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1.
FASEB J ; 37(10): e23206, 2023 10.
Article in English | MEDLINE | ID: mdl-37718485

ABSTRACT

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.


Subject(s)
Fibronectins , Head and Neck Neoplasms , Humans , Squamous Cell Carcinoma of Head and Neck/genetics , Fibronectins/genetics , Endothelial Cells , Epidermal Growth Factor , ErbB Receptors/genetics , 5' Untranslated Regions , Integrins , Head and Neck Neoplasms/genetics
3.
World J Gastroenterol ; 22(18): 4515-28, 2016 May 14.
Article in English | MEDLINE | ID: mdl-27182161

ABSTRACT

AIM: To discover novel biomarkers for early diagnosis, prognosis or treatment of human colorectal cancer. METHODS: iTRAQ 2D LC-MS/MS analysis was used to identify differentially expressed proteins (DEPs) in the human colonic epithelial carcinogenic process using laser capture microdissection-purified colonic epithelial cells from normal colon, adenoma, carcinoma in situ and invasive carcinoma tissues. RESULTS: A total of 326 DEPs were identified, and four DEPs (DMBT1, S100A9, Galectin-10, and S100A8) with progressive alteration in the carcinogenic process were further validated by immunohistochemistry. The DEPs were involved in multiple biological processes including cell cycle, cell adhesion, translation, mRNA processing, and protein synthesis. Some of the DEPs involved in cellular process such as "translation" and "mRNA splicing" were progressively up-regulated, while some DEPs involved in other processes such as "metabolism" and "cell response to stress" was progressively down-regulated. Other proteins with up- or down-regulation at certain stages of carcinogenesis may play various roles at different stages of the colorectal carcinogenic process. CONCLUSION: These findings give insights into our understanding of the mechanisms of colorectal carcinogenesis and provide clues for further investigation of carcinogenesis and identification of biomarkers.


Subject(s)
Adenoma/chemistry , Biomarkers, Tumor/analysis , Carcinoma in Situ/chemistry , Carcinoma/chemistry , Cell Transformation, Neoplastic/chemistry , Colorectal Neoplasms/chemistry , Adenoma/pathology , Calcium-Binding Proteins , Calgranulin A/analysis , Calgranulin B/analysis , Carcinoma/pathology , Carcinoma in Situ/pathology , Cell Transformation, Neoplastic/pathology , Chromatography, Liquid , Colorectal Neoplasms/pathology , Computational Biology , DNA-Binding Proteins , Early Detection of Cancer/methods , Galectins/analysis , Humans , Immunohistochemistry , Laser Capture Microdissection , Predictive Value of Tests , Proteomics/methods , Receptors, Cell Surface/analysis , Tandem Mass Spectrometry , Tumor Suppressor Proteins
5.
J Biol Chem ; 290(22): 14192-207, 2015 May 29.
Article in English | MEDLINE | ID: mdl-25770214

ABSTRACT

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.


Subject(s)
Proteomics/methods , Spider Venoms/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cysteine/chemistry , DNA, Complementary/metabolism , Expressed Sequence Tags , Gene Deletion , Molecular Sequence Data , Mutation , Neurons/metabolism , Neurotoxins/chemistry , Patch-Clamp Techniques , Peptides/chemistry , Phylogeny , Protein Processing, Post-Translational , Rats , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Spiders , Transcription, Genetic
6.
PLoS One ; 10(2): e0117099, 2015.
Article in English | MEDLINE | ID: mdl-25647561

ABSTRACT

Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.


Subject(s)
Peptides/genetics , Peptides/isolation & purification , Spider Venoms/genetics , Spider Venoms/isolation & purification , Spiders/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cloning, Molecular , Escherichia coli/genetics , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Genetic Vectors/genetics , Molecular Sequence Data , Peptides/chemistry , Peptides/pharmacology , Rats, Sprague-Dawley , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Spider Venoms/chemistry , Spider Venoms/pharmacology , Spiders/chemistry
7.
Sci Rep ; 4: 4569, 2014 Apr 02.
Article in English | MEDLINE | ID: mdl-24691553

ABSTRACT

The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.


