UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

[Space flight/bedrest immobilization and bone. Space flight and bed rest-mediated muscle atrophy].

Muscle atrophy caused by unloading stress is a challenging problem for bed-rested patients or astronauts. However, countermeasures against these muscle atrophy have not been developed yet. Under unloading conditions, skeletal muscle mass is rapidly lost by the increase in protein breakdown and the decrease in protein synthesis. It has been shown that this enhancement of proteolysis in atrophying muscles results mainly from activation of the ubiquitin-proteasome proteolytic pathway. Previous our studies revealed that unloading stress led to skeletal muscle atrophy through the induction of ubiquitin ligase, Cbl-b (Casitas B-lineage lymphoma b) expression. Thus, Cbl-b inhibiters may be potent therapeutic and preventive sources against skeletal muscle atrophy caused by unloading stress.

Isolation of phospholipase D mutants having phosphatidylinositol-synthesizing activity with positional specificity on myo-inositol.

ENZYME-MEDIATED SYNTHESIS OF PHOSPHATIDYLINOSITOL: Engineered phospholipase D enzymes enable the synthesis of phosphatidylinositol by transphosphatidylation. The 1- or 3-hydroxy group of myo-inositol is selectively reacted. Phospholipase D (PLD) mutants that have phosphatidylinositol (PI)-synthesizing activity with positional selectivity towards 1- or 3-OH groups of myo-inositol have been isolated. A mutant PLD library, in which site-directed saturation mutations were introduced in vitro at positions 187, 191, and 385 of the wild-type PLD of Streptomyces antibioticus, was screened for PI-synthesizing mutants. TLC and HPLC analyses of the PI synthesized by the isolated mutant PLDs revealed that three mutants, namely 187D/191Y/385R (DYR), 187A/191Y/385R (AYR), and 187M/191Y/385R (MYR), selectively generated 1- or 3-PI among the other possible PI positional isomers. Taking into account the consensus sequence of the three mutants, a series of mutants, 187X/191Y/385R (XYR), was constructed and analyzed. Almost all the XYR mutants generated 1(3)-PI selectively, thus suggesting that the Y385R mutation contributed to the selectivity for the 1(3)-PI synthesis. The XYR mutants showed similar phosphatidylcholine-hydrolyzing activity among the mutants, but the PI-synthesizing activities were different depending on the amino acid at position 187. In particular, aromatic amino acids at position 187 greatly reduced the PI-synthesizing activity. The ratios of 1-PI versus 3-PI in the PIs synthesized with the XYR mutants were analyzed by selective hydrolysis with PI-specific phospholipase C. It was found that 187H/191Y/385R (HYR) generated 1-PI more than 3-PI (ratio=7:3), whereas 187T/191Y/385R (TYR) generated 1-PI less than 3-PI (ratio=2:8). This confirmed that the amino acid at position 187 determined the selectivity between 1-PI and 3-PI formation.

Probucol attenuates hyperoxia-induced lung injury in mice.

Hyperoxic lung injury is pathologically characterized by alveolar edema, interlobular septal edema, hyaline membrane disease, lung inflammation, and alveolar hemorrhage. Although the precise mechanism by which hyperoxia causes lung injury is not well defined, oxidative stress, epithelial cell death, and proinflammatory cytokines are thought to be involved. Probucol-a commercially available drug for treating hypercholesterolemia-has been suggested to have antioxidant and antiapoptotic effects. This study aimed to assess whether probucol could attenuate hyperoxic lung injury in mice. Mice were exposed to 95% O2 for 72 h, with or without pre-treatment with 130 µg/kg probucol intratracheally. Probucol treatment significantly decreased both the number of inflammatory cells in the bronchoalveolar lavage fluid and the degree of lung injury in hyperoxia-exposed mice. Probucol treatment reduced the number of cells positive for 8-hydroxyl-2'-deoxyguanosine or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and suppressed NF-κB activation, Bax expression, and caspase-9 activation in lung tissues from hyperoxia-exposed mice. These results suggest that probucol can reduce oxidative DNA damage, apoptotic cell death, and inflammation in lung tissues. Intratracheal administration of probucol may be a novel treatment for lung diseases induced by oxidative stress, such as hyperoxic lung injury and acute respiratory distress syndrome.

Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation.

Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)-α treatment. In C2C12 myotubes, TNF-α treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFκB, a downstream target of the TNF-α signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF-α in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography.

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.