Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson's disease.

Inhibitory PAS domain protein (IPAS), a repressor of hypoxia-inducible factor-dependent transcription under hypoxia, was found to exert pro-apoptotic activity in oxidative stress-induced cell death. However, physiological and pathological processes associated with this activity are not known. Here we show that IPAS is a key molecule involved in neuronal cell death in Parkinson's disease (PD). IPAS was ubiquitinated by Parkin for proteasomal degradation following carbonyl cyanide m-chlorophenyl hydrazone treatment. Phosphorylation of IPAS at Thr12 by PTEN-induced putative kinase 1 (PINK1) was required for ubiquitination to occur. Activation of the PINK1-Parkin pathway attenuated IPAS-dependent apoptosis. IPAS was markedly induced in the midbrain following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and IPAS-deficient mice showed resistance to MPTP-induced degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). A significant increase in IPAS expression was found in SNpc neurons in patients with sporadic PD. These results indicate a mechanism of neurodegeneration in PD.

Development of specific inhibitors of CYP707A, a key enzyme in the catabolism of abscisic acid.

Abscisic acid (ABA) is a plant hormone involved in stress tolerance, stomatal closure, seed dormancy, and other physiological events. Although ABA is registered as a farm chemical (plant growth regulator), its practical use has been limited, mainly due to its weak effect in field trials, which is considered to be due to its rapid inactivation through biodegradation. Catabolic inactivation of ABA is mainly controlled by ABA 8'-hydroxylase (CYP707A), which is the cytochrome P450 catalyzing the C8'-hydroxylation of ABA into 8'-hydroxy-ABA and its more stable tautomer, phaseic acid, which has much lower hormonal activity than ABA. Thus, a specific inhibitor of CYP707A is promising not only as a chemical probe for the mechanism of ABA action, but also because of its potential use in agriculture and horticulture. This review article focuses on our recent research on the development of two types of specific inhibitors of CYP707A: the ABA analogue-type inhibitors that were designed on the basis of differences between the structural requirements for CYP707A and for ABA activity, and the azole-type P450 inhibitors that were developed by conformational restriction or enlargement of the plant growth retardant uniconazole.

Biosynthesis of abscisic acid by the non-mevalonate pathway in plants, and by the mevalonate pathway in fungi.

The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-(13)C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4', 7' and 9' of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1', 3', 5', 7', 8' and 9' of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled beta-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway.

3'-Azidoabscisic acid as a photoaffinity reagent for abscisic acid binding proteins.

3'-Azidoabscisic acid was synthesized as a potential photoaffinity reagent for abscisic acid binding proteins. This compound was stable in organic and aqueous solutions in the dark, but was decomposed by UV irradiation. Its biological activity was equivalent to that of abscisic acid, suggesting that it may be an effective photoaffinity reagent.

Synthesis, Biological Activity, and Metabolism of 8',8',8'-Trideuteroabscisic Acid.

An 8',8',8'-trideuterated analog of abscisic acid (ABA) was diastereoselectively synthesized as a new analog of ABA that is resistant to 8'-hydroxyIation, the first metabolic reaction of ABA, owing to the primary kinetic isotope effect. (+)-8',8',8'-Trideutero-ABA showed long-term activity in the rice elongation assay. The rate of metabolism of this analog in rice cell suspension culture was about two fold slower than that of (+)-ABA. The concentration of 8',8'-dideuterophaseic acid produced was about 1/3 that of phaseic acid converted from (+)-ABA. This result indicated that the long-lasting activity of the (+)-trideutero-ABA in the rice assay was the result of the delayed 8'-hydroxylation as expected.

Synthesis and Biological Activities of 8'-Methylene- and 8'-Methylidyneabscisic Acids.

8'-Methylene- and 8'-methylidyneabscisic acids which might act as suicide inhibitors of the 8'-hydroxylase of abscisic acid, were designed, synthesized, and optically resolved. The (+)-isomers showed stronger inhibitory activity in rice elongation and in lettuce seed germination than (+)-abscisic acid. The activity of (+)-8'-methylidyneabscisic acid was the strongest of the analogues synthesized to date, 40-fold stronger than abscisic acid.

Effects of (+)-8',8',8'-trifluoroabscisic acid on alpha-amylase expression and sugar accumulation in rice cells.

The effects of (+)-8',8',8'-trifluoroabscisic acid (trifluoro-ABA) on alpha-amylase expression were studied in rice embryoless half-seeds, scutella, and suspension-cultured cells derived from the embryo, and the effects of the analog on sugar accumulation were also studied in scutella and suspension-cultured cells. Treatment with (+)-trifluoro-ABA strongly inhibited the gibberellic acid-inducible expression of alpha-amylase I-1 encoded by RAmy1A in the aleurone layers of embryoless half-seeds at the levels of transcription, protein synthesis, and enzyme activity. It was also found that (+)-trifluoro-ABA stimulated (i) the uptake of glucose from the incubation medium and (ii) the synthesis of sucrose in scutellar tissues and suspension-cultured cells of rice. The biological activity of (+)-trifluoro-ABA was found to be more potent and persistent than that of natural ABA. We further examined the effects of trifluoro-ABA on the expression of alpha-amylase I-1 in scutellar tissues and suspension-cultured cells. It was found that (+)-trifluoro-ABA did not inhibit the formation of alpha-amylase I-1 in the absence of external glucose. However, glucose and (+)-trifluoro-ABA cooperatively suppressed the formation of alpha-amylase I-1. Judging from these results, we conclude that the regulatory mechanism for the expression of alpha-amylase I-1 in the scutellar epithelium is distinguishable from that operating in the aleurone layer.