Subject(s)
Membrane Transport Modulators/metabolism , Peptides/metabolism , Voltage-Gated Sodium Channels/metabolism , Amino Acid Sequence , Animals , Cell Line , Extracellular Matrix/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Neurotoxins/metabolism
8.
Sheng Li Xue Bao ; 62(3): 255-60, 2010 Jun 25.
Article in Chinese | MEDLINE | ID: mdl-20571743

ABSTRACT

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.


Subject(s)
Ganglia, Spinal/cytology , Neurons/drug effects , Neurotoxins/pharmacology , Shal Potassium Channels/metabolism , Spider Venoms/pharmacology , Animals , Oocytes , Patch-Clamp Techniques , Peptides/pharmacology , Potassium Channel Blockers/pharmacology , Rats , Xenopus laevis
9.
J Zhejiang Univ Sci B ; 11(6): 451-7, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20506577

ABSTRACT

Selenocosmia huwena and Selenocosmia hainana are two tarantula species found in southern China. Their venoms contain abundant peptide toxins. Two new neurotoxic peptides, huwentoxin-III (HWTX-III) and hainantoxin-VI (HNTX-VI), were obtained from the venom using ion-exchange chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The mechanism of action of HWTX-III and HNTX-VI on insect neuronal voltage-gated sodium channels (VGSCs) was studied via whole-cell patch clamp techniques. In a fashion similar to delta-atracotoxins, HNTX-VI can induce a slowdown of current inactivation of the VGSC and reduction in the peak of Na+ current in cockroach dorsal unpaired median (DUM) neurons. Meanwhile, 10 micromol/L HNTX-IV caused a positive shift of steady-state inactivation of sodium channel. HWTX-III inhibited VGSCs on DUM neurons (concentration of toxin at half-maximal inhibition (IC(50)) approximately 1.106 micromol/L) in a way much similar to tetrodotoxin (TTX). HWTX-III had no effect on the kinetics of activation and inactivation. The shift in the steady-state inactivation curve was distinct from other depressant spider toxins. The diverse effect and the mechanism of action of the two insect toxins illustrate the diverse biological activities of spider toxins and provide a fresh theoretical foundation to design and develop novel insecticides.


Subject(s)
Ion Channel Gating/physiology , Membrane Potentials/physiology , Neurons/physiology , Sodium Channels/physiology , Sodium/metabolism , Spider Venoms/administration & dosage , Animals , Cells, Cultured , Cockroaches , Dose-Response Relationship, Drug , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Neurons/drug effects , Sodium Channels/drug effects
10.
PLoS One ; 3(10): e3414, 2008.
Article in English | MEDLINE | ID: mdl-18923708

ABSTRACT

BACKGROUND: Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking) were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle. PRINCIPAL FINDINGS: Here we report the presence of a new superfamily of ktts in spiders (TARANTULAS: Ornithoctonus huwena and Ornithoctonus hainana), which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI) as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (omega) for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins) derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications. CONCLUSIONS/SIGNIFICANCE: These finding illustrate that the two activity sites of Kunitz-type toxins are functionally and evolutionally independent and provide new insights into effects of Darwinian selection pressures on KTT evolution, and mechanisms by which new functions can be grafted onto old protein scaffolds.


Subject(s)
Spider Venoms/pharmacology , Animals , Binding Sites , Evolution, Molecular , Kv1.1 Potassium Channel/antagonists & inhibitors , Protein Conformation , Selection, Genetic , Spider Venoms/chemistry , Spider Venoms/genetics , Spiders , Trypsin Inhibitors
11.
Proteomics ; 8(20): 4259-72, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18924182

ABSTRACT

Osteoporosis (OP) is a major public health problem, mainly characterized by low bone mineral density (BMD). Circulating monocytes (CMCs) may serve as progenitors of osteoclasts and produce a wide variety of factors important to bone metabolism. However, the specific action mechanism of CMCs in the pathogenesis of OP is far from clear. We performed a comparative protein expression profiling study of CMCs in Chinese premenopausal females with extremely discordant BMD, identified a total of 38 differentially expressed proteins, and confirmed with Western blotting five proteins: ras suppressor protein1 (RSU1), gelsolin (GSN), manganese-containing superoxide dismutase (SOD2), glutathione peroxidase 1(GPX1), and prolyl 4-hydroxylase beta subunit (P4HB). These proteins might affect CMCs' trans-endothelium, differentiation, and/or downstream osteoclast functions, thus contribute to differential osteoclastogenesis and finally lead to BMD variation. The findings promote our understanding of the role of CMCs in BMD determination, and provide an insight into the pathogenesis of human OP.


