAIR and HOXA7 could increase the chemosensitivity of ovarian cancer cells, thus suppressing tumor development.

Serum and liver selenium levels were studied during the pathogenesis of N -nitrosodimethylamine (NDMA) induced hepatic fibrosis in rats. The degree of fibrosis was assessed with Masson’s trichrome staining and quantifying collagen content in the liver. Lipid peroxides were measured in blood and liver samples and total glutathione and glutathione peroxidase were assayed in the liver tissue to evaluate oxidative stress. Interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1) were measured in the serum. Selenium levels were determined using inductively coupled plasma-mass spectrometry (ICP-MS) after acid digestion and hydride generation of selenium. Serial administrations of NDMA produced well-developed fibrosis and early cirrhosis in the liver with 4-fold increase of total collagen content and deposition of collagen fibers. Blood and hepatic lipid peroxides, serum IL-6 and TGF-β1 were significantly increased. There was significant reduction in hepatic glutathione and glutathione peroxidase levels. Serum and liver selenium were remarkably decreased on all the days studied. The results suggest that decreased selenium and glutathione peroxidase contribute to the impairment of cellular antioxidant defense, which in turn results in oxidative stress and trigger pathogenesis of hepatic fibrosis. The study further demonstrated that ICP-MS with hydride generation technique is a reliable and sensitive method for determination of selenium in biological samples.