Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.     

The devil in the details of life-history evolution: Instability and reversal of genetic correlations during selection on<Emphasis Type="Italic">Drosophila</Emphasis> development

The evolutionary relationships between three major components of Darwinian fitness, development rate, growth rate and preadult survival, were estimated using a comparison of 55 distinct populations ofDrosophila melanogaster variously selected for age-specific fertility, environmental-stress tolerance and accelerated development. Development rate displayed a strong net negative evolutionary correlation with weight at eclosion across all selection treatments, consistent with the existence of a size-versus-time tradeoff between these characters. However, within the data set, the magnitude of the evolutionary correlation depended upon the particular selection treatments contrasted. A previously proposed tradeoff between preadult viability and growth rate was apparent only under weak selection for juvenile fitness components. Direct selection for rapid development led to sharp reductions in both growth rates and viability. These data add to the mounting results from experimental evolution that illustrate the sensitivity of evolutionary correlations to (i) genotype-by-environment (G X E) interaction, (ii) complex functional-trait interactions, and (iii) character definition. Instability, disappearance and reversal of patterns of genetic covariation often occur over short evolutionary time frames and as the direct product of selection, rather than some stochastic process. We suggest that the functional architecture of fitness is a rapidly evolving matrix with reticulate properties, a matrix that we understand only poorly.     

Structurally distinct recognition motifs in lymphotoxin-beta receptor and CD40 for tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.     

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.     

Role of XRCC1 in the coordination and stimulation of oxidative DNA damage repair initiated by the DNA glycosylase hOGG1

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase beta. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.