Subject(s)
Bone Density/physiology , Gene Expression Profiling , Monocytes/metabolism , Premenopause/physiology , Adult , Asian People , China , Female , Gelsolin/metabolism , Glutathione Peroxidase/metabolism , Humans , Osteoporosis/etiology , Procollagen-Proline Dioxygenase/metabolism , Protein Disulfide-Isomerases/metabolism , Superoxide Dismutase/metabolism , Transcription Factors/metabolism , Glutathione Peroxidase GPX1
12.
Nucleic Acids Res ; 36(Database issue): D293-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17933766

ABSTRACT

Venomous animals possess an arsenal of toxins for predation and defense. These toxins have great diversity in function and structure as well as evolution and therefore are of value in both basic and applied research. Recently, toxinomics researches using cDNA library sequencing and proteomics profiling have revealed a large number of new toxins. Although several previous groups have attempted to manage these data, most of them are restricted to certain taxonomic groups and/or lack effective systems for data query and access. In addition, the description of the function and the classification of toxins is rather inconsistent resulting in a barrier against exchanging and comparing the data. Here, we report the ATDB database and website which contains more than 3235 animal toxins from UniProtKB/Swiss-Prot and TrEMBL and related toxin databases as well as published literature. A new ontology (Toxin Ontology) was constructed to standardize the toxin annotations, which includes 745 distinct terms within four term spaces. Furthermore, more than 8423 TO terms have been manually assigned to 2132 toxins by trained biologists. Queries to the database can be conducted via a user-friendly web interface at http://protchem.hunnu.edu.cn/toxin.


Subject(s)
Databases, Protein , Toxins, Biological/chemistry , Animals , Internet , Peptides/chemistry , Protein Sorting Signals , Proteins/chemistry , Proteins/genetics , Toxins, Biological/genetics , User-Computer Interface , Vocabulary, Controlled
13.
Mol Plant ; 1(1): 167-77, 2008 Jan.
Article in English | MEDLINE | ID: mdl-20031923

ABSTRACT

Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and blue-light stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition. However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis, the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light, although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced. These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.


Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Cryptochromes/genetics , Cryptochromes/metabolism , Gene Expression Regulation, Plant/radiation effects , Light , RNA, Messenger/genetics , RNA, Plant/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/radiation effects , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Carbonic Anhydrases/radiation effects , Cryptochromes/radiation effects , Deoxyribodipyrimidine Photo-Lyase/genetics , Deoxyribodipyrimidine Photo-Lyase/metabolism , Deoxyribodipyrimidine Photo-Lyase/radiation effects , Kinetics , Mutagenesis , Mutation , Seedlings/genetics , Seedlings/metabolism , Seedlings/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Zhonghua Zhong Liu Za Zhi ; 28(4): 274-9, 2006 Apr.
Article in Chinese | MEDLINE | ID: mdl-16875628

