Hideto Miyoshi

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.

Mutations in theclk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ9) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ9. Instead, the UQ9 biosynthesis intermediate, demethoxyubiquinone (DMQ9), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome creductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ2 was chemically synthesized. We find that DMQ2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ2, cannot. The accumulation of DMQ9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues.

Identification of receptors of main sex-pheromone components of three Lepidopteran species

Male moths discriminate conspecific female‐emitted sex pheromones. Although the chemical components of sex pheromones have been identified in more than 500 moth species, only three components in Bombyx mori and Heliothis virescens have had their receptors identified. Here we report the identification of receptors for the main sex‐pheromone components in three moth species, Plutella xylostella, Mythimna separata and Diaphania indica. We cloned putative sex‐pheromone receptor genes PxOR1, MsOR1 and DiOR1 from P. xylostella, M. separata and D. indica, respectively. Each of the three genes was exclusively expressed with an Or83b orthologous gene in male olfactory receptor neurons (ORNs) that are surrounded by supporting cells expressing pheromone‐binding‐protein (PBP) genes. By two‐electrode voltage‐clamp recording, we tested the ligand specificity of Xenopus oocytes co‐expressing PxOR1, MsOR1 or DiOR1 with an OR83b family protein. Among the seven sex‐pheromone components of the three moth species, the oocytes dose‐dependently responded only to the main sex‐pheromone component of the corresponding moth species. In our study, PBPs were not essential for ligand specificity of the receptors. On the phylogenetic tree of insect olfactory receptors, the six sex‐pheromone receptors identified in the present and previous studies are grouped in the same subfamily but have no relation with the taxonomy of moths. It is most likely that sex‐pheromone receptors have randomly evolved from ancestral sex‐pheromone receptors before the speciation of moths and that their ligand specificity was modified by mutations of local amino acid sequences after speciation.

Structure-activity relationships of some complex I inhibitors.

A wide variety of complex I inhibitors act at or close to the ubiquinone reduction site. Identification of the structural factors required for exhibiting inhibitory actions on the basis of structure-activity relationships is useful to elucidate the manner in which inhibitors interact with the enzyme. This review summarizes studies on the structure-activity relationship of rotenoids, piericidins, capsaicins, pyridinium-type inhibitors and modern synthetic agrochemicals acting at mitochondrial complex I.

Essential structural factors of annonaceous acetogenins as potent inhibitors of mitochondrial complex I

The annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act, at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors. A systematic set of isolated natural acetogenins was prepared and examined for their inhibitory actions with bovine heart mitochondrial complex I to identify the essential structural factors of these inhibitors for the exhibition of potent activity. Despite their very potent activity, the structural requirements of the acetogenins are not particularly rigid and remain somewhat ambiguous. The most common structural units, such as adjacent bis-tetrahydrofuran (THF) rings and hydroxyl groups in the 4- and/or 10-positions, were not essential for exhibiting potent activity. The stereochemistry surrounding the THF rings, surprisingly, seemed to be unimportant, which was corroborated by an exhaustive conformational space search analysis, indicating that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Proper length and flexibility of the alkyl spacer moiety, which links the THF and the alpha, beta-unsaturated gamma-lactone ring moieties, were essential for the potent activity. This probably results from some sort of specific conformation of the spacer moiety which regulates the two ring moieties to locate into an optimal spatial position on the enzyme. It is, therefore, suggested that the structural specificity of the acetogenins, required for optimum inhibition, differs significantly from that of the common complex I inhibitors in which essential structural units are compactly arranged and conveniently defined. The structure-activity profile for complex I inhibition is discussed in comparison with those for other biological activities.

Comparison of the inhibitory action of synthetic capsaicin analogues with various NADH-ubiquinone oxidoreductases.

