Nitric oxide confers cisplatin resistance in human lung cancer cells through upregulation of aldo-keto reductase 1B10 and proteasome.

In this study, we show that exposure of human lung cancer A549 cells to cisplatin (cis-diamminedichloroplatinum, CDDP) promotes production of nitric oxide (NO) through generation of reactive oxygen species (ROS) and resulting upregulation of inducible NO synthase (iNOS). The incubation of the cells with a NO donor, diethylenetriamine NONOate, not only reduced the CDDP-induced cell death and apoptotic alterations (induction of CCAAT-enhancer-binding protein homologous protein and caspase-3 activation), but also elevated proteolytic activity of 26S proteasome, suggesting that the activation of proteasome function contributes to the reduction of CDDP sensitivity by NO. Monitoring expression levels of six aldo-keto reductases (AKRs) (1A1, 1B1, 1B10, 1C1, 1C2, and 1C3) during the treatment with the NO donor and subsequent CDDP sensitivity test using the specific inhibitors also proposed that upregulation of AKR1B10 by NO is a key process for acquiring the CDDP resistance in A549 cells. Treatment with CDDP and NO increased amounts of nitrotyrosine protein adducts, indicative of peroxynitrite formation, and promoted the induction of AKR1B10, inferring a relationship between peroxynitrite formation and the enzyme upregulation in the cells. The treatment with CDDP or a ROS-related lipid aldehyde, 4-hydroxy-2-nonenal, facilitated the iNOS upregulation, which was restored by increasing the AKR1B10 expression. In contrast, the facilitation of NO production by CDDP treatment was hardly observed in AKR1B10-overexpressing A549 cells and established CDDP-resistant cancer cells (A549, LoVo, and PC3). Collectively, these results suggest the NO functions as a key regulator controlling AKR1B10 expression and 26S proteasome function leading to gain of the CDDP resistance.

Structure/function analysis of a critical disulfide bond in the active site of L-xylulose reductase.

L-xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138 and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible disulfide-bond formation and by S-cysteinylation.

Crystallization and preliminary X-ray investigation of factor D of human complement.

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.

Tertiary structure of histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

The tertiary structure of the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been determined by x-ray diffraction at 2.8-A resolution. Initially, a partial structure was fitted to the multiple isomorphous replacement map and then least-squares refined by the Konnert/Hendrickson restrained parameter method (Konnert, J. H., and Hendrickson, W. A. (1980) Acta Crystallogr. A36, 344-350) and finally, a subsequent map was computed by use of the phase combination method of Read (Read, R. J. (1986) Acta Crystallogr. A42, 140-149). More of the protein structure was located in the latter map. The procedure of model building, least-squares refinement, and electron density map recalculation was repeated until the tertiary structure of HPr was obtained. The overall structure of HPr consists of four beta-strands, three helical regions, and four beta-turns. At the active center, the His15 imidazole interacts with one oxygen atom of the alpha-carboxyl C terminus of the polypeptide chain; the conserved Arg17 side chain interacts with the other oxygen atom of the alpha-carboxyl C terminus as well as with the side chain of Glu85. This is the first x-ray analysis of a protein of the phosphoenolpyruvate:sugar phosphotransferase system. Furthermore, this work represents a protein structure which has been solved by starting with a model that represented only one-third of the scattering matter.