Pheromone Metabolism Research Articles

Pheromone Guidance of Polarity Site Movement in Yeast.

Cells’ ability to track chemical gradients is integral to many biological phenomena, including fertilization, development, accessing nutrients, and combating infection. Mating of the yeast Saccharomyces cerevisiae provides a tractable model to understand how cells interpret the spatial information in chemical gradients. Mating yeast of the two different mating types secrete distinct peptide pheromones, called a-factor and α-factor, to communicate with potential partners. Spatial gradients of pheromones are decoded to guide mobile polarity sites so that polarity sites in mating partners align towards each other, as a prerequisite for cell-cell fusion and zygote formation. In ascomycetes including S. cerevisiae, one pheromone is prenylated (a-factor) while the other is not (α-factor). The difference in physical properties between the pheromones, combined with associated differences in mechanisms of secretion and extracellular pheromone metabolism, suggested that the pheromones might differ in the spatial information that they convey to potential mating partners. However, as mating appears to be isogamous in this species, it is not clear why any such signaling difference would be advantageous. Here we report assays that directly track movement of the polarity site in each partner as a way to understand the spatial information conveyed by each pheromone. Our findings suggest that both pheromones convey very similar information. We speculate that the different pheromones were advantageous in ancestral species with asymmetric mating systems and may represent an evolutionary vestige in yeasts that mate isogamously.

Expression Levels of Detoxification Enzyme Genes from Dendroctonus armandi (Coleoptera: Curculionidae) Fed on a Solid Diet Containing Pine Phloem and Terpenoids.

Simple SummaryThe bark beetle is the most well-known pest in coniferous trees worldwide. These insects only leave the host pine bark when they disperse to locate a new host. Determining how Dendroctonus armandi overcome the trees’ terpene-based defense systems has been the key problem in the study of bark beetles. Therefore, the aim of this study was to discover the molecular mechanism of insect detoxification enzymes’ ability to confer resistance to terpenes. For this purpose, the genes of cytochrome P450s, glutathione S-transferases, and carboxylesterases were studied in beetles given diets containing terpenes. The results suggest that beetles express different genes in response to terpenoids, and the responses of multiple detoxifying enzymes indicate these insects’ adaption to their chemical environment.Bark beetles overcome the toxic terpenoids produced by pine trees by both detoxifying and converting them into a pheromone system. Detoxification enzymes such as cytochrome P450s, glutathione S-transferases, and carboxylesterases are involved in the ability of Dendroctonus armandi to adapt to its chemical environment. Ten genes from these three major classes of detoxification enzymes were selected to study how these enzymes help D. armandi to respond to the host defenses. The expression profile of these detoxification enzyme genes was observed in adult beetles after feeding on different types of diet. Significant differences were observed between two types of seminatural diet containing the phloem of pines, and a purely artificial diet containing five monoterpenes ((−)-α-pinene, (−)-β-pinene, (+)-3-carene, (±)-limonene, and turpentine oil) also caused differential transcript levels in the detoxification enzyme genes. The results suggest that monoterpenes enter the beetles through different routes (i.e., respiratory and digestive systems) and cause the expression of different genes in response, which might be involved in pheromone metabolism. In addition, the xenobiotic metabolism in bark beetles should be considered as a system comprising multiple detoxifying enzymes.

Faecal microbiome analysis reveals Clostridiales and Bacteroidales as signature gut microbes during estrus of buffalo

The non-pathogenic intestinal microbes that conquer our intestines are not an accidental jumble of organisms, but rather a disparate community of microbes that coexist, and sustain a mutualistic and symbiotic relationship with the host. The gut microbiome has been shown to be influenced by animal physiology and vice versa. However, information is still scanty. The present study aimed to analyse the variation between faecal bacteria of three different stages (proestrus, estrus and postestrus) of the estrous cycle of Murrah buffalos. A phylogenetic study of buffalo faeces derived from three different stages of estrous cycle was conducted in order to compare the bacterial diversity among these three stages. We performed an exploratory microbiome analysis of buffalo faeces using 16S rRNA sequencing during these stages of the buffalo estrous cycle. A total of three bacterial phyla with six different bacterial orders and twenty-three different genera were identified among all the three comparative phases of the estrous cycle. Among them, the Clostridiales were found to be the most abundant, and Bacteroidales were present exclusive during the estrus phase. As faeces is a source of gut microbes and a non-invasive representative of the metabolic steroids and perceptible pheromones, the profiling of gut microbes during estrous cycle would provide clues towards the major microbes contributing to the perceptible pheromones during estrus stage. To the best of our knowledge, this is the first ever report describing the faecal bacterial diversity during estrous cycle of any ruminant species. Although future studies are required to understand the role of Clostridiales and Bacteroidales in faecal pheromone metabolism.

