Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Photoresists as a high spatial resolution autoradiography substrate for quantitative mapping of intra- and sub-cellular distribution of Auger electron emitting radionuclides.

PURPOSE: To explore poly(methyl methacrylate) (PMMA950) as an autoradiography substrate. MATERIALS AND METHODS: PMMA950 was spin coated onto a silicon substrate. Resists were exposed to either a 25 or 50 keV electron beam (e-beam) with fluences of 0.1-33.6 µC/cm(2). The resulting patterns were analyzed by atomic force microscopy (AFM). The dependence of pattern sensitivity and resolution on resist thickness, development time and electron energy was evaluated and correlated with Monte Carlo (MC) modeling. Conventional micro-autoradiography (MAR) images were compared to AFM images of photoresist patterns obtained following exposure from (111)In-diethylenetriaminepentaacetic acid (DTPA)-human epidermal growth factor (hEGF) (4-6 MBq/µg, 40 nM DTPA-hEGF)-treated human breast cancer cells MDA-MB-468. RESULTS: MC simulation results confirmed the similarity of particle transport in PMMA950 exposed to either an (111)In point source or a 25 keV e-beam. Sensitivity was inversely related to resist thickness. Development conditions of the resists greatly affected image quality. Sensitivity of PMMA950 was similar to the UVIII™ resist (consisting of a copolymer of 4-hydroxystyrene and t- butylacrylate) at low electron fluence for both 25 and 50 keV e-beam exposure. AFM evaluation of the exposure patterns from (111)In-DTPA-hEGF treated cells and nuclei provides more detailed information in comparison with that from MAR. CONCLUSIONS: Photoresist autoradiography can provide information on both the distribution of radiation sources and their strengths within a biological sample; however, the choice of photoresist material and processing conditions greatly affects the outcome.

A structure-based sliding-rebinding mechanism for catch bonds.

Catch bonds, whose lifetimes are prolonged by force, have been observed in selectin-ligand interactions and other systems. Several biophysical models have been proposed to explain this counterintuitive phenomenon, but none was based on the structure of the interacting molecules and the noncovalent interactions at the binding interface. Here we used molecular dynamics simulations to study changes in structure and atomic-level interactions during forced unbinding of P-selectin from P-selectin glycoprotein ligand-1. A mechanistic model for catch bonds was developed based on these observations. In the model, "catch" results from forced opening of an interdomain hinge that tilts the binding interface to allow two sides of the contact to slide against each other. Sliding promotes formation of new interactions and even rebinding to the original state, thereby slowing dissociation and prolonging bond lifetimes. Properties of this sliding-rebinding mechanism were explored using a pseudoatom representation and Monte Carlo simulations. The model has been supported by its ability to fit experimental data and can be related to previously proposed two-pathway models.

Computational modeling reveals molecular details of epidermal growth factor binding.

BACKGROUND: The ErbB family of receptors are dysregulated in a number of cancers, and the signaling pathway of this receptor family is a critical target for several anti-cancer drugs. Therefore a detailed understanding of the mechanisms of receptor activation is critical. However, despite a plethora of biochemical studies and recent single particle tracking experiments, the early molecular mechanisms involving epidermal growth factor (EGF) binding and EGF receptor (EGFR) dimerization are not as well understood. Herein, we describe a spatially distributed Monte Carlo based simulation framework to enable the simulation of in vivo receptor diffusion and dimerization. RESULTS: Our simulation results are in agreement with the data from single particle tracking and biochemical experiments on EGFR. Furthermore, the simulations reveal that the sequence of receptor-receptor and ligand-receptor reaction events depends on the ligand concentration, receptor density and receptor mobility. CONCLUSION: Our computer simulations reveal the mechanism of EGF binding on EGFR. Overall, we show that spatial simulation of receptor dynamics can be used to gain a mechanistic understanding of receptor activation which may in turn enable improved cancer treatments in the future.

E1-INT (Transition Therapeutics/Novo Nordisk).

Transition Therapeutics (through its acquisition of Waratah Pharmaceuticals), in collaboration with Novo Nordisk, is developing E1-INT, an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog, for the treatment of insulin-dependent (type 1) and non-insulin-dependent (type 2) diabetes. The compound is currently undergoing phase II clinical trials.

Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Multiple alignment using hidden Markov models.

A simulated annealing method is described for training hidden Markov models and producing multiple sequence alignments from initially unaligned protein or DNA sequences. Simulated annealing in turn uses a dynamic programming algorithm for correctly sampling suboptimal multiple alignments according to their probability and a Boltzmann temperature factor. The quality of simulated annealing alignments is evaluated on structural alignments of ten different protein families, and compared to the performance of other HMM training methods and the ClustalW program. Simulated annealing is better able to find near-global optima in the multiple alignment probability landscape than the other tested HMM training methods. Neither ClustalW nor simulated annealing produce consistently better alignments compared to each other. Examination of the specific cases in which ClustalW outperforms simulated annealing, and vice versa, provides insight into the strengths and weaknesses of current hidden Markov model approaches.