Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: In vivo and modeling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programs. Diacylhydrazine (DAH)-based ecdysone agonists also known as molting-accelerating compounds (MACs) are considered a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project in vivo toxicity assays demonstrated that the DAH-based RH-5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand-binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced and a homology protein model was constructed which confirmed a cavity structure with 12 ?-helixes, harboring the natural insect molting hormone 20-hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH-based insecticides due to a restricted extent of the ligand-binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modeling/docking experiments are indications that these pesticides do not bind with the LBD-EcR of O. laevigatus and support that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programs. Keywords: Orius laevigatus, selectivity, diacylhydrazine insecticides, ecdysone receptor, homology modelling, docking studies.

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR)might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine-based compounds methoxyfenozide, tebufenozide and RH-5849 showed different results. Methoxyfenozide and tebufenozide did not provoke anynegative effectsontheadults,but RH-5849 killed98-100%of the treated insects15 days after treatment. Theligand-binding domain (LBD) of the EcR of B. oleae (BoEcR-LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand-binding cavity of the BoEcR-LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH-5849. CONCLUSION: IGR molecules similar to the RH-5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest.

Compatibilidad de Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) y Nesidiocoris tenuis (Reuter)(Hemiptera: Miridae), depredadores importantes en cultivos hortícolas protegidos, con nuevas barreras físicas selectivas y modernos plaguicidas

