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.

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.

Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins.

Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs.

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-Based Genetic Interaction Analysis of Human Kinome.

Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human-yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role.

Base-pair Opening Dynamics of Nucleic Acids in Relation to Their Biological Function.

Base-pair opening is a conformational transition that is required for proper biological function of nucleic acids. Hydrogen exchange, observed by NMR spectroscopic experiments, is a widely used method to study the thermodynamics and kinetics of base-pair opening in nucleic acids. The hydrogen exchange data of imino protons are analyzed based on a two-state (open/closed) model for the base-pair, where hydrogen exchange only occurs from the open state. In this review, we discuss examples of how hydrogen exchange data provide insight into several interesting biological processes involving functional interactions of nucleic acids: i) selective recognition of DNA by proteins; ii) regulation of RNA cleavage by site-specific mutations; iii) intermolecular interaction of proteins with their target DNA or RNA; iv) formation of PNA:DNA hybrid duplexes.

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.

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.

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.

NMR study of the antifreeze activities of active and inactive isoforms of a type III antifreeze protein.

The quaternary-amino-ethyl 1 (QAE1) isoforms of type III antifreeze proteins (AFPs) prevent the growth of ice crystals within organisms living in polar regions. We determined the antifreeze activity of wild-type and mutant constructs of the Japanese notched-fin eelpout (Zoarces elongates Kner) AFP8 (nfeAFP8) and characterized the structural and dynamics properties of their ice-binding surface using NMR. We found that the three constructs containing the V20G mutation were incapable of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 310 helix (residues 18-22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane-binding surface.

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.

NMR investigation on the DNA binding and B-Z transition pathway of the Zα domain of human ADAR1.

Human ADAR1, which has two left-handed Z-DNA binding domains, preferentially binds Z-DNA rather than B-DNA with a high binding affinity. Z-DNA can be induced in long genomic DNA by Z-DNA binding proteins through the formation of two B-Z junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of Zα(ADAR1) with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the Zα(ADAR1) first binds to an 8-bp CG-rich DNA segment via a unique conformation during B-Z transition and the neighboring AT-rich region becomes destabilized. We also found that, when DNA duplexes have only 6-bp CG-rich segment, the interaction with Zα(ADAR1) did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two A·T base pairs. These results indicate that four Zα(ADAR1) proteins interact with the 8-bp DNA sequence containing a 6-bp CG-repeat segment as well as a dinucleotide step, even though the dinucleotid step contains A∙T base pairs. Thus this study suggests that the length of the CG-rich region is more important than the specific DNA sequence for determining which base-pair is extruded from the B-Z junction structure. This study also found that the Zα(ADAR1) in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free Zα(ADAR1) and the initial contact conformations of Zα(ADAR1) complexed with 13-bp DNA duplexes.

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.

The Zß domain of human DAI binds to Z-DNA via a novel B-Z transition pathway.

The human DNA-dependent activator of IFN-regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z-DNA binding domains (Zα and Zß) at the NH(2) terminus. The hZß(DAI) structure is similar to other Z-DNA binding proteins, although it demonstrates an unusual Z-DNA recognition. We performed NMR experiments on complexes of hZß(DAI) with DNA duplex, d(CGCGCG)(2), at a variety of protein-to-DNA molar ratios. The results suggest that hZß(DAI) binds to Z-DNA via an active-di B-Z transition mechanism, where two hZß(DAI) proteins bind to B-DNA to form the hZß(DAI)-B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. This novel mechanism of DNA binding and B-Z conversion is distinct from Z-DNA binding of the human ADAR1 protein.

NMR study of hydrogen exchange during the B-Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L.

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα(E3L)) bound to Z-DNA revealed that the overall structure of yabZα(E3L) and its interaction with Z-DNA are very similar to those of hZα(ADAR1). Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα(E3L) and d(CGCGCG)(2) with a variety of protein-to-DNA molar ratios. This study revealed that yabZα(E3L) could efficiently change the B-form helix of the d(CGCGCG)(2) to left-handed Z-DNA via the active-mono B-Z transition pathway like hZα(ADAR1).

Sequence discrimination of the Zα domain of human ADAR1 during B-Z transition of DNA duplexes.

The Zα domain of human ADAR1 (Zα(ADAR1)) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Zα(ADAR1) binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)(2) duplex similarly. We performed NMR experiments on complexes between the Zα(ADAR1) and non-CG-repeat DNA duplexes, d(CACGTG)(2) or d(CGTACG)(2), with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)(2) in the previous study suggests that Zα(ADAR1) exhibits the sequence preference of d(CGCGCG)(2)≫d(CACGTG)(2)>d(CGTACG)(2) through multiple sequence discrimination steps during the B-Z transition.

NMR spectroscopic elucidation of the B-Z transition of a DNA double helix induced by the Z alpha domain of human ADAR1.

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.