Differential Interactions of the Catalytic Subunits of Adenylyl Cyclase with Forskolin Analogs

The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I–VIII. Diterpenes without C1-OH group do not activate ACs. The C1-OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′-O-(N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS ~ 7-deacetyl-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS > 1-deoxy-FS ~ 1,9-dideoxy-FS ~ 7-deacetyl-1-deoxy-FS ~ 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS > 7-deacety-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS ≫ 1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C1-OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2\u27,3\u27-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5\u27-Triphosphates

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70: 878-886, 2006), we reported that 2\u27,3\u27-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5\u27-triphosphates (TNP-NTPs) are potent inhibitors (K(i) values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1: IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1: IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1: IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K(i), 3.7 nM) and sGC (K(i), 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K(i), 7.1 nM) and ACII (K(i), 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1: IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1: IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far

Exploring Government Officials’ Data Collection Process in Open Data Initiatives

The implementation of open government data (OGD) initiatives has become a worldwide phenomenon over the last decade. While there have been many studies exploring this phenomenon, few have focused on data collection phase in data opening process, during which government officials seek government-owned datasets for OGD purposes. To establish a model for the process of data collection phase, this study conducted semi-structured interviews with fifteen participants recruited through purposive and snowball sampling methods from eleven central and local Taiwanese government agencies. This paper discusses the research findings regarding (1) why government officials undertake tasks of data preparation, (2) how potential datasets are discovered, selected, and obtained from multiple government agencies, and (3) how information is sought to facilitate tasks of data preparation. These findings bridged the research gap in the study of data opening process and verified previous information seeking models. Suggestions for future practitioners and topics worthy of further exploration are enumerated in the conclusion section

Identification and characterization of a yeast iso-1-cytochrome c C-terminal domain swapped dimer

Domain swapped protein dimers consist of a swapped domain linked by a hinge loop. They have been proposed as a means of achieving larger assemblies potentially contributing to biological cellular activity or conferring disease. Here we present a crystal structure of a C-terminal domain swapped dimer in yeast iso-1-cytochrome c. In this structure the C-terminal alpha helix from one monomer positions itself in the native position of the opposite monomer and vice versa. The highly dynamic heme crevice loop, the most highly conserved portion of the cytochrome c sequence, spans the gap acting as the hinge loop. Interestingly, conversion of the heme crevice loop to the hinge loop results in a loss of the native Met80-heme ligation. This produces an open heme coordination site on each subunit of the dimer. As cytochrome c requires an open heme coordination site to act as a peroxidase, to oxidize cardiolipin and initiate the intrinsic apoptotic pathway, this dimer structure could potentially be a structure particularly suited to function in oxidizing cardiolipin. In fact, a recently reported C-terminal domain swapped dimer of horse cytochrome c demonstrates increased peroxidase activity relative to the monomer. Although the yeast and horse dimer are similar, the hinge loop orientations differ. The hinge loop is two residues longer in the yeast dimer resulting in an increased distance between the heme groups and an altered angle of the hinge loops relative to the horse cytochrome c dimer. Even though they contain similar structure and sequence, the domain swapped yeast iso-1-cytochrome c dimer demonstrates decreased stability compared to the horse cytochrome c dimer

Identification and characterization of a yeast iso-1-cytochrome c C-terminal domain swapped dimer

Domain swapped protein dimers consist of a swapped domain linked by a hinge loop. They have been proposed as a means of achieving larger assemblies potentially contributing to biological cellular activity or conferring disease. Here we present a crystal structure of a C-terminal domain swapped dimer in yeast iso-1-cytochrome c. In this structure the C-terminal alpha helix from one monomer positions itself in the native position of the opposite monomer and vice versa. The highly dynamic heme crevice loop, the most highly conserved portion of the cytochrome c sequence, spans the gap acting as the hinge loop. Interestingly, conversion of the heme crevice loop to the hinge loop results in a loss of the native Met80-heme ligation. This produces an open heme coordination site on each subunit of the dimer. As cytochrome c requires an open heme coordination site to act as a peroxidase, to oxidize cardiolipin and initiate the intrinsic apoptotic pathway, this dimer structure could potentially be a structure particularly suited to function in oxidizing cardiolipin. In fact, a recently reported C-terminal domain swapped dimer of horse cytochrome c demonstrates increased peroxidase activity relative to the monomer. Although the yeast and horse dimer are similar, the hinge loop orientations differ. The hinge loop is two residues longer in the yeast dimer resulting in an increased distance between the heme groups and an altered angle of the hinge loops relative to the horse cytochrome c dimer. Even though they contain similar structure and sequence, the domain swapped yeast iso-1-cytochrome c dimer demonstrates decreased stability compared to the horse cytochrome c dimer

2′-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2′-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 µM. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN·RNA > normal DNA·RNA > S-ODN·RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2′-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects

Crystal structure of the major quadruplex formed in the promoter region of the human c-MYC oncogene.

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds. Previous solution NMR studies of the c-MYC quadruplex have assigned the major conformer and topology of this important target, however, regions outside the G-quartet core were not as well-defined. Here, we report a high-resolution crystal structure (2.35 Å) of the major quadruplex formed in the NHE III1 region of the c-MYC promoter. The crystal structure is in general agreement with the solution NMR structure, however, key differences are observed in the position of nucleotides outside the G-quartet core. The crystal structure provides an alternative model that, along with comparisons to other reported quadruplex crystal structures, will be important to the rational design of selective compounds. This work will aid in development of ligands to target the c-MYC promoter quadruplex with the goal of creating novel anticancer therapies