Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.     

Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt-expressing tobacco are eliminated by short-term exposure to benzyladenine

Using a novel system for expressing ipt gene from Agrobacterium tumefaciens in tobacco ( Nicotiana tabacum L., cv. Petit Havana SR1), we were able to grow seedlings and teratoma-like tissue with increased content of cytokinins. This material enabled us to investigate new regulatory aspects of nitrate reduction. We grew control plants and plants with elevated cytokinins on MS media, with or without nitrate and benzyladenine (BA). We determined in vitro nitrate reductase (EC 1.6.6.1) activity (NRA) in this plant material. Initially, we found that ipt -expressing plants always displayed lowered levels of NRA when compared to wild-type SR1 plants. We determined that long-term exposure of tobacco plants and tissue to cytokinins caused up to 60% decrease in NRA. Exposure to 40 m M nitrate was able to induce the activity in such plants 3-fold, increasing the activity in SR1 plants more than 5-fold. We were able to restore wild-type levels of NRA in ipt -expressing plants by simultaneous induction of NR with BA and nitrate. Our results suggest that regulation of NR by nitrate and cytokinin is a result of overlaying cytokinin-driven regulatory processes, with those acting in the short-term having a positive effect on NRA, and those acting over extended periods of time having inhibitory effects on NRA.     

Binding to the open conformation of HIV-1 protease

A recent crystal structure of HIV-1 protease (HIVp) was the first to experimentally observe a ligand targeting an open-flap conformation. Researchers studying a symmetric pyrrolidine inhibitor found that two ligands cocrystallized with the protease, forcing an unusual configuration and unique crystallographic contacts. One molecule is centered in the traditional binding site (α pose) and the other binds between the flaps (β pose). The ligands stack against each other in a region termed the "eye" site. Ligands bound to the eye site should prevent flap closure, but it is unclear if the pyrrolidine inhibitors or the crystal packing are causing the open state. Molecular dynamics simulations were used to examine the solution-state behavior of three possible binding modes: the ternary complex of HIVp+αβ and the binary complexes, HIVp+α and HIVp+β. We show that HIVp+α is the most stable of the three states. During conformational sampling, α takes an asymmetric binding pose, with one naphthyl ring occupying the eye site and the other reoriented down to occupy positions seen with traditional inhibitors. This finding supports previous studies that reveal a requirement for asymmetric binding at the eye site. In fact, if the α pose is modified to splay both naphthyl rings across the binding site like traditional inhibitors, one ring consistently flips to occupy the eye site. Our simulations reveal that interactions to the eye site encourage a conformationally restrained state, and understanding those contacts may aid the design of ligands to specifically target alternate conformations of the protease.     

A Structure-Based Model for Predicting Serum Albumin Binding

One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data.