neil dixon

The Escherichia coli thiM riboswitch forms specific contacts with its natural ligand, thiamine pyrophosphate (TPP or thiamine diphosphate), allowing it to generate not only nanomolar binding affinity, but also a high degree of discrimination against similar small molecules. A range of synthetic TPP analogues have been used to probe each of the riboswitch–ligand interactions.

[摘要]: 正Small molecule modulators of gene expression which offer rapid, temporal and spatial control of gene expression would be tremendously valuable tools in biological and biomedical sciences. The ability to exert differential control on the expression of multiple genes simultaneously, in prokaryotic and eukaryotic cells using distinct small molecule inhibitors or inducers, remains extremely limited.

[摘要]: 正Aberrant splicing of mRNA is implicated in various genetic disease states and thus is a target for potential small molecule therapeutic intervention. The genetic message that is transcribed into mRNA is processed before being translated into the required protein. In eukaryotic cells this processing involves various different stages including editing, 5′ cap addition, transport from the nucleus to the

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumour cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21.

The osteoclast variant of the vacuolar H+-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5, 6-dichloro-2-indolyl)-2-methoxy-2, 4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases.

Post-translational modification of proteins by poly (ADP-ribosyl) ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis 1. Poly (ADP-ribose)(PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases 1, 2.

The selective inhibitor of osteoclastic V-ATPase (2Z, 4E)-5-(5, 6-dichloro-2-indolyl)-2-methoxy-N-(1, 2, 2, 6, 6-pentamethylpiperidin-4-yl)-2, 4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study of the interaction of this inhibitor with the lipidic environment is an essential step in the understanding of the mechanism of inhibition.

The macrolide antibiotic bafilomycin and the related synthetic compound SB 242784 are potent inhibitors of the vacuolar H+-ATPases (V-ATPase). It is currently believed that the site of action of these inhibitors is located on the membrane bound c-subunits of V-ATPases.

Abstract—This paper focuses on the problem of detecting and locating the position of water leaks in water distribution Medium Density Polyethylene (MDPE) pipes using passive acoustic detection methods. A leaking water pipe generates noise which depends primarily on water pressure, pipe characteristics and the leak size and shape. This leak noise comprises vibration and acoustic signals, which can be detected using non-invasive accelerometers and invasive hydrophone sensors respectively.

The macrolide antibiotic concanamycin is a potent and specific inhibitor of the vacuolar H(+)-ATPase (V-ATPase), binding to the V(0) membrane domain of this eukaryotic acid pump. Although binding is known to involve the 16 kDa proteolipid subunit, contributions from other V(0) subunits are possible that could account for the apparently different inhibitor sensitivities of pump isoforms in vertebrate cells. In this study, we used a fluorescence quenching assay to directly examine the roles of V(0) subunits in inhibitor binding. Pyrene-labeled V(0) domains were affinity purified from Saccharomyces vacuolar membranes, and the 16 kDa proteolipid was subsequently extracted into chloroform and methanol and purified by size exclusion chromatography. Fluorescence from the isolated proteins was strongly quenched by nanomolar concentrations of both concanamycin and an indolyl pentadieneamide compound, indicating high-affinity binding of both natural macrolide and synthetic inhibitors. Competition studies showed that these inhibitors bind to overlapping sites on the proteolipid. Significantly, the 16 kDa proteolipid in isolation was able to bind inhibitors as strongly as V(0) did. In contrast, proteolipids carrying mutations that confer resistance to both inhibitors showed no binding. We conclude that the extracted 16 kDa proteolipid retains sufficient fold to form a high-affinity inhibitor binding site for both natural and synthetic V-ATPase inhibitors and that the proteolipid contains the major proportion of the structural determinants for inhibitor binding. The role of membrane domain subunit a in concanamycin binding and therefore in defining the inhibitor binding properties of tissue-specific V-ATPases is critically re-assessed in light of these data.

The osteoclast variant of the vacuolar H+-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC50 in the 10-nanomolar range.

Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae.