Indraneel Ghosh

DCX, a new mediator of the JNK pathway

Mutations in the X‐linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c‐Jun N‐terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus‐end directed molecular motor) versus dynein (a minus‐end directed molecular motor).

Selective amplification by auto- and cross-catalysis in a replicating peptide system

Self-replication has been demonstrated in synthetic chemical systems based on oligonucleotides, peptides and complementary molecules without natural analogues. However, within a living cell virtually no molecule catalyses its own formation, and the search for chemical systems in which both auto- and cross-catalysis can occur has therefore attracted wide interest. One such system, consisting of two self-replicating peptides that catalyse each others production, has been reported. Here we describe a four-component peptide system that is capable of auto- and cross-catalysis and allows for the selective amplification of one or more of the products by changing the reaction conditions. The ability of this system selectively to amplify one or more molecules in response to changes in environmental conditions such as pH or salt concentration supports the suggestion that self-replicating peptides may have played a role in the origin of life.

Split-protein systems: beyond binary protein-protein interactions.

It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches.

Molecules that target beta-amyloid.

The devastating effects of Alzheimer’s and related amyloidogenic diseases have inspired the synthesis and evaluation of numerous ligands to understand the molecular mechanism of the aggregation of the beta‐amyloid peptide. Our review focuses on the current knowledge in this field with respect to molecules that have been demonstrated to interact with either oligomeric or fibrillar forms of the beta‐amyloid peptide. We describe natural proteins, peptides, peptidomimetics, and small molecules that have been found to interfere with beta‐amyloid aggregation. We also detail recent efforts in selecting molecules that target beta‐amyloid isolated from antibody, protein, and peptide libraries. These new molecules will likely aid in deciphering the details of the aggregation pathway for the beta‐amyloid peptide and provide reagents that may stabilize relevant oligomeric intermediates which likely have bearing on the pathophysiology of Alzheimer’s disease. Moreover, the described anti‐amyloid molecular toolbox will also provide an avenue for designing new diagnostic and therapeutic reagents.

Water-Based Route to Ligand-Selective Synthesis of ZnSe and Cd-Doped ZnSe Quantum Dots with Tunable Ultraviolet A to Blue Photoluminescence

A water-based route has been demonstrated for synthesizing ZnSe and Cd-doped ZnSe (Zn(x)Cd(1-x)Se, 0 < x < 1) quantum dots (QDs) that have tunable and narrow photoluminescence (PL) peaks from the ultraviolet A (UVA) to the blue range (350-490 nm) with full-width at half-maximum (fwhm) values of 24-36 nm. Hydrazine (N(2)H(4)) was used to maintain oxygen-free conditions, allowing the reaction vessel to be open to air. The properties of the QDs were controlled using the thiol ligands, 3-mercaptopropionic acid (MPA), thiolglycolic acid (TGA), and l-glutathione (GSH). On the basis of optical spectra, linear three-carbon MPA attenuated nucleation and growth, yielding small ZnSe QDs with a high density of surface defects. In contrast, TGA and GSH produced larger ZnSe QDs with lower surface defect densities. The absorption spectra show that growth was more uniform and better controlled with linear two-carbon TGA than branched bifunctional GSH. After 20 min of growth TGA-capped ZnSe had an average diameter of 2.5 nm based on high-resolution transmission electron microscopy images; these nanocrystals had an absorbance peak maximum of approximately 340 nm (3.65 eV) and a band gap PL emission peak at 372 nm (3.34 eV). Highly fluorescent Zn(x)Cd(1-x)Se QDs were fabricated by adding a Cd-thiol complex directly to ZnSe QD solutions; PL peaks were tuned in the blue range (400-490 nm) by changing the Zn to Cd ratio. The Cd-bearing nanocrystals contained proportionally more Se based on X-ray photoelectron spectroscopy, and Cd-Se bonds had ionic character, in contrast to primarily covalent Zn-Se bonds.

New Directions in Targeting Protein Kinases: Focusing Upon True Allosteric and Bivalent Inhibitors

Over the past decade, therapeutics that target subsets of the 518 human protein kinases have played a vital role in the fight against cancer. Protein kinases are typically targeted at the adenosine triphosphate (ATP) binding cleft by type I and II inhibitors, however, the high sequence and structural homology shared by protein kinases, especially at the ATP binding site, inherently leads to polypharmacology. In order to discover or design truly selective protein kinase inhibitors as both pharmacological reagents and safer therapeutic leads, new efforts are needed to target kinases outside the ATP cleft. Recent advances include the serendipitous discovery of type III inhibitors that bind a site proximal to the ATP pocket as well as the truly allosteric type IV inhibitors that target protein kinases distal to the substrate binding pocket. These new classes of inhibitors are often selective but usually display moderate affinities. In this review we will discuss the different classes of inhibitors with an emphasis on bisubstrate and bivalent inhibitors (type V) that combine different inhibitor classes. These inhibitors have the potential to couple the high affinity and potency of traditional active site targeted small molecule inhibitors with the selectivity of inhibitors that target the protein kinase surface outside ATP cleft.

High Specificity in Protein Recognition by Hydrogen-Bond-Surrogate α-Helices: Selective Inhibition of the p53/MDM2 Complex

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors.[1–8] Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity.[9–10] The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. We recently introduced a new strategy for the preparation of stabilized α-helices, termed hydrogen bond surrogate (HBS) helices, which involves replacement of one of the main chain hydrogen bonds with a covalent linkage (Figure 1A).[11] The salient feature of the HBS approach is its ability to constrain very short peptides into highly stable α-helical conformation without blocking any molecular recognition surfaces. We have extensively analyzed the conformation adopted by HBS α-helices with 2D NMR, X-ray, and circular dichroism spectroscopies.[12–14] In addition, HBS helices have been shown to target their expected protein partners with high affinity in cell-free and cell culture assays.[15–17]

A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

A self-replicating peptide under ionic control

The chemical coupling of two peptide fragments to give the peptide K1 K2 (shown in the helical wheel diagram on the right) is autocatalytic at high NaClO4 concentrations (1 M). Under these conditions K1 K2 assumes a coiled-coil conformation, which can function as a template for the coupling. Autocatalysis is not observed under conditions that prevent formation of the coiled-coil conformation.

An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors

Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.