Co-reporter:Shi-Jin Yin, Ling Jiang, Hong Yi, Song Han, Dai-Wen Yang, Mai-Li Liu, Hui Liu, Zhi-Jian Cao, Ying-Liang Wu and Wen-Xin Li
Journal of Proteome Research November 7, 2008 Volume 7(Issue 11) pp:4890-4897
Publication Date(Web):November 7, 2008
DOI:10.1021/pr800494a
The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.Keywords: ADWX-1 peptide selectivity; channel turret; Kv1.1 channel; Kv1.3 channel; structural basis;
Co-reporter:Xiaobo Xu, Zhigui Duan, Zhiyong Di, Yawen He, Jianglin Li, Zhongjie Li, Chunliang Xie, Xiongzhi Zeng, Zhijian Cao, Yingliang Wu, Songping Liang, Wenxin Li
Journal of Proteomics 2014 Volume 106() pp:162-180
Publication Date(Web):25 June 2014
DOI:10.1016/j.jprot.2014.04.032
•The venom of M. martensii is firstly analyzed by combined proteomic approaches.•A large number of venom proteins are unambiguously identified.•Seven novel toxin sequences are reported in this study.•We obtain a mass fingerprint with more than 200 different molecular masses.•We contribute 93 unknown protein sequences with potential biological functions.The scorpion Mesobuthus martensii is the most populous species in eastern Asian countries, and several toxic components have been identified from their venoms. Nevertheless, a complete proteomic profile of the venom of M. martensii is still not available. In this study, the venom of M. martensii was analyzed by comprehensive proteomic approaches. 153 fractions were isolated from the M. martensii venom by 2-DE, SDS-PAGE and RP-HPLC. The ESI-Q-TOF MS results of all fractions were used to search the scorpion genomic and transcriptomic databases. Totally, 227 non-redundant protein sequences were unambiguously identified, composed of 134 previously known and 93 previously unknown proteins. Among 134 previously known proteins, 115 proteins were firstly confirmed from the M. martensii crude venom and 19 toxins were confirmed once again, involving 43 typical toxins, 7 atypical toxins, 12 venom enzymes and 72 cell associated proteins. In typical toxins, 7 novel-toxin sequences were identified, including 3 Na+-channel toxins, 3K+-channel toxins and 1 no-annotation toxin. These results increased 230% (115/50) venom components compared with previous studies from the M. martensii venom, especially 50% (24/