Capsaicin is a new naturally occurring inhibitor of proton-pumping NADH-ubiquinone oxidoreductase (NDH-1), that competitively acts against ubiquinone. A series of capsaicin analogues was synthesized to examine the structural factors required for the inhibitory action and to probe the structural property of the ubiquinone catalytic site of various NADH-ubiquinone reductases, including non-proton-pumping enzyme (NDH-2), from bovine heart mitochondria, potato tuber (Solanum tuberosum, L) mitochondria and Escherichia coli (GR 19N) plasma membranes. Some synthetic capsaicins were fairly potent inhibitors of each of the three NDH-1 compared with the potent rotenone and piericidin A. Synthetic capsaicin analogues inhibited all three NDH-1 activities in a competitive manner against an exogenous quinone. The modification both of the substitution pattern and of the number of methoxy groups on the benzene ring, which may be superimposable on the quinone ring of ubiquinone, did not drastically affect the inhibitory potency. In addition, alteration of the position of dipolar amide bond unit in the molecule and chemical modifications of this unit did not change the inhibitory potency, particularly with bovine heart and potato tuber NDH-1. These results might be explained assuming that the ubiquinone catalytic site of NDH-1 is spacious enough to accommodate a variety of structurally different capsaicin analogues in a dissimilar manner. Regarding the moiety corresponding to the alkyl side chain, a rigid diphenyl ether structure was more inhibitory than a flexible alkyl chain. Structure-activity studies and molecular orbital calculations suggested that a bent form is the active conformation of capsaicin analogues. On the other hand, poor correlations between the inhibitory potencies determined with the three NDH-1 suggested that the structural similarity of the ubiquinone catalytic sites of these enzymes is rather poor. The sensitivity to the inhibition by synthetic capsaicins remarkably differed between NDH-1 and NDH-2, supporting the notion that the sensitivity against capsaicin inhibition correlates well with the presence of an energy coupling site in the enzyme (Yagi, T. (1990) Arch. Biochem. Biophys. 281, 305-311). It is noteworthy that several synthetic capsaicins discriminated between NDH-1 and NDH-2 much better than natural capsaicin.

Regulation of the Mitochondrial Permeability Transition Pore by Ubiquinone Analogs. A Progress Report

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca 2+ -sensitive channel that plays a key role in different models of cell death. In a series of recent studies we have shown that the PTP is modulated by quinones, and we have identified three functional classes: (i) PTP inhibitors; (ii) PTP inducers; and (iii) PTP-inactive quinones that compete with both inhibitors and inducers. Here, we review our current understanding of pore regulation by quinones, and present the results obtained with a new series of structural variants. Based on the effects of the compounds studied so far, we confirm that minor structural changes profoundly modify the effects of quinones on the PTP. On the other hand, quinones with very different structural features may have qualitatively similar effects on the PTP. Taken together, these results support our original proposal that quinones affect the PTP through a common binding site whose occupancy modulates its open-closed transitions, possibly through secondary changes of the Ca 2+ -binding affinity.

Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana

Hydroxycinnamic acid amides (HCAAs) are secondary metabolites involved in the defense of plants against pathogens. Here, we report the first identification of HCAAs, p-coumaroylagmatine, feruloylagmatine, p-coumaroylputrescine and feruloylputrescine, in Arabidopsis thaliana rosette leaves infected with Alternaria brassicicola and the assignment of At5g61160 as the agmatine coumaroyltransferase (AtACT) that catalyzes the last reaction in the biosynthesis of the HCAAs. Feeding experiments with putative labeled precursors revealed that the four HCAAs were synthesized from hydroxycinnamic acids and agmatine or putrescine. AtACT gene function was identified from an analysis of a mutant that did not accumulate HCAAs. In wild-type Arabidopsis, AtACT transcripts markedly increased in response to A. brassicicola infection. Enzymatic activity that catalyzes the synthesis of the HCAAs was confirmed in vitro by using a recombinant AtACT expressed in Escherichia coli. The Atact mutant was susceptible to infection by A. brassicicola, indicating that HCAAs are responsible for defense against pathogens in A.thaliana.

Uncoupling activity of a newly developed fungicide, fluazinam [3-chloro-N-(3-chloro-2,6-dinitro-4-trifluoromethylphenyl)-5-trifluoromethyl-2-pyridinamine]

A very unusual uncoupling activity was found in a newly developed phenylpyridylamine fungicide for agricultural use, fluazinam. The compound had extraordinarily strong uncoupling activity, but the activity rapidly disappeared with rat-liver mitochondria isolated by the usual method with centrifugation. Treatment that lowered the concentration of glutathione (GSH), in the mitochondrial matrix prevented the disappearance of the uncoupling activity. The activity of an analog of fluazinam in which the 3-chloro substituent on the phenyl moiety, probably works as a leaving substituent, was replaced by the i -propoxy group lacking such a function, did not disappear. These results suggest that fluazinam was metabolically transformed on the mitochondrial level, probably by a GSH conjugation mechanism. When GSH was completely eliminated, fluazinam had powerful uncoupling potency, greater than that of SF6847, the most potent acidic uncoupler known until now.

Characterization of the inhibitor binding site in mitochondrial NADH-ubiquinone oxidoreductase by photoaffinity labeling using a quinazoline-type inhibitor.

The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.