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Charge density and molecular coverage on the surface of electrode play major roles in the science and technology of surface chemistry and biochemical sensing. However, there has been no easy and direct method to characterize these quantities. By extending the method of Transient Induced Molecular Electronic Signal (TIMES) which we have used to measure molecular interactions, we are able to quantify the amount of charge in the double layers at the solution/electrode interface for different buffer strengths, buffer types, and pH values. Most uniquely, such capabilities can be applied to study surface coverage of immobilized molecules. As an example, we have measured the surface coverage for thiol-modified single-strand deoxyribonucleic acid (ssDNA) as anchored probe and 6-Mercapto-1-hexanol (MCH) as blocking agent on the platinum surface. Through these experiments, we demonstrate that TIMES offers a simple and accurate method to quantify surface charge and coverage of molecules on a metal surface, as an enabling tool for studies of surface properties and surface functionalization for biochemical sensing and reactions.

SUMMARYWNTs play key roles in development and disease, by binding both Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors that include the ROR and RYK receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2). RORs bind WNTs though a FZD-related cysteine-rich domain (CRD), and RYKs through a WNT-inhibitory factor (WIF) domain. Our structures suggest that neither the Nrk CRD nor the Drl-2 WIF domain can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable with a WNT acyl chain. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also show that DWnt-5, which regulates Drosophila ROR and RYK orthologs, lacks an acyl chain. Together with analysis of WNT/receptor interaction sites, these structures provide new insight into how WNTs recruit their RTK co-receptors into signaling complexes.

Quantitative information about protein-ligand interactions is central to drug discovery. To obtain the quintessential reaction dissociation constant, ideally measurements of reactions should be performed without perturbations by molecular labeling or immobilization. The technique of transient induced molecular electrical signal (TIMES) has provided a promising technique to meet such requirements, and its performance in a microfluidic environment further offers the potential for high throughput and reduced consumption of reagents. In this work, we further the development by using integrated TIMES signal (i-TIMES) to greatly enhance the accuracy and reproducibility of the measurement. While the transient response may be of interest, the integrated signal directly measures the total amount of surface charge density resulted from molecules near the surface of electrode. The signals enable quantitative characterization of protein-ligand interactions. We have demonstrated the feasibility of i-TIMES technique using different biomolecules including lysozyme, N,N',N″-triacetylchitotriose (TriNAG), aptamer, p-aminobenzamidine (pABA), bovine pancreatic ribonuclease A (RNaseA), and uridine-3'-phosphate (3'UMP). The results show i-TIMES is a simple and accurate technique that can bring tremendous value to drug discovery and research of intermolecular interactions.

As with other groups of protein kinases, approximately 10% of the RTKs (receptor tyrosine kinases) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the EGFR (epidermal growth factor receptor) family, and a series of unconventional Wnt receptors. We showed previously that, despite its reputation as a pseudokinase, the ErbB3 TKD (tyrosine kinase domain) does retain significant, albeit weak, kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3′s weak kinase activity have not so far been found to exhibit signalling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signalling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies, particularly of the pseudokinase RTKs involved in Wnt signalling, are required to shed new light on these intriguing signalling mechanisms.

The Drosophila protein Argos is an antagonist of epidermal growth factor receptor (EGFR) signalling that functions by binding and sequestering EGFR ligands. Here the structure of Argos bound to the EGFR ligand Spitz reveals that Argos engulfs the ligand using three related domains with structural resemblance to receptors for TGF-β and urokinase plasminogen activator. These results suggest that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anticancer therapeutics. Drosophila Argos is an antagonist of EGF receptor signalling that functions by binding and sequestering EGFR ligands. The structure of Argos bound to the EGFR ligand Spitz reveals that Argos engulfs the ligand using three related domains with structural resemblance to receptors for TGF-β and urokinase plasminogen activator. Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes1,2. Inappropriate activation of these receptors is a key feature of many human cancers3, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila4. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR5. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations4,6, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold7) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-β family receptors8. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA9. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.

Candidate plasticity-related gene 15 (cpg15) encodes a protein that regulates dendritic and axonal arbor growth and synaptic maturation. In the present study, we investigated the potential role of CPG15 in regulating the neuronal network re-establishment after ischemic brain injury. In the mouse model with transient global ischemia (TGI), CPG15 transcripts and proteins were determined using RT-PCR and Western blot analyses. Cell proliferation was observed using 5'-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) labeling. Double immunostaining and depletion of soluble CPG15 proteins were performed to examine the cellular distribution of CPG15 and the role of soluble CPG15 in the neurite outgrowth during the neuronal network re-establishment in primarily cultured hippocampal cells after glutamate-induced injury. We demonstrated that CPG15 expression in the hippocampus was upregulated at 1-2 weeks after TGI. In the dentate gyrus, the number of CPG15 and BrdU positive cells increased concurrently after the injury. During the neuronal network re-establishment after the glutamate-induced injury of primarily cultured hippocampal cells, CPG15 was mainly located at the ends and turn-off regions of the growth cones and in the vesicles. Depletion of soluble CPG15 proteins secreted from the hippocampal cells in the culture media significantly reduced the neurite outgrowth and neuron-neuron connection. The results indicate that CPG15 may function as a new factor required in re-establishment of neuronal network after the injury. Our findings will be important in developing a new strategy to enhance endogenous neurogenesis after an ischemic brain injury.

Organization of the bewildering array of known cell signaling proteins into coherent networks is facilitated by “scaffold” proteins that guide local information flow by binding multiple signaling molecules ( 1 ). Recent studies on one such scaffold protein called kinase suppressor of RAS (KSR), however, blur distinctions between the scaffold and the “scaffolded” by indicating that KSR itself has enzymatic activity that controls the signaling pathway assembled upon it ( 2 – 4 ).

Technology transfer (TT) from academia to industry is one of the key components that is required in the translation of basic research discoveries into commercial opportunities that benefit humanity. This paper examines the TT process at four premier universities and research institutions across the USA: Harvard University, Emory University, the University of Wisconsin-Madison, and The Scripps Research Institute. In this inaugural publication in a series of related publications studying multiple aspects of TT, we develop a variety of initial metrics and ratios measuring critical factors contributing to the success of TT and provide a generic model that can address certain bottlenecks in the process and enhance its efficiency.