Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs

It's all about the children: a participant-driven photo-elicitation study of Mexican-origin mothers' food choices

Abstract Background There is a desperate need to address diet-related chronic diseases in Mexican-origin women, particularly for those in border region colonias (Mexican settlements) and other new destination communities in rural and non-rural areas of the U.S. Understanding the food choices of mothers, who lead food and health activities in their families, provides one way to improve health outcomes in Mexican-origin women and their children. This study used a visual method, participant-driven photo-elicitation, and grounded theory in a contextual study of food choices from the perspectives of Mexican-origin mothers. Methods Teams of trained promotoras (female community health workers from the area) collected all data in Spanish. Ten Mexican-origin mothers living in colonias in Hidalgo County, TX completed a creative photography assignment and an in-depth interview using their photographs as visual prompts and examples. English transcripts were coded inductively by hand, and initial observations emphasized the salience of mothers' food practices in their routine care-giving. This was explored further by coding transcripts in the qualitative data analysis software Atlas.ti. Results An inductive conceptual framework was created to provide context for understanding mothers' daily practices and their food practices in particular. Three themes emerged from the data: 1) a mother's primary orientation was toward her children; 2) leveraging resources to provide the best for her children; and 3) a mother's daily food practices kept her children happy, healthy, and well-fed. Results offer insight into the intricate meanings embedded in Mexican-origin mothers' routine food choices. Conclusions This paper provides a new perspective for understanding food choice through the eyes of mothers living in the colonias of South Texas -- one that emphasizes the importance of children in their routine food practices and the resilience of the mothers themselves. Additional research is needed to better understand mothers' perspectives and food practices with larger samples of women and among other socioeconomic groups

Olfactory Response of the Predatory Bug Orius laevigatus (Hemiptera:Anthocoridae) to the Aggregation Pheromone of Its Prey, Frankliniella occidentalis (Thysanoptera: Thripidae)

Herbivore natural enemies base their foraging decision on information cues from different trophic levels but mainly from plant odors. However, the second trophic level (i.e., the herbivorous prey) may also provide reliable infochemical cues for their natural enemies. We have evaluated the role of the aggregation pheromone from Frankliniella occidentalis (Pergande) as a potential kairomone for its natural enemy, the predatory bug Orius laevigatus (Fieber). For this purpose, we have analyzed the response of O. laevigatus to (R)-lavandulyl acetate and neryl (S)-2-methylbutanoate, the two major components of the thrips aggregation pheromone. These compounds have been offered to O. laevigatus adult females and nymphs of the predatory bugs both in separate and as specific (1:1 or 1:2.3) blends, in experiments involving a dual choice Y-tube olfactometer. None of the compounds attracted adults or nymphs when they were individually supplied. Conversely, they were significantly attracted to both adults and nymphs when offered as a blend. A 1:2.3 (R)-lavandulyl acetate:neryl (S)-2-methylbutanoate blend was attractive to both nymphs and adults, while a 1:1 blend elicited response only in nymphs. These results suggest that specific blends of these compounds from the aggregation pheromone may be used as an attractant to O. laevigatus. The results of this work highlight the importance of studying olfactory responses of natural enemies for a better understanding of their foraging behavior. Potential uses of these results in future studies are discussed

The N-terminal helix of MarA as a key element in the mechanism of DNA binding

Inputs to run regular and GaMD of free MarA, MarA-mar and Rob-mar complexes, as well as the starting structures used in all the simulations

Modulation of the glycine response by Ca2+-permeable AMPA receptors in rat spinal neurones

In acutely isolated rat sacral dorsal commisural nucleus (SDCN) neurones, application of kainate (KA) reversibly potentiated glycine-evoked Cl− currents (IGly) in a concentration-dependent manner.The cellular events underlying the interaction between non-NMDA receptors and glycine receptors were studied by using nystatin-perforated patch and cell-attached single-channel recording modes.The action of KA was not accompanied by a shift in the reversal potential for IGly. In dose-response curves, KA potentiated IGly without significantly changing glycine binding affinity.GYKI 52466 blocked while NS-102 had no effect on the KA-induced potentiation of IGly.The potentiation was reduced when KA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels.Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the action of KA. Inhibition of calcineurin converted the KA-induced potentiation to a sustained one.Single-channel recordings revealed that KA decreased the mean closing time of glycine-gated single-channel activity, resulting in an increase in the probability of channel opening.It is proposed that Ca2+ entry through AMPA receptors modulates the glycine receptor function via coactivation of CaMKII and calcineurin in SDCN neurones. This interaction may provide a new postsynaptic mechanism for control of inhibitory synaptic signalling and represent one of the important regulatory mechanisms of spinal nociception

Binding site stoichiometry and the effects of phosphorylation on human α1 homomeric glycine receptors

The kinetic properties of the human α1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are ‘silent’, possibly due to strong negative cooperativity