La nueva legislación en materia fitosanitaria se dirige hacia una Gestión Integrada de Plagas (GIP). Estos programas dan preferencia a aquellos métodos más respetuosos y sostenibles con el medio ambiente, siendo piezas claves en ellos el control biológico, el físico y otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos es a veces necesario para el adecuado manejo de plagas en cultivos hortícolas. Por ello, el objetivo general de este estudio es aportar conocimientos para mejorar el control de plagas en cultivos hortícolas, mediante la integración de tres estrategias de lucha: biológica, física y química. Una parte de este trabajo ha consistido en el estudio de los posibles efectos que mallas tratadas con insecticida (bifentrin) pudieran provocar mediante diferentes ensayos de laboratorio, invernadero y campo, en los enemigos naturales Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) (depredador de trips), Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) (depredador de mosca blanca y Tuta absoluta (Meirick) (Lepidoptera: Gelechiidae)), y otros agentes de biocontrol comúnmente usados en cultivos hortícolas protegidos. Este tipo de mallas se han empleado con éxito en entomología médica para controlar mosquitos vectores de la malaria, y actualmente se está trabajando en su desarrollo para uso agrícola como método de exclusión, y método directo de control de plagas. En los ensayos realizados en laboratorio, O. laevigatus y N. tenuis no fueron capaces de detectar la presencia de bifentrin en el ensayo de preferencia. Además, no se produjo mortalidad a corto plazo (72 horas) en ambos chinches depredadores. Por el contrario, se registró una elevada mortalidad cuando se expusieron por contacto a la malla tratada durante 72 horas en cajas de dimensiones reducidas (10 cm de diámetro X 3 cm de altura). En ensayos llevados a cabo bajo condiciones más reales de exposición, en un invernadero experimental con jaulas de 25 X 25 X 60 cm de altura, no se produjo ningún efecto en la mortalidad a corto plazo (72 horas) o en los parámetros reproductivos de O. laevigatus y N. tenuis. Finalmente, en ensayos de campo realizados en túneles semi-comerciales (8 m de largo X 6,5 m de ancho X 2,6 m de altura), ni las condiciones ambientales [temperatura, humedad relativa, radiación ultravioleta (UV) y fotosintéticamente activa (PAR)], ni los enemigos naturales, se vieron afectados por la presencia de la malla tratada con bifentrin en el cultivo. Sin embargo, los resultados no fueron concluyentes, debido al bajo establecimiento de los agentes de biocontrol liberados. Por lo tanto, más estudios son necesarios en invernaderos comerciales para confirmar los resultados preliminares de compatibilidad. Además, en este trabajo se han evaluado los efectos letales (mortalidad) y subletales (parámetros reproductivos) de seis modernos insecticidas sobre los chinches depredadores O. laevigatus y N. tenuis, mediante ensayos de laboratorio y persistencia. Los ensayos se realizaron por contacto residual, aplicando los insecticidas a la dosis máxima de campo sobre placas de cristal (laboratorio) o plantas (persistencia). Los productos fitosanitarios se seleccionaron por representar a un grupo de modernos plaguicidas con modos de acción en principio más selectivos para los enemigos naturales que antiguos plaguicidas como organoclorados, oroganofosforados o carbamatos, y por su uso frecuente en cultivos hortícolas donde O. laevigatus y N. tenuis están presentes. Todos ellos están incluidos o en proceso de inclusión en la lista comunitaria de sustancias activas para uso agrícola, Anexo I de la Directiva 91/414/CEE: abamectina y emamectina (avermectinas neurotóxicas, activadoras del canal del cloro), deltametrina (piretroide neurotóxico, modulador del canal del sodio, control positivo), flubendiamida (neurotóxico, modulador del receptor de rianodina), spinosad (naturalito neurotóxico, agonistas/antagonistas del receptor de nicotínico acetilcolina) y spiromesifen (inhibidor de la acetil CoA carboxilasa). El estudio mostró que O. laevigatus fue más susceptible a los insecticidas que N. tenuis. Además, los resultados revelaron que flubendiamida y spiromesifen fueron compatibles con los dos enemigos naturales estudiados, y por tanto se podrían usar en programas de GIP. Por el contrario, los insecticidas abamectina, deltametrina, emamectina y spinosad no fueron selectivos para ninguno de los chinches depredadores. Sin embargo, los estudios de persistencia demostraron que a pesar de que estos insecticidas no proporcionaron selectividad fisiológica, pueden proporcionar selectividad ecológica en algunos casos. Abamectina, deltametrina, emamectina y spinosad podrían ser compatibles con N. tenuis si el enemigo natural es introducido en el cultivo 4 días después de su aplicación. En el caso de O. laevigatus, abamectina, deltametrina y spinosad se clasificaron como persistentes, por lo tanto es necesario completar el estudio con experimentos de semi-campo y campo que determinen si es posible su uso conjunto en programas de GIP. Por otro lado, emamectina podría ser compatible con O. laevigatus si el enemigo natural es introducido en el cultivo 7 días después de su aplicación. Por último, se ha comprobado la selectividad de tres insecticidas aceleradores de la muda (MACs) (metoxifenocida, tebufenocida y RH-5849) sobre O. laevigatus y N. tenuis. Además de realizar estudios para evaluar la toxicidad en laboratorio de los insecticidas por contacto residual e ingestión (principal modo de acción de los MAC´s), se extrajo RNA de los insectos y con el cDNA obtenido se secuenció y clonó el dominio de unión al ligando (LBD) del receptor de ecdisona correspondiente a O. laevigatus (OlEcR-LBD) y N. tenuis (NtEcR-LBD). Posteriormente, se obtuvo la configuración en tres dimensiones del LBD y se estudió el acoplamiento de las moléculas de los tres insecticidas en la cavidad que forman las 12 α-hélices que constituyen el EcR-LBD. En el caso de N. tenuis se debe mencionar que no fue posible la obtención de la secuencia completa del LBD. Sin embargo, se obtuvo una secuencia parcial (hélice 6-hélice 11), que mostró una alta conservación de aminoácidos con respecto a la obtenida en O. laevigatus. Los ensayos de toxicidad mostraron que metoxifenocida, tebufenocida y RH-5849 no produjeron ningún efecto nocivo en ambos depredadores. Además, los estudios de modelado por homología y acoplamiento molecular llevados a cabo con O. laevigatus, también indicaron que los MACs no produjeron ningún efecto deletéreo en este enemigo natural. Por lo tanto, estos compuestos pueden ser aplicados de manera segura en programas de GIP en los cuales O. laevigatus y N. tenuis estén presentes. ABSTRACT The new pesticide legislation on pest control is aimed at integrated pest management (IPM). These programs are based on the most environmentally sustainable approaches, where biological, physical control and other non-chemical methods are the cornerstone. However, selective pesticides are often required for pest management on horticultural crops. Therefore, the main goal of this study is to provide knowledge to improve pest control on horticultural crops through the integration of three strategies: biological, physical and chemical. Firstly, the effects of insecticide treated nets (bifenthrin) were evaluated in different laboratory, greenhouse and field experiments on the natural enemies Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) (predator of thrips), Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) (predator of whiteflies and Tuta absoluta (Meirick) (Lepidoptera: Gelechiidae)), and other biocontrol agents commonly used on protected horticultural crops. These types of nets have been successfully used in medical entomology to control mosquito malaria vectors, and work is currently being done on their use as exclusion barriers and as a direct method of pest control in agriculture. In experiments made under laboratory conditions, O. laevigatus and N. tenuis were not able to detect the presence of bifenthrin in a dual-choice test. Furthermore, no shortterm mortality (72 hours) was recorded on both predatory bugs. In contrast, a high mortality rate was found when they were exposed by contact to the bifenthrin-treated net for 72 hours in small cages (10 cm diameter X 3 cm high). In assays carried out under more realistic conditions of exposure, in an experimental greenhouse with cages of 25 X 25 X 60 cm high, short-term mortality (72 hours) and reproductive parameters were not affected. Lastly, in field experiments carried out in semi-commercial tunnels (8 m long X 6.5 m width X 2.6 m high), neither environmental conditions [temperature, relative humidity, ultraviolet (UV) and photosynthetically active radiation (PAR)] nor natural enemies were affected by the presence of the bifenthrin-treated net on the crop. However, results were not conclusive, mainly due to a low settlement of the released biocontrol agents, and further studies are needed in commercial greenhouses to confirm our preliminary results of compatibility. Secondly, the lethal (mortality) and sublethal effects (reproductive parameters) of six modern pesticides on the predatory bugs O. laevigatus and N. tenuis has been evaluated through laboratory and persistence experiments. Trials were carried out by residual contact, applying the insecticides to the maximum field recommended concentration on glass plates (laboratory) or plants (persistence). Insecticides were chosen as representatives of modern pesticides with a more selective mode of action on natural enemies than organochlorine, organophosphorus and carbamate insecticides. Moreover, they were also chosen because of their frequent use on horticultural crops where O. laevigatus and N. tenuis are present. All of them have been included or have been requested for inclusion in the community list of active substances on the agricultural market, Annex I of the European Directive 91/414/EEC: abamectin and emamectin (neurotoxic avermectins, chloride channel activators), deltamethrin (neutotoxic pyrethroid, sodium channel modulator, positive commercial standard), flubendiamide (neurotoxic, rianodine receptor modulator), spinosad (neurotoxic naturalyte, nicotinic acetylcholine receptor allosteric activator) and spiromesifen (inhibitors of acetyl CoA carboxylase). The study showed that O. laevigatus was more susceptible to all the studied pesticides than N. tenuis. In addition, the research results indicated no impact of flubendiamide and spiromesifen on the two natural enemies studied under laboratory conditions. Consequently, both pesticides are candidates to be included in IPM programmes where these biocontrol agents are present. On the other hand, abamectin, deltamethrin, emamectin and spinosad were not selective for both predatory bugs in laboratory experiments. However, persistence test demonstrated that in spite of the lack of physiological selectivity, these pesticides can provide ecological selectivity in some cases. Abamectin, deltamethrin, emamectin and spinosad could be compatible with N. tenuis if the mirid bug is released 4 days after the insecticide treatment on the crop. With regard to O. laevigatus, abamectin, deltamethrin and spinosad were classified as persistent in our assays, thus the study should be completed with semi-field and field experiments in order to ascertain their possible joint use in IPM programs. In contrast, emamectin could be compatible with O. laevigatus if the pirate bug is released 7 days after the insecticide treatment on the crop. Finally, the selectivity of three moulting accelerating compounds (MACs) (methoxyfenozide, tebufenozide and RH-5849) has also been evaluated on O. laevigatus and N. tenuis. In addition to laboratory experiments to evaluate the toxicity of the insecticides by residual contact and ingestion, molecular approaches were used as well. RNA of both insects was isolated, cDNA was subsequently synthesized and the complete sequence of the ligand binding domain (LBD) of the ecdysone receptor of O. laevigatus (OlEcR-LBD) and N. tenuis (NtEcR-LBD) were determined. Afterwards, the three dimensional structure of LBD was constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the EcRLBD was performed. In the case of N. tenuis, it should be noted that in spite of intensive efforts, we did not manage to complete the sequence for the LBD.However, a partial sequence of the LBD was obtained (helix 6-helix 11), and a strong conservation between the amino acids of N. tenuis and O. laevigatus was observed. Results showed no biological activity of methoxyfenozide, tebufenozide and RH-5849, on both predatory bugs. Moreover, modeling of the OlEcR-LBD and docking experiments also suggested that MACs were devoid of any deleterious effect on O. laevigatus. Therefore, our results indicate that these compounds could be safely applied in IPM programs in which O. laevigatus and N. tenuis are present.

