Proteomic study on X-irradiation-responsive proteins and ageing: search for responsible proteins for radiation adaptive response

We investigated high- or low-dose irradiation-responsive proteins using proteomics on two-dimensional (2D) PAGE, and the effects of ageing on cell responses to radiation in variously aged rat astrocytes. After 5 Gy irradiation, the relative abundance of peroxiredoxin 2, an antioxidant enzyme, and latexin, an inhibitor of carboxypeptidase, increased. The induction of these proteins was suppressed by ageing, suggesting that the response to high-dose radiation decreased with ageing. The relative abundance of elongation factor 2 (EF-2) fragment increased 3 h and reduced 24 h after 0.1 Gy irradiation. Temporal enhancement of the EF-2 fragment due to low-dose irradiation was suppressed by ageing. Since radiation adaptive response in cultured astrocytes was observed 3 h but not 24 h after 0.1 Gy irradiation and suppressed by ageing as previously reported, alteration of the EF-2 fragment corresponded to the radiation adaptive response. We also examined phospho-protein profiles, resulting in the relative abundance of phospho-EF-1beta and phospho-beta-actin being altered by 0.1 Gy irradiation; however, ageing did not affect the alteration of phospho-EF-1beta and phospho-beta-actin, unlike the EF-2 fragment. The results suggested that the EF-2 fragment was a possible candidate for the protein responsible for the radiation adaptive response in cultured astrocytes.     

Calcineurin potentiates the activation of procaspase-3 by accelerating its proteolytic maturation

We have previously shown that procaspase-3 exists in a high molecular weight complex in neonatal rat brain. Here, we purify and identify the protein that interacts with procaspase-3 from rat neonatal cortex. We searched binding proteins to procaspase-3 from a cytosolic extract of neonatal rat brain using chromatogram, two-dimensional gel electrophoresis, and far Western immunoblot. Analysis by tandem mass spectrometry identified the protein as a regulatory subunit of calcineurin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-alpha and cycloheximide treatment. In a cell-free system, overexpression of calcineurin B in HEK293 cells markedly increased processing of caspase-3 by cytochrome c. Immunodepletion of calcineurin B from cytosolic extracts from Jurkat cells decreased processing of caspase-3 by cytochrome c. Knockdown of calcineurin B by RNA interference resulted in reduced apoptosis in HEK293 cells but not in caspase-3-deficient MCF-7 cells. These results suggest that calcineurin B potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.     

Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.     

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening

The Na(+)/H(+) exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca(2+) overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca(2+) overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and the mitochondrial membrane potential (DeltaPsi(m)) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 microM) prevented ouabain-induced hypercontracture (from 40 +/- 2 to 24 +/- 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca(2+)](m) overload (from 149 +/- 6 to 121 +/- 5% of the baseline value, P < 0.05) but did not affect DeltaPsi(m). These results indicate that cariporide attenuates the [Ca(2+)](m) overload without the accompanying depolarization of DeltaPsi(m). Moreover, cariporide increased the time taken to induce the MPT (from 79 +/- 11 to 137 +/- 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 +/- 3 to 50 +/- 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca(2+)](m) overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.     

Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction

Several studies have demonstrated that NF-kappaB is substantially involved in the progression of cardiac remodeling; however, it remains uncertain whether the continuous inhibition of NF-kappaB is effective for the prevention of myocardial remodeling. Myocardial infarction (MI) was produced by ligation of the left anterior coronary artery of rats. IMD-0354 (10 mg/kg per day), a novel phosphorylation inhibitor of IkappaB that acts via inhibition of IKK-beta, was injected intraperitoneally starting 24 h after induction of MI for 28 days. After 28 days, the IMD-0354-treated group showed significantly improved survival rate compared with that of the vehicle-treated group (P < 0.05). Although infarct size was similar in both groups, improved left ventricular (LV) remodeling and diastolic dysfunction, as indicated by smaller LV cavity (LV end-diastolic area: vehicle, 74.13 +/- 3.57 mm(2); IMD-0354, 55.00 +/- 3.73 mm(2); P < 0.05), smaller peak velocity of early-to-late filling wave (E/A) ratio (vehicle, 3.87 +/- 0.26; IMD-0354, 2.61 +/- 0.24; P < 0.05), and lower plasma brain natriuretic peptide level (vehicle, 167.63 +/- 14.87 pg/ml; IMD-0354, 110.75 +/- 6.41 pg/ml; P < 0.05), were observed in the IMD-0354-treated group. Moreover, fibrosis, accumulation of macrophages, and expression of several factors (transforming growth factor-beta1, monocyte chemoattractant protein-1, matrix metalloproteinase-9 and -2) in the noninfarcted myocardium was remarkably inhibited by IMD-0354. In conclusion, inhibition of NF-kappaB activation may reduce the proinflammatory reactions and modulate the extracellular matrix and provide an effective approach to prevent adverse cardiac remodeling after MI.     

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.     

Fission yeast Cut8 is required for the repair of DNA double-strand breaks, ribosomal DNA maintenance, and cell survival in the absence of Rqh1 helicase

Schizosaccharomyces pombe Rqh1 is a member of the RecQ DNA helicase family. Members of this protein family are mutated in cancer predisposition diseases, causing Bloom's, Werner, and Rothmund-Thomson syndromes. Rqh1 forms a complex with topoisomerase III and is proposed to process or disrupt aberrant recombination structures that arise during S phase to allow proper chromosome segregation during mitosis. Intriguingly, in the absence of Rqh1, processing of these structures appears to be dependent on Rad3 (human ATR) in a manner that is distinct from its role in checkpoint control. Here, we show that rad3 rqh1 mutants are normally committed to a lethal pathway of DNA repair requiring homologous recombination, but blocking this pathway by Rhp51 inactivation restores viability. Remarkably, viability is also restored by overexpression of Cut8, a nuclear envelope protein involved in tethering and proper function of the proteasome. In keeping with a recently described function of the proteasome in the repair of DNA double-strand breaks, we found that Cut8 is also required for DNA double-strand break repair and is essential for proper chromosome segregation in the absence of Rqh1, suggesting that these proteins might function in a common pathway in homologous recombination repair to ensure accurate nuclear division in S. pombe.     

Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium

The trace amine-associated receptors (TAARs) form a specific family of G protein-coupled receptors in vertebrates. TAARs were initially considered neurotransmitter receptors, but recent study showed that mouse TAARs function as chemosensory receptors in the olfactory epithelium. To clarify the evolutionary dynamics of the TAAR gene family in vertebrates, near-complete repertoires of TAAR genes and pseudogenes were identified from the genomic assemblies of 4 teleost fishes (zebrafish, fugu, stickleback, and medaka), western clawed frogs, chickens, 3 mammals (humans, mice, and opossum), and sea lampreys. Database searches revealed that fishes had many putatively functional TAAR genes (13-109 genes), whereas relatively small numbers of TAAR genes (3-22 genes) were identified in tetrapods. Phylogenetic analysis of these genes indicated that the TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence of ray-finned fishes and tetrapods. In tetrapods, virtually all TAAR genes were located in 1 specific region of their genomes as a gene cluster; however, in fishes, TAAR genes were scattered throughout more than 2 genomic locations. This possibly reflects a whole-genome duplication that occurred in the common ancestor of ray-finned fishes. Expression analysis of zebrafish and stickleback TAAR genes revealed that many TAARs in these fishes were expressed in the olfactory organ, suggesting the relatively high importance of TAARs as chemosensory receptors in fishes. A possible evolutionary history of the vertebrate TAAR gene family was inferred from the phylogenetic and comparative genomic analyses.