Effects of carboxylesterase gene silence on wheat aphid Sitobion avenae (Fabricius)

Multifunctional carboxylesterase (CarE) has been found in all animals, plants and microbes, and belongs to a superfamily enzyme of serine hydrolase involved in detoxification, allelochemical tolerance and some specific hormone or pheromone metabolism. Insects usually utilize carboxylesterases to detoxify xenobiotics, and positively correlated with insect resistance to some insecticides. Despite the importance of CarEs in insects, carboxylesterases and their functions in wheat aphid Sitobion avenae (Fabricius) have not been clear. In this study, a sequence that encodes a carboxylesterase protein from S. avenae (SaCarE) was sequenced and cloned. After aligning the encoded amino acid sequence of the SaCarE gene with other known CarEs of insects, we found that the CarE gene was highly conserved in insects. The SaCarE mRNA levels at different developmental stages of S. avenae were gradually increased from the first instar of nymphs to adult stage. RNAi was employed to further explore its functions, in which oral ingestion of SaCarE double-stranded RNA from the third instar nymph significantly knocked down SaCarE expression, and significantly decreased ecdysis index in S. avenae. These results indicate that SaCarE is functional in S. avenae and could serve one of the potential target genes for management of S. avenae.

Metabolic enzymes associated with xenobiotic and chemosensory responses inNasonia vitripennis

The numbers of glutathione S-transferase, cytochrome P450 and esterase genes in the genome of the hymenopteran parasitoid Nasonia vitripennis are about twice those found in the genome of another hymenopteran, the honeybee Apis mellifera. Some of the difference is associated with clades of these families implicated in xenobiotic resistance in other insects and some is in clades implicated in hormone and pheromone metabolism. The data support the hypothesis that the eusocial behaviour of the honeybee and the concomitant homeostasis of the nest environment may obviate the need for as many gene/enzyme systems associated with xenobiotic metabolism as are found in other species, including N. vitripennis, that are thought to encounter a wider range of potentially toxic xenobiotics in their diet and habitat.

Evolving protein functional diversity in new genes of Drosophila

The mechanism by which protein functional diversity expands is an important evolutionary issue. Studies of recently evolved chimeric genes permit direct investigation of the origin of new protein functions before they become obscured by subsequent evolution. Found in several African Drosophila species, jingwei (jgw), a recently evolved gene with a domain derived from the still extant short-chain alcohol dehydrogenase (ADH) through retroposition, provides an opportunity to examine this previously undescribed process directly. We expressed JGW proteins in a microbial expression system and, after purification, investigated their enzymatic properties. We found that, unexpectedly, positive Darwinian selection for amino acid replacements outside the active site of JGW produced a novel dehydrogenase with altered substrate specificity compared with the ancestral ADH. Instead of detoxifying and assimilating ethanol like its Adh parental gene, we observe that JGW efficiently utilizes long-chain primary alcohols found in hormone and pheromone metabolism. These data suggest that protein functional diversity can expand rapidly under the joint forces of exon shuffling, gene duplication, and natural selection.

Synthesis and Inhibitory Properties of Pheromone Analogs for the Epoxide Hydrolase of the Gypsy Moth

A series of analogues of disparlure, the gypsy moth (Lymantria dispar) sex attractant, was synthesized, and the potency of these inhibitors in suppressing the metabolism of disparlure by the L. dispar epoxide hydrolase (EH) was determined. The analogues substituted at the 6-position (6-hydroxy-, 6-oxo-, and 6,6-difluorodisparlure; (±)-threo,cis-11, (±)-13, and (±)-17, respectively), along with 9,9-difluorodisparlure [(±)-26], were the most potent inhibitors (IC 50 values of 4-9 μM). Two other 9-substituted analogues, 9-hydroxydisparlure [(±)-threo,cis-21] and 9-oxodisparlure [(±)-22], were slightly less potent (IC 50 values of 18 and 30 μM, respectively). Analogues substituted at both the 6- and 9-positions (threo,erythro-6,9-dihydroxy-, threo,threo-6,9-dihydroxy-, and 6,9-dioxodisparlure; (±)-threo,erythro-32, (±)-33, respectively) were generally the least potent inhibitors (IC 50 values of 27-200 μM). On the basis of a model of the EH active site, a hypothesis is advanced to rationalize the higher potencies of the 6-substituted analogues.Pheromone metabolism plays a key role in pheromone perception, and the potential consequences of inhibition of pheromone metabolism are discussed

Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone

A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes.

