[reaction: see text] The synthesis and characterization of a new leucine-zipper dendrimer (LZD) is reported that displays four copies of the peptide corresponding to the coiled-coiled dimerization domain of Fos. Circular dichroism spectroscopy, fluorescence titration, and sedimentation equilibrium experiments demonstrate that Fos-LZD can noncovalently assemble four copies of the peptide corresponding to the coiled-coil domain of Jun. This work provides the basis for the future construction of noncovalently assembled multivalent protein assemblies displaying any protein of interest.
PMID: 14765123;PMCID: PMC380994;Abstract:
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).
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. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.
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. Copyright © 2011 Elsevier Ltd. All rights reserved.
Virtually all methods for reading the sequence of bases in DNA rely on the ability to denature double-stranded DNA into single strands and then use Watson-Crick base-pairing rules to hybridize the strands with high specificity to another DNA primer or probe. However, nature frequently uses an alternative method, reading the sequence information directly from double-stranded DNA using sequence-specific DNA-binding proteins. Here we describe methods for the construction and testing of sequence probes based on engineered zinc finger DNA-binding proteins. Background is reduced using split-reporter molecules, and signal is amplified using enzymatic reporters. The resulting sequence-enabled reassembly (SEER) probes can be configured to detect DNA sequence (genetic) or DNA methylation (epigenetic) information.