Xiaocheng Gu

PlantTFDB 2.0: update and improvement of the comprehensive plant transcription factor database

We updated the plant transcription factor (TF) database to version 2.0 (PlantTFDB 2.0, http://planttfdb.cbi.pku.edu.cn) which contains 53 319 putative TFs predicted from 49 species. We made detailed annotation including general information, domain feature, gene ontology, expression pattern and ortholog groups, as well as cross references to various databases and literature citations for these TFs classified into 58 newly defined families with computational approach and manual inspection. Multiple sequence alignments and phylogenetic trees for each family can be shown as Weblogo pictures or downloaded as text files. We have redesigned the user interface in the new version. Users can search TFs with much more flexibility through the improved advanced search page, and the search results can be exported into various formats for further analysis. In addition, we now provide web service for advanced users to access PlantTFDB 2.0 more efficiently.

PlantTFDB: a comprehensive plant transcription factor database

Transcription factors (TFs) play key roles in controlling gene expression. Systematic identification and annotation of TFs, followed by construction of TF databases may serve as useful resources for studying the function and evolution of transcription factors. We developed a comprehensive plant transcription factor database PlantTFDB (http://planttfdb.cbi.pku.edu.cn), which contains 26 402 TFs predicted from 22 species, including five model organisms with available whole genome sequence and 17 plants with available EST sequences. To provide comprehensive information for those putative TFs, we made extensive annotation at both family and gene levels. A brief introduction and key references were presented for each family. Functional domain information and cross-references to various well-known public databases were available for each identified TF. In addition, we predicted putative orthologs of those TFs among the 22 species. PlantTFDB has a simple interface to allow users to search the database by IDs or free texts, to make sequence similarity search against TFs of all or individual species, and to download TF sequences for local analysis.

DRTF: a database of rice transcription factors

SUMMARY DRTF contains 2025 putative transcription factors (TFs) in Oryza sativa L. ssp. indica and 2384 in ssp. japonica, distributed in 63 families, identified by computational prediction and manual curation. It includes detailed annotations of each TF including sequence features, functional domains, Gene Ontology assignment, chromosomal localization, EST and microarray expression information, as well as multiple sequence alignment of the DNA-binding domains for each TF family. The database can be browsed and searched with a user-friendly web interface. AVAILABILITY DRTF is available at http://drtf.cbi.pku.edu.cn

SPD—a web-based secreted protein database

With the improved secreted protein prediction approach and comprehensive data sources, including Swiss-Prot, TrEMBL, RefSeq, Ensembl and CBI-Gene, we have constructed secretomes of human, mouse and rat, with a total of 18 152 secreted proteins. All the entries are ranked according to the prediction confidence. They were further annotated via a proteome annotation pipeline that we developed. We also set up a secreted protein classification pipeline and classified our predicted secreted proteins into different functional categories. To make the dataset more convincing and comprehensive, nine reference datasets are also integrated, such as the secreted proteins from the Gene Ontology Annotation (GOA) system at the European Bioinformatics Institute, and the vertebrate secreted proteins from Swiss-Prot. All these entries were grouped via a TribeMCL based clustering pipeline. We have constructed a web-based secreted protein database, which has been publicly available at http://spd.cbi.pku.edu.cn. Users can browse the database via a GO assignment or chromosomal-location-based interface. Moreover, text query and sequence similarity search are also provided, and the sequence and annotation data can be downloaded freely from the SPD website.

Genome-wide identification and evolutionary analysis of the plant specific SBP-box transcription factor family.

We made genome-wide analyses to explore the evolutionary process of the SBP-box gene family. We identified 120 SBP-box genes from nine species representing the main green plant lineages: green alga, moss, lycophyte, gymnosperm and angiosperm. A maximum-likelihood phylogenetic tree was constructed using the protein sequences of the DNA-binding domain of SBP-box genes (SBP-domain). Our results revealed that all SBP-box genes of green alga clustered into a single clade (CR group), while all genes from land-plants fell into two distinct groups. Group I had a single copy in each species except for poplar while group II had several members in each species and can be divided into several subgroups. The SBP-domain encoded by all SBP-box genes possesses two zinc fingers. The C-terminal zinc finger of both group I and group II had the same C2HC motif while their N-terminal zinc finger showed different signatures, C4 in group I and C3H in group II. The patterns of exon-intron structure in Arabidopsis and rice SBP-box genes were consistent with the phylogenetic results. A target site of microRNA miR156 was highly conserved among land-plant SBP-box genes. Our results suggested that the SBP-box gene family might have originated from a common ancestor of green plants, followed by duplication and divergence in each lineage including exon-intron loss processes.

A nonsynonymous SNP in human cytosolic sialidase in a small Asian population results in reduced enzyme activity: potential link with severe adverse reactions to oseltamivir

The use of oseltamivir, widely stockpiled as one of the drugs for use in a possible avian influenza pandemic, has been reported to be associated with neuropsychiatric disorders and severe skin reactions, primarily in Japan. Here we identified a nonsynonymous SNP (single nucleotide polymorphism) in dbSNP database, R41Q, near the enzymatic active site of human cytosolic sialidase, a homologue of virus neuraminidase that is the target of oseltamivir. This SNP occurred in 9.29% of Asian population and none of European and African American population. Our structural analyses and Ki measurements using in vitro sialidase assays indicated that this SNP could increase the unintended binding affinity of human sialidase to oseltamivir carboxylate, the active form of oseltamivir, thus reducing sialidase activity. In addition, this SNP itself results in an enzyme with an intrinsically lower sialidase activity, as shown by its increased Km and decreased Vmax values. Theoretically administration of oseltamivir to people with this SNP might further reduce their sialidase activity. We note the similarity between the reported neuropsychiatric side effects of oseltamivir and the known symptoms of human sialidase-related disorders. We propose that this Asian-enriched sialidase variation caused by the SNP, likely in homozygous form, may be associated with certain severe adverse reactions to oseltamivir.

Structure-Activity Relationships of Hainantoxin-IV and Structure Determination of Active and Inactive Sodium Channel Blockers

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The sequence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH2. In the present study, to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. 1H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore, in the determined structures of K27A and R29A, the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV. These results suggested that residues Ser12, Arg26, Lys27, and Arg29 were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys27 and Arg29 were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys27 and Arg29 with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys27, Arg29, His28, Lys32, Phe5, and Trp30 clustered on one face of HNTX-IV were responsible for ligand binding.

Proton Nuclear Magnetic Resonance Studies on Huwentoxin-I from the Venom of the Spider Selenocosmia Huwena:2.Three-Dimensional Structure in Solution

The three-dimensional structure in aqueous solution of native huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena, has been determined from two-dimensional 1H NMR data recorded at 500 and 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin–spin coupling constants were used as input for distance geometry calculation with the program XPLOR 3.1. The best 10 structures have NOE violations <0.3 Å, dihedral violations <2°, and pairwise root-mean-square differences of 1.08 (±0.20) Å over backbone atoms (N, Cα;, C). The molecule adopts a compact structure consisting of a small triple-stranded antiparallel β-sheet and five β-turns. A small hydrophobic patch consisting of Phe 6, Trp 28, and Trp 31 is located on one side of the molecule. All six lysine residues are distributed on the molecular surface. The three disulfidc bridges are buried within the molecule. The structure contains an “inhibitor cystine knot motif” which is adopted by several other small proteins, such as ω-conotoxin, agatoxin IVA, and gurmarin.

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-Å resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an α-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis.

The structure of spider toxin huwentoxin-II with unique disulfide linkage: Evidence for structural evolution

The three‐dimensional structure of huwentoxin‐II (HWTX‐II), an insecticidal peptide purified from the venom of spider Selenocosmia huwena with a unique disulfide bond linkage as I‐III, II‐V, and IV‐VI, has been determined using 2D 1H‐NMR. The resulting structure of HWTX‐II contains two β‐turns (C4‐S7 and K24‐W27) and a double‐stranded antiparallel β‐sheet (W27‐C29 and C34‐K36). Although the C‐terminal double‐stranded β‐sheet cross‐linked by two disulfide bonds (II‐V and IV‐VI in HWTX‐II, II‐V and III‐VI in the ICK molecules) is conserved both in HWTX‐II and the ICK molecules, the structure of HWTX‐II is unexpected absence of the cystine knot because of its unique disulfide linkage. It suggests that HWTX‐II adopts a novel scaffold different from the ICK motif that is adopted by all other spider toxin structures elucidated thus far. Furthermore, the structure of HWTX‐II, which conforms to the disulfide‐directed β‐hairpin (DDH) motif, not only supports the hypothesis that the ICK is a minor elaboration of the more ancestral DDH motif but also suggests that HWTX‐II may have evolved from the same structural ancestor.