ABSTRACT

OBJECTIVE: This study was designed to establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous cell carcinoma tissue and paired tumor-adjacent normal bronchial epithelial tissue, and to identify differential expression of tumor-associated proteins by using proteome analysis. METHODS: Comparative proteome analysis of human lung squamous carcinoma and paired normal bronchial mucosa adjacent to tumors from 20 cases were carried out. Total proteins of the carcinoma tissue and normal bronchial mucosa were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: (1) Seventy-six differentially expressed proteins were screened by analyzing the electrophoretic maps of the 20 carcinoma and control mucosa tissues. (2) Sixty-eight differential proteins were identified by peptide mass fingerprinting (PMF). Some proteins were products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. (3) The expression of three proteins mdm2, c-Jun and EGFR, correlated with lung squamous carcinoma, were detected by immunohistochemical staining and Western blot analysis. The results showed that the expression of mdm2, c-Jun and EGFR were up-regulated in lung squamous carcinomas, whereas down-regulated in control normal mucosa. It was consistent with our proteome analysis results. Those results suggested that those proteins may play roles in the carcinogenesis of lung squamous carcinoma. CONCLUSION: sixty-eight differentially expressed proteins were successfully characterized by comparative proteome analysis. Those results may provide scientific foundation for screening the molecular biomarkers which can be used in diagnosis and treatment of lung squamous carcinoma, as well as to improve patients' prognosis and provide a new clue for carcinogenesis research of lung squamous cell carcinoma.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Lung Neoplasms/metabolism , Proteome , Adult , Aged , Biomarkers, Tumor , Carcinoma, Squamous Cell/pathology , Electrophoresis, Gel, Two-Dimensional , ErbB Receptors/metabolism , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Proto-Oncogene Proteins c-jun/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Respiratory Mucosa/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Up-Regulation
15.
Acta Biochim Biophys Sin (Shanghai) ; 38(7): 457-66, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16820861

ABSTRACT

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin with both antiprotease activity and potassium channel blocking activity. To determine its solution structure, huwentoxin-XI was expressed in a yeast eukaryotic expression system and studied by NMR. The 15N labeling strategy was used to facilitate the process of resonance assignments. The nearly complete sequence-specific assignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional (2D) and three-dimensional (3D) spectra, including DQF-COSY, TOCSY, NOESY, 15N-1H HSQC, 15N-1H HNHA, 15N-1H HNHB, 15N-1H TOCSY-HSQC and 15N-1H NOESY-HSQC spectra. Secondary structure analysis of huwentoxin-XI showed that it mainly contains an N-terminal 310-helix from Thr3 to Arg5 and a C-terminal alpha-helix from Gln45 to Cys52, plus a triple-stranded antiparallel beta-sheet of Glu18-Asn23, Thr26-Ile31 and Asn40-Lys41. These studies provide a solid basis for the final structure determination of huwentoxin-XI.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Spider Venoms/chemistry , Animals , Hydrogen Bonding , Potassium Channel Blockers/chemistry , Protease Inhibitors/chemistry , Protein Structure, Secondary , Recombinant Proteins/chemistry , Spiders/chemistry
16.
Bioinformatics ; 22(18): 2189-91, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16632497

ABSTRACT

UNLABELLED: We report a novel protein domain-G8-which contains five repeated beta-strand pairs and is present in some disease-related proteins such as PKHD1, KIAA1199, TMEM2 as well as other uncharacterized proteins. Most G8-containing proteins are predicted to be membrane-integral or secreted. The G8 domain may be involved in extracellular ligand binding and catalysis. It has been reported that mis-sense mutations in the two G8 domains of human PKHD1 protein resulted in a less stable protein and are associated with autosomal-recessive polycystic kidney disease, indicating the importance of the domain structure. G8 is also present in the N-terminus of some non-syndromic hearing loss disease-related proteins such as KIAA1109 and TMEM2. Discovery of G8 domain will be important for the research of the structure/function of related proteins and beneficial for the development of novel therapeutics. CONTACT: liangsp@hunnu.edu.cn


Subject(s)
Hearing Loss, Sensorineural/metabolism , Membrane Proteins/chemistry , Polycystic Kidney Diseases/metabolism , Proteins/chemistry , Receptors, Cell Surface/chemistry , Sequence Analysis, Protein/methods , Amino Acid Sequence , Animals , Conserved Sequence , Evolution, Molecular , Hearing Loss, Sensorineural/genetics , Hyaluronoglucosaminidase , Membrane Proteins/genetics , Molecular Sequence Data , Polycystic Kidney Diseases/genetics , Protein Structure, Tertiary , Proteins/genetics , Receptors, Cell Surface/genetics , Sequence Alignment/methods , Sequence Homology, Amino Acid , Species Specificity
17.
Acta Biochim Biophys Sin (Shanghai) ; 37(8): 567-72, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16077905

