The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins

Pairwise sequence alignment of mammalian JIP1 and Drosophila melanogaster APLIP1 [UniProt:Q9UQF2 and UniProt:Q9W0K0, respectively]. (DOCX 24 kb

Micro-plasticity of genomes as illustrated by the evolution of glutathione transferases in 12 Drosophila species.

Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family

A sensitive core region in the structure of glutathione S-transferases.

A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, site-directed mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25 degrees C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater K (m) towards GSH and a differing K (m) towards CDNB. The V (max) ranged from 44.7 to 87.0 micromol/min per mg (wild-type, 44.7 micromol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu-103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit-subunit interface. Finally, the data suggest that Leu-103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability

Structural Biology and Its Applications to the Health Sciences

Part of the decipherment of genomic information lies in understanding the structure and function of the protein products of these genes. Protein structure is of further importance because of the molecular basis of many diseases. Structural biology is the field of research focusing on the experimental determination of the structure of biological molecules. We review the field of structural biology and its application to medical research and drug discovery, and describe the structural results recently obtained in our laboratory for the detoxifying enzyme glutathione S-transferase from the Asian mosquito Anopheles dirus species B, an important malaria vector. These enzymes have detoxifying activity toward pesticides and thus contribute to pesticide resistance in insects. Since the first protein structure (of sperm whale myoglobin) was determined (1) and Watson and Crick discovered the double helix structure of DNA (2), there has been an ever-increasing research effort in the field of structural biology. Broadly, structural biology is defined as the investigation of the structure and function of biological systems at the molecular level. The significance of this field of research in part derives from the fact that macromolecular structure is important to many disease states. Sickle-cell anemia was recognized to be a result of a mutation in hemoglobin, causing it to polymerize into long rod-shape complexes that distort and destroy red blood cells (3). The structural consequence of this mutation is now understood (4). Today, many molecular diseases, such as cancer, are known to result from mutations of genes that affect the gene product, altering its function. This is invariably caused by changes in the structure and function of the protein product of the gene. Understanding these processes can be important for treating the disease. Information about protein structure can be used in so-called structure-based drug design, where a macromolecular structure is used as a template for drug design. This review examines the field of structural biology and its relevance to medicine and drug discovery. Typically, protein crystallography and nuclear magnetic resonance (NMR) spectroscopy have been the tools of choice for the structural biologist. Briefly described here are the two main techniques for macromolecular structure determination. X-ray Crystallography X-ray crystallography can give atomic resolution structure of proteins and other macromolecules, such as DNA and their complexes. The technique requires the availability of milligram quantities of >99% pure macromolecule, usually produced through cloning and overexpression in bacterial plasmids, and then purified through the standard techniques of biochemistry, such as gel filtration, affinity chromatography, and ion exchange chromatography. The macromolecule must then be crystalized. This is usually achieved through the addition of the protein to a precipitant, such as ammonium sulfate or polyethylene glycol. Extensive trials of numerous potential conditions are often required to find a condition that gives crystals of sufficiently high quality for X-ray analysis. The next step is to place the crystal in an X-ray beam produced by a laboratory source or at a synchrotron radiation source, such as those located in Trieste (Italy) or Argonne (USA). Crystals diffract X-rays, and the resulting pattern of scattered X-rays is processed computationally to reveal the electron density of the molecule subject. This technique is reviewed in detail elsewhere (5). Nuclear Magnetic Resonance Spectroscopy NMR is another important tool for probing the structure of biological molecules. Like X-ray crystallography, this technique can provide three-dimensional structural information, but the underlying method is completely different. Like X-ray crystallography, the technique requires milligram quantities of www.cmj.hr 37

Phylogenetic tree analysis.

<p>The phylogenetic tree analysis used sequences from the Omega class from the 12 <i>Drosophila</i> species. Each isoform is designated by their FlyBase symbol followed by the <i>D. melanogaster</i> ortholog name in parenthesis. For example, Dana\GF10159(O1), where O1 refers to the DmelGST Omega 1 ortholog.</p

The effects of differential induction of cytochrome P-450, carboxylesterase and glutathione S-transferase activities on malathion toxicity in mice

The organophosphorous pesticide malathion is metabolized by three hepatic enzyme systems: the microsomal cytochrome P-450-dependent monooxygenase system, the microsomal carboxylesterases, and the cytosolic glutathione S-transferases. We produced differential induction of these three enzyme systems in mice with phenobarbital and 2(3)-tert-butyl-4-hydroxyanisole (BHA) and examined the effects of the induction on the inhibition of acetylcholinesterases by malathion. Phenobarbital not only significantly induced hepatic microsomal cytochrome P-450 (p < 0.05) but also increased microsomal carboxylesterase activity (p < 0.05). BHA not only increased the activity of microsomal carboxylesterases (p < 0.05) but also substantially increased cytosolic glutathione S-transferase activity (p < 0.05). Despite the differential effects of phenobarbital and BHA on the three enzyme systems, neither agent protected the mice against malathion toxicity

Glutathione transferases in the 12 <i>Drosophila</i> species.

<p>The numbers of genes and proteins transcribed are from FlyBase annotations and the present curation. The present curation numbers include proposed corrections for genes and protein expressions.</p><p>Glutathione transferases in the 12 <i>Drosophila</i> species.</p