The family of DOF transcription factors in Brachypodium distachyon: BdDof24 in germinating seeds

The DOF (DNA binding with One Finger) transcription factor (TF) family is characterized by a binding domain of 52 amino acid residues that is structured as a Cys2/Cys2 Zn2+ finger that recognizes the common core 5?-T/AAAAG-3? in the promoter regions of their target genes. DOF TFs have been associated with biological processes exclusive to higher plants and their close ancestors (algae, mosses and ferns).

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

ADD1/SREBP1 activates PPARγ through the production of endogenous ligand

Adipose differentiation is an important part of the energy homeostasis system of higher organisms. Recent data have suggested that this process is controlled by an interplay of transcription factors including PPARγ, the C/EBPs, and ADD1/SREBP1. Although these factors interact functionally to initiate the program of differentiation, there are no data concerning specific mechanisms of interaction. We show here that the expression of ADD1/SREBP1 specifically increases the activity of PPARγ but not other isoforms, PPARα, or PPARδ. This activation occurs through the ligand-binding domain of PPARγ when it is fused to the DNA-binding domain of Gal4. The stimulation of PPARγ by ADD1/SREBP1 does not require coexpression in the same cells; supernatants from cultures that express ADD1/SREBP1 augment the transcriptional activity of PPARγ. Finally, we demonstrate directly that cells expressing ADD1/SREBP1 produce and secrete lipid molecule(s) that bind directly to PPARγ, displacing the binding of radioactive thiazolidinedione ligands. These data establish that ADD1/SREBP1 can control the production of endogenous ligand(s) for PPARγ and suggest a mechanism for coordinating the actions of these adipogenic factors.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Structural flexibility in transcription complex formation revealed by protein–DNA photocrosslinking

The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16. The POU domain contains two separate DNA-binding domains joined by a flexible linker. By protein–DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target. On a single VP16-responsive element, the POU domain adopts multiple conformations. To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain–DNA complexes and found that VP16 can associate with multiple POU-domain conformations. These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new myc collaborating oncogene

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2αA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2αA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2αA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic β-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The β-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-l-ornithine (PALO) has been determined at 2.8-Å resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein–protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762–10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an l-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.