Showing papers in "Journal of Chemical Biology in 2010"

Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status

Abstract: Because understanding amyloid fibrillation in molecular detail is essential for development of strategies to control amyloid formation and overcome neurodegenerative disorders, increased understanding of present molecular probes as well as development of new probes are of utmost importance. To date, the binding modes of these molecular probes to amyloid fibrils are by no means adequately described or understood, and the large number of studies on Thioflavin T (ThT) and Congo Red (CR) binding have resulted in models that are incomplete and conflicting. Different types of binding sites are likely to be present in amyloid fibrils with differences in binding modes. ThT may bind in channels running parallel to the long axis of the fibril. In the channels, ThT may bind in either a monomeric or dimeric form of which the molecular conformation is likely to be planar. CR may bind in grooves formed along the β-sheets as a planar molecule in either a monomeric or supramolecular form.

Protein myristoylation in health and disease

Abstract: N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.

Hill coefficients, dose-response curves and allosteric mechanisms.

Abstract: Hill coefficients (nH) derived from four parameter logistic fits to dose–response curves were compared to calculated realistic reaction schemes and related to experimental data: (1) Hill coefficients may give information on the number of interacting sites but cannot distinguish between competitive, non-competitive or ortho-, iso-, or allosteric mechanisms. (2) For enzymatic dose–inhibition curves, Hill coefficients smaller than one do not indicate anticooperative binding but show that at least one ternary complex has enzymatic activity. (3) Hill coefficients different from one are proof for multiple ligand binding. The large variations of reported Hill coefficients corresponds to multiple allosteric binding, where induced conformational changes cause loss of the active conformation. Such a denaturation mechanism is in stark contrast to the desired specificity of drugs. The discussion is open.

In silico identification of common putative drug targets in Leptospira interrogans

Abstract: Infectious diseases are the leading causes of death worldwide. Hence, there is a need to develop new antimicrobial agents. Traditional method of drug discovery is time consuming and yields a few drug targets with little intracellular information for guiding target selection. Thus, focus in drug development has been shifted to computational comparative genomics for identifying novel drug targets. Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. Availability of L. interrogans serovars and human genome sequences facilitated to search for novel drug targets using bioinformatics tools. The genome sequence of L. interrogans serovar Copenhageni has 5,124 genes while that of serovar Lai has 4,727 genes. Through subtractive genomic approach 218 genes in serovar Copenhageni and 158 genes in serovar Lai have been identified as putative drug targets. Comparative genomic approach had revealed that 88 drug targets were common to both the serovars. Pathway analysis using the Kyoto Encyclopaedia of Genes and Genomes revealed that 66 targets are enzymes and 22 are non-enzymes. Sixty two common drug targets were predicted to be localized in cytoplasm and 16 were surface proteins. The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic compounds against Leptospira.

Characterisation of the PTEN inhibitor VO-OHpic.

Abstract: PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K(m) and V(max) are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K(ic) and K(iu) were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP(3)) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.

Conserved negative charges in the transmembrane segments of subunit K of the NADH:ubiquinone oxidoreductase determine its dependence on YidC for membrane insertion.

Abstract: All members of the Oxa1/Alb3/YidC family have been implicated in the biogenesis of respiratory and energy transducing proteins. In Escherichia coli, YidC functions together with and independently of the Sec system. Although the range of proteins shown to be dependent on YidC continues to increase, the exact role of YidC in insertion remains enigmatic. Here we show that YidC is essential for the insertion of subunit K of the NADH:ubiquinone oxidoreductase and that the dependence is due to the presence of two conserved glutamate residues in the transmembrane segments of subunit K. The results suggest a model in which YidC serves as a membrane chaperone for the insertion of the less hydrophobic, negatively charged transmembrane segments of NuoK.

Towards native-state imaging in biological context in the electron microscope.

Abstract: Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.

