Salt Dependence of DNA Binding Activity of Human Transcription Factor Dlx3.

Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5'-TAATT-3') of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.

Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

Yeast genetic interaction screen of human genes associated with amyotrophic lateral sclerosis: identification of MAP2K5 kinase as a potential drug target.

To understand disease mechanisms, a large-scale analysis of human-yeast genetic interactions was performed. Of 1305 human disease genes assayed, 20 genes exhibited strong toxicity in yeast. Human-yeast genetic interactions were identified by en masse transformation of the human disease genes into a pool of 4653 homozygous diploid yeast deletion mutants with unique barcode sequences, followed by multiplexed barcode sequencing to identify yeast toxicity modifiers. Subsequent network analyses focusing on amyotrophic lateral sclerosis (ALS)-associated genes, such as optineurin (OPTN) and angiogenin (ANG), showed that the human orthologs of the yeast toxicity modifiers of these ALS genes are enriched for several biological processes, such as cell death, lipid metabolism, and molecular transport. When yeast genetic interaction partners held in common between human OPTN and ANG were validated in mammalian cells and zebrafish, MAP2K5 kinase emerged as a potential drug target for ALS therapy. The toxicity modifiers identified in this study may deepen our understanding of the pathogenic mechanisms of ALS and other devastating diseases.

Thermodynamic Model for B-Z Transition of DNA Induced by Z-DNA Binding Proteins.

Z-DNA is stabilized by various Z-DNA binding proteins (ZBPs) that play important roles in RNA editing, innate immune response, and viral infection. In this review, the structural and dynamics of various ZBPs complexed with Z-DNA are summarized to better understand the mechanisms by which ZBPs selectively recognize d(CG)-repeat DNA sequences in genomic DNA and efficiently convert them to left-handed Z-DNA to achieve their biological function. The intermolecular interaction of ZBPs with Z-DNA strands is mediated through a single continuous recognition surface which consists of an α3 helix and a ß-hairpin. In the ZBP-Z-DNA complexes, three identical, conserved residues (N173, Y177, and W195 in the Zα domain of human ADAR1) play central roles in the interaction with Z-DNA. ZBPs convert a 6-base DNA pair to a Z-form helix via the B-Z transition mechanism in which the ZBP first binds to B-DNA and then shifts the equilibrium from B-DNA to Z-DNA, a conformation that is then selectively stabilized by the additional binding of a second ZBP molecule. During B-Z transition, ZBPs selectively recognize the alternating d(CG)n sequence and convert it to a Z-form helix in long genomic DNA through multiple sequence discrimination steps. In addition, the intermediate complex formed by ZBPs and B-DNA, which is modulated by varying conditions, determines the degree of B-Z transition.

NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration.

A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two ß-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Molecular basis of facilitated target search and sequence discrimination of TALE homeodomain transcription factor Meis1.

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD-DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.

Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer.

Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ(3)T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ(3)T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.

NMR Titration Studies in Z-DNA Dynamics.

Chemical shift perturbation (CSP) is a simple NMR technique for studying the DNA binding of proteins. Titration of the unlabeled DNA into the 15N-labeled protein is monitored by acquiring a two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectrum at each step of the titration. CSP can also provide information on the DNA-binding dynamics of proteins, as well as protein-induced conformational changes in DNA. Here, we describe the titration of DNA for the 15N-labeled Z-DNA-binding protein, monitored via 2D HSQC spectra. NMR titration data can be analyzed with the active B-Z transition model to provide the protein-induced B-Z transition dynamics of DNA.

Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord.

Preconditioning nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these macrophages influence the neuronal capacity of axon regeneration remains elusive. We report that oncomodulin (ONCM) is produced from the regeneration-associated macrophages and strongly influences regeneration of DRG sensory axons. We also attempted to promote sensory axon regeneration by nanogel-mediated delivery of ONCM to DRGs. Methods:In vitro neuron-macrophage interaction model and preconditioning sciatic nerve injury were used to verify the necessity of ONCM in preconditioning injury-induced neurite outgrowth. We developed a nanogel-mediated delivery system in which electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) enabled a controlled release of ONCM. Results: Sciatic nerve injury upregulated ONCM in DRG macrophages. ONCM in macrophages was necessary to produce pro-regenerative macrophages in the in vitro model of neuron-macrophage interaction and played an essential role in preconditioning-induced neurite outgrowth. ONCM increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. Increasing extracellularly secreted ONCM in DRGs sufficiently enhanced the capacity of neurite outgrowth. Intraganglionic injection of REPL-NG/ONCM complex allowed sustained ONCM activity in DRG tissue and achieved a remarkable long-range regeneration of dorsal column sensory axons beyond spinal cord lesion. Conclusion: NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.