Neural mechanisms of context-dependent processing of CO2 avoidance behavior in fruit flies

The fruit fly, Drosophila melanogaster, innately avoids even low levels of CO₂. CO₂ is part of the so-called Drosophila stress odor produced by stressed flies, but also a byproduct of fermenting fruit, a main food source, making the strong avoidance behavior somewhat surprising. Therefore, we addressed whether feeding states might influence the fly’s behavior and processing of CO₂. In a recent report, we showed that this innate behavior is differentially processed and modified according to the feeding state of the fly. Interestingly, we found that hungry flies require the function of the mushroom body, a higher brain center required for olfactory learning and memory, but thought to be dispensable for innate olfactory behaviors. In addition, we anatomically and functionally characterized a novel bilateral projection neuron connecting the CO₂ sensory input to the mushroom body. This neuron was essential for processing of CO₂ in the starved fly but not in the fed fly. In this Extra View article, we provide evidence for the potential involvement of the neuromodulator dopamine in state-dependent CO₂ avoidance behavior. Taken together, our work demonstrates that CO₂ avoidance behavior is mediated by alternative neural pathways in a context-dependent manner. Furthermore, it shows that the mushroom body is not only involved in processing of learned olfactory behavior, as previously suggested, but also in context-dependent innate olfaction.     

Assessment of changes in the brca2 and p53 genes in breast invasive ductal carcinoma in northeast Brazil

Background

BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models.

Results

BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant.

Conclusions

There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.     

Characterization of oxylipins and dioxygenase genes in the asexual fungus Aspergillus niger

Background  

Aspergillus niger is an ascomycetous fungus that is known to reproduce through asexual spores, only. Interestingly, recent genome analysis of A. niger has revealed the presence of a full complement of functional genes related to sexual reproduction [1]. An example of such genes are the dioxygenase genes which in Aspergillus nidulans, have been shown to be connected to oxylipin production and regulation of both sexual and asexual sporulation [2–4]. Nevertheless, the presence of sex related genes alone does not confirm sexual sporulation in A. niger.     

Structural and Functional Analysis of Campylobacter jejuni PseG: A UDP-SUGAR HYDROLASE FROM THE PSEUDAMINIC ACID BIOSYNTHETIC PATHWAY*

Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His¹⁷-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His¹⁷ functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C–O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.The gastrointestinal pathogens Campylobacter jejuni and Helicobacter pylori have been shown to modify their flagellins with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid or pseudaminic acid (Pse),³ via O-linkage at up to 19 sites per flagellin monomer (1, 2). Not only is this sialic acid-like modification necessary for flagellar assembly and motility (1, 2), it has also been shown to be important for C. jejuni virulence (3). In addition to its role in autoagglutination of bacterial cells, Pse and related derivatives may also influence pathogenesis through bacterial adhesion, invasion, and immune evasion (4, 5), since sialic acids in humans have been shown to mediate a myriad of cell-cell and cell-molecule interactions (6). As flagellin glycosylation in these organisms is required for host colonization and ultimately virulence (3, 7, 8), these novel sugar biosynthetic pathways provide an excellent platform for therapeutic development.The reliance of H. pylori pathogenicity on Pse biosynthesis, in combination with the prevalence of H. pylori resistance to existing antibiotic treatments (9), prompted and led to the complete elucidation of the CMP-pseudaminic acid (CMP-Pse) biosynthetic pathway in both C. jejuni and H. pylori (10–15). The CMP-Pse biosynthetic pathway (Fig. 1) is similar to that of CMP-sialic acid, involving condensation of an N-acetylhexosamine intermediate with the three-carbon pyruvate molecule forming a nine-carbon sialic acid-like nonulosonate, although in contrast the CMP-Pse pathway consists of several more steps between the initial building block UDP-GlcNAc and the condensation reaction. PseG, a UDP-sugar hydrolase, produces the final 6-deoxy-N-acetylhexosamine intermediate in the CMP-Pse pathway by removing the nucleotide moiety from UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose or UDP-6-deoxy-AltdiNAc (Fig. 1). This sort of single enzymatic function is rare in nature, with the only other similar example being a GDP-mannose/GDP-glucose hydrolase (16), which belongs to the metal-dependent Nudix family of enzymes. In an elegant study, Liu and Tanner (11) demonstrated that PseG catalyzes nucleotide removal by a metal-independent C–O bond cleavage mechanism resulting in inversion of stereochemistry at C-1 of the product 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose or 6-deoxy-AltdiNAc, similar to the catalytic properties of some GT-B glycosyltransferases.Open in a separate windowFIGURE 1.Role of PseG within the CMP-pseudaminic acid biosynthetic pathway of C. jejuni and H. pylori. The biosynthetic step involving PseG is highlighted in blue. The enzymes and biosynthetic intermediates of the CMP-pseudaminic acid pathway are, in the following order, PseB (Cj1293/HP0840), NADP-dependent dehydratase/epimerase; PseC (Cj1294/HP0366), pyridoxal phosphate-dependent aminotransferase; PseH (Cj1313/HP0327), N-acetyltransferase; PseG (Cj1312/HP0326B), NDP-sugar hydrolase; PseI (Cj1317/HP0178), pseudaminic acid synthase; PseF (Cj1311/HP0326A), CMP-pseudaminic acid synthetase; and I, UDP-GlcNAc; II, UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose; III, UDP-4-amino-4,6-dideoxy-β-l-AltNAc; IV, UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose; V, 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose; VI, pseudaminic acid; and VII, CMP-pseudaminic acid. Here, PEP refers to phosphoenolpyruvate. Pyranose rings are shown as their predominant chair conformation in solution as determined from nuclear Overhauser effects and J_H,H coupling constants (13).Together, glycosyltransferases and glycoside hydrolases compose the majority of enzymes in both eukaryotes and prokaryotes that manipulate glycosidic bonds. Glycosyltransferases of the Leloir classification use sugar-nucleotide derivatives as glycosyl donors resulting in transfer to acceptors such as a monosaccharide, oligosaccharide, or polysaccharide. It is therefore plausible that a “glycosyltransferase fold” in PseG has evolved to efficiently utilize water as an acceptor, instead of another carbohydrate, consequently behaving as a hydrolase (11). Based on structure, most glycosyltransferases fall into two groups, GT-A and GT-B, that exhibit different folds, respectively (17). For both families, depending on the particular enzyme, the outcome may result in either inversion or retention of stereochemistry for the donor anomeric carbon (see Fig. 2). In addition, GT-B family enzymes are metal-independent, lacking an important DXD motif present in most GT-A members. Based on the novelty of PseG and its role in H. pylori pathogenicity, we sought a greater structural and mechanistic understanding of this important enzyme.Open in a separate windowFIGURE 2.Functional comparison of enzymes belonging to the GT-B superfamily. A, UDP-sugar hydrolase PseG catalyzes the removal of UDP from UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-Alt or UDP-6-deoxy-AltdiNAc. B, UDP-GlcNAc hydrolyzing 2-epimerase NeuC catalyzes the removal of UDP and the formation of ManNAc from UDP-GlcNAc. C, GlcNAc transferase MurG catalyzes the formation of undecaprenyl-phosphoryl-muramyl-pentapeptide-GlcNAc via formation of a glycosidic linkage between UDP-GlcNAc and undecaprenyl-phosphoryl-muramyl-pentapeptide. R represents the phosphoryl-undecaprenyl moiety, with the pentapeptide having the specific sequence l-Ala-d-γGlu-l-Lys-d-Ala-d-Ala. Both A and C activities result in an initial inversion of stereochemistry at C-1 for the donor substrate. In contrast, the activity for B results in an initial retention of C-1 stereochemistry. Enzymatically altered anomeric bonds are indicated in red.Here we report the crystal structure of PseG alone at 1.8 Å resolution and in complex with UDP, a product of the reaction, at 1.85 Å resolution. Although very few homologs have been identified based on sequence similarity alone, PseG bears the closest structural similarity to MurG, a GT-B family member (18). In addition, computational docking and molecular dynamics simulations were performed to gain insight into the binding mode of the PseG substrate UDP-6-deoxy-AltdiNAc. Based on the crystallographic and modeled structures, several potential active site residues were selected for mutagenesis and kinetic analyses to further characterize the PseG active site. The relevance of these findings to the structurally related MurG family of enzymes is discussed.     

Mechanistic studies on PseB of pseudaminic acid biosynthesis: a UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase

UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5″ stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP⁺ to oxidize UDP-GlcNAc at C-4″, enabling dehydration. The α,β unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6″ and a proton to C-5″. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5″ position of product during catalysis in D₂O. Likewise, PseB catalyzes solvent isotope exchange into the H-5″ position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.     

Conserved SUN-KASH Interfaces Mediate LINC Complex-Dependent Nuclear Movement and Positioning

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Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.     

An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function

The energy-dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild-type cells but not in cells producing either protein alone. Radiolabelling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non-ionic detergents. A G229D substitution in the kinase-3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase-1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA–ExeB complex, but overexpression of the mutant complex until wild-type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA–ExeB complex formation, while hydrolysis is required for its function in secretion once established.