Virtual screening for potential inhibitors of homology modeled Leptospira interrogans MurD ligase

Abstract: The life-threatening infections caused by Leptospira serovars remain a global challenge since long time. Prevention of infection by controlling environmental factors being difficult to practice in developing countries, there is a need for designing potent anti-leptospirosis drugs. ATP-dependent MurD involved in biosynthesis of peptidoglycan was identified as common drug target among pathogenic Leptospira serovars through subtractive genomic approach. Peptidoglycan biosynthesis pathway being unique to bacteria and absent in host represents promising target for antimicrobial drug discovery. Thus, MurD 3D models were generated using crystal structures of 1EEH and 2JFF as templates in Modeller9v7. Structural refinement and energy minimization of the model was carried out in Maestro 9.0 applying OPLS-AA 2001 force field and was evaluated through Procheck, ProSA, PROQ, and Profile 3D. The active site residues were confirmed from the models in complex with substrate and inhibitor. Four published MurD inhibitors (two phosphinics, one sulfonamide, and one benzene 1,3-dicarbixylic acid derivative) were queried against more than one million entries of Ligand.Info Meta-Database to generate in-house library of 1,496 MurD inhibitor analogs. Our approach of virtual screening of the best-ranked compounds with pharmacokinetics property prediction has provided 17 novel MurD inhibitors for developing anti-leptospirosis drug targeting peptidoglycan biosynthesis pathway.

Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies.

Abstract: Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH2. One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells.

An intercalation mechanism as a mode of action exerted by psychotropic drugs: results of altered phospholipid substrate availabilities in membranes?

Abstract: Patients respond differently to psychotropic drugs, and this is currently a controversial theme among psychiatrists. The effects of 16 psychotropics on cell membrane parameters have been reported. These drugs belong to three major groups used in therapeutic psychiatry: antipsychotics, antidepressants, and anxiolytic/hypnotics. Human platelets, lacking dopamine (D2) receptors (proposed targets of most psychotropics), have been used as a cell model. Here we discuss the effects of these drugs on three metabolic phenomena and also results from Langmuir experiments. Diazepam, in contrast to the remaining drugs, had negligible effects on metabolic phenomena and had no effects in Langmuir experiments. Psychotropic drugs may work through intercalation in membrane phospholipids. It is possible that the fluidity of membranes, rich in essential fatty acids, the content being influenced by diet, could be a contributing factor to the action of psychotropics. This might in turn explain the observed major differences in therapeutic response among patients.

Chemical synthesis and biological activities of 3-alkyl pyridinium polymeric analogues of marine toxins.

Abstract: Two new large poly-1,3-dodecylpyridinium salts, APS12 and APS12-2 of 12.5- and 14.7-kDa size, respectively, were synthesised and tested for their pore-forming and transfection capabilities in HEK 293 and undifferentiated mouse ES cells using patch-clamp recording, Ca2+ imaging and flow cytometry. Polymerisation reactions were enhanced by microwaves, and the product sizes were controlled by altering the irradiation time. This method can also be applied to obtain polymers with variable linking chains as shown by the preparation of poly-(1,3-octylpyridinium) salt of 11.9-kDa size. Molecular weights of the final products were determined using ESIMS analysis, which also indicated the products to be amongst the largest macro-cycles ever recorded, up to a 900-membered ring. Anti-bacterial, haemolytic and anti-acetylcholinesterase activities were also reported for the two dodecyl pyridinium polymers. These biological activities are characteristic to the structurally related marine toxin, poly-APS.

The two-mutant problem: clonal interference in evolutionary graph theory

Abstract: In large asexual populations, clonal interference, whereby different beneficial mutations compete to fix in the population simultaneously, may be the norm. Results extrapolated from the spread of individual mutations in homogeneous backgrounds are found to be misleading in such situations: clonal interference severely inhibits the spread of beneficial mutations. In contrast with results gained in systems with just one mutation striving for fixation at any one time, the spatial structure of the population is found to be an important factor in determining the fixation probability when there are two beneficial mutations.

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

Abstract: The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 A), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 A) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.