ABSTRACT

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Peptide Mapping/methods , Peptides/analysis , Peptides/chemistry , Sequence Analysis, Protein/methods , Spider Venoms/analysis , Spider Venoms/chemistry , Amino Acid Sequence , Molecular Sequence Data , Protein Structure, Secondary , Protons
18.
Bioinformatics ; 21(18): 3694-6, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-16020467

ABSTRACT

SUMMARY: The Differentially Expressed Protein Database was designed to store the output of comparative proteomics studies and provides a publicly available query and analysis platform for data mining. The database contains information about more than 3000 differentially expressed proteins (DEPs) manually extracted from the published literature, including relevant biological, experimental and methodological elements. Tools for visualization and functional analysis of DEPs are provided via a user-friendly webinterface. AVAILABILITY: http://protchem.hunnu.edu.cn/depd/.


Subject(s)
Computational Biology/methods , Databases, Protein , Proteomics/methods , Computational Biology/instrumentation , Humans , Information Storage and Retrieval , Internet , Programming Languages , Sequence Analysis, Protein , Software , User-Computer Interface
19.
Sheng Wu Gong Cheng Xue Bao ; 21(1): 92-6, 2005 Jan.
Article in Chinese | MEDLINE | ID: mdl-15859335

ABSTRACT

Hainantoxin-IV (HNTX-IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin-sensitive (TrX-S) sodium channels. It is a 35-residue polypeptide and includes three disulfide bridges. In order to investigate the structure-function relationship of HNTX-IV, two mutants (S12A-HNTX-IV and R29A-HNTX-IV) of HNTX-TV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX-IV (nHNTX-IV) have almost identical three-dimensional structures. The bioactivity level of S12A-HNTX-IV is also about the same as that of nHNTX-IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A-HNTX-IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX-IV. It is presumed that the decrease in activity of R29A-HNTX-IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.


Subject(s)
Mutation , Sodium Channel Blockers , Sodium Channels/drug effects , Spider Venoms/chemical synthesis , Amino Acid Substitution , Animals , Sodium Channels/physiology , Spider Venoms/genetics , Structure-Activity Relationship , Tetrodotoxin/pharmacology
20.
Di Yi Jun Yi Da Xue Xue Bao ; 25(1): 10-4, 2005 Jan.
Article in Chinese | MEDLINE | ID: mdl-15683987

ABSTRACT

OBJECTIVE: To study the antinociceptive effect of intrathecally administered huwentoxin-I (HWTX-I or HWAP-I) on visceral pain in rats with acute colon inflammation. METHODS: Nociceptive behaviors was induced by formalin injected into the submucosa of the sigmoid colon in rats and the typical behavioral patterns induced served as the indexes for visceral nociception scoring. HWAP-I (0.1-1.0 microg/kg x b x w.), SNX-111 (0.2, 0.5 and 0.75 microg/kg.b.w.) and morphine hydrochloride (0.05, 0.1 and 0.2 microg/kg x b x w.) were administered subarachnoidally 30 min before formalin injection. RESULTS: Similar to SNX-111 and hydrochloride morphine, HWAP-I administered subarachnoidally significantly reduced nociceptive responses in a dose-dependent manner in the rat model of visceral pain (P<0.05). Both HWAP-I and SNX-111 produced pain suppression effect at the dosage of 0.2 microg/kg x b x w., and in spite of the stronger antinociceptive effect of SNX-111 than HWAP-I at the same doses, SNX-111 caused obvious motor dysfunction in the rats at the doses higher than 0.5 microg/kg x b x w., which was not observed with HWAP-1 at such doses. The antinociceptive effect of morphine hydrochloride was initiated faster but lasted for a shorter period of time than the effects of HWAP-I and SNX-111. CONCLUSION: As a potent blocker of neuronal N-type voltage-sensitive calcium channel, HWAP-I induces remarkably dose-dependent inhibitory effect similar to SNX-111 and morphine on visceral pain induced by sigmoid colon submucosal injection of formalin in conscious rats.


Subject(s)
Analgesics, Non-Narcotic/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type , Nociceptors/drug effects , Reptilian Proteins/pharmacology , Spider Venoms/pharmacology , Animals , Dose-Response Relationship, Drug , Female , Injections, Spinal , Male , Pain Measurement , Rats , Rats, Sprague-Dawley
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