The catabolism of endogenous adenine nucleotides in rat liver mitochondria

Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [¹⁴C]-adenosine yielded no [¹⁴C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.     

Cardiac dysfunction in spontaneously hypertensive old rats is associated with a significant decrease of SUR2 expression

Molecular and Cellular Biochemistry - ATP-sensitive potassium (KATP) channels are participants of mechanisms of pathological myocardial remodeling containment. The aim of our work was to find the...     

Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides

Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O₂-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O₂ monitoring in live cells and in tissue, and an alternative to the existing O₂ probes which require facilitated transport into the cell.     

Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr³⁴ in RhoA. Tyr³⁴ is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr³⁴ against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr³⁴ nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI.     

The interactive roles of zinc and calcium in mitochondrial dysfunction and neurodegeneration

Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons – normal (2 mM) calcium plus elevated (> 100 μM) exogenous zinc – mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium‐containing media. This contrasts with the lack of any mitochondrial response in zinc‐containing but calcium‐free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore‐induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium‐ and zinc‐containing media, depolarization‐induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium.

     

Toll-like receptors: linking innate and adaptive immunity

Detection of and response to microbial infections by the immune system depends largely on a family of pattern-recognition receptors called Toll-like receptors (TLRs). These receptors recognize conserved molecular products derived from various classes of pathogens, including Gram-positive and -negative bacteria, DNA and RNA viruses, fungi and protozoa. Recognition of ligands by TLRs leads to a series of signaling events resulting in induction of acute responses necessary to kill the pathogen. TLRs are also responsible for the induction of dendritic cell maturation, which is responsible and necessary for initiation of adaptive immune responses. Although TLRs control induction of adaptive immunity, it is not clear at this point how responses are appropriately tailored by individual TLRs to the advantage of the host.     

Improved <Emphasis Type="Italic">A. faecalis</Emphasis> Penicillin Amidase Mutant Retains the Thermodynamic and pH Stability of the Wild Type Enzyme

Penicillin amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic β-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of penicillin G was 140 s⁻¹, about twice of the value for the wild-type enzyme (80 s⁻¹). At the same time the specificity constant was improved about three-fold. The wild-type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.     

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.     

Asymmetric Dimethylarginine Induces Endothelial Nitric-oxide Synthase Mitochondrial Redistribution through the Nitration-mediated Activation of Akt1

We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser⁶¹⁷ and Ser¹¹⁷⁹. Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr³⁵⁰) located within the client-binding domain. Replacement of Tyr³⁵⁰ with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser⁶¹⁷ and Ser¹¹⁷⁹ by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr³⁵⁰.     

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.