Binding and hydrolysis of radiolabeled pheromone and several analogs by male-specific antennal proteins of the mothAntheraea polyphemus

Sensory hair proteins from antennae of males of the wild silk moth,Antheraea polyphemus (Lepidoptera, Saturniidae) were incubated with radiolabeled 6E,11Z-hexadecadienyl acetate in the presence of unlabeled pheromone analogs as competitive inhibitors. The two extracellular proteins of importance, a highly active sensillar esterase and an abundant 15,000 mol wt binding protein, interact to degrade labeled pheromone less efficiently in the presence of certain unsaturated acetate analogs of the natural pheromone.Enzymatic hydrolysis of the acetate (or diazoacetate) was also examined for three pheromone analogs: [11,12-(3)H2]-6E,11Z-hexadecadienyl diazoacetate, [11,12-(3)H2]-hexadecyl acetate, and [9,10-(3)H2]-9Z-tetradecenyl acetate. The former two are poor substrates at concentrations over four orders of magnitude. The 9Z-14:Ac, however, is the best alternative substrate for this in vitro pheromone metabolism system. Unlabeled 9Z-14: Ac is also the best competitive inhibitor of the hydrolysis of labeled 6E, 11Z-16: Ac. Whereas the tritiated natural pheromone shows a flat response (ca. 40% conversion) to increasing concentrations from 3 × 10(-9) to 3 × 10(-6) M, tritiated 9Z-14: Ac is degraded more rapidly at higher concentrations.

Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.

Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides.

A trypsin-type endopeptidase (Kamiya et al., Biochem. Biophys. Res. Commun. 94:855-860, 1980) responsible for the metabolism of rhodotorucine A, the farnesyl undecapeptide mating pheromone secreted by mating type A cells of Rhodosporidium toruloides, was biologically characterized. Metabolic activity was found to be present exclusively on the cell surface of the pheromone target cell. The activity was highly specific to the pheromone, and a biologically inactive analog which has the complete amino acid sequence of rhodotorucine A but lacks the farnesyl residue was not metabolized by intact cells. Pheromone metabolism was inhibited by trypsin substrates such as tosyl-L-arginine methyl ester. The presence of tosyl-L-arginine methyl ester strongly inhibited the sexual differentiation induced by the pheromone at a concentration which did not affect the vegetative growth of R. toruloides. Pheromone-induced sexual differentiation was also strongly inhibited by a metabolizable analog, rhodotorucine A S-oxide, but not by a non-metabolizable one. In mutants defective in early processes of mating, the decrease in the pheromone metabolic activity correlated well with the extent of loss of sensitivity to the pheromone. Both the pheromone metabolism and the capacity for sexual differentiation of a sterile mutant were restored concomitantly with reversion from the sterile to the fertile phenotype. These results suggested that metabolism of the mating pheromone plays an essential role in the process of sexual differentiation in R. toruloides.

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster Substrate specificity of the alleloenzymes Adh S and Adh UF

The substrate specificity of the two alleloenzymes Adh S and Adh UF from Drosophila melanogaster has been studied and found to be similar. With most of the secondary alcohols, the V m value is essentially the same, and indicative of a Theorell-Chance mechanism with rate-limiting enzyme-coenzyme dissociation. The experiments indicate that the enzyme-coenzyme complex formed with Adh UF dissociates at a faster rate than the corresponding complex with Adhs s. For primary alcohols the V m value is much lower than for secondary alcohols, varies with the type of alcohol and the dissociation of the enzyme-coenzyme complex is not rate limiting. For these alcohols a primary isotope effect with deuteroethanol indicates that it is the interconversion of the ternary complexes that is rate determining. Studies with the enantiomers of butan-2-ol and octan-2-ol show that both alkyl groups in the secondary alcohols interact hydrophobically with the alcohol-binding region of the active site. However, the two parts of the alcohol-binding region that interact with the two alkyl groups are of different size. The high activity observed with secondary alcohols and especially with ( R)-(+)- cis-verbenol, indicates that these flies can metabolize terpenes. Such compounds may be part of the pheromone system in the flies with D. melanogaster alcohol dehydrogenase playing a role in pheromone metabolism.

The secretory cycle of a gland involved in pheromone production in the noctuid moth, Pseudaletia separata

The structure of the pheromone producing gland is briefly described. Examination of variations in gland volume, nucleus to cytoplasm ratio, and distribution and form of nucleic acids suggests that the secretory cycle of this gland consists of three phases. Two types of small vesicle are found and suggested as possible sites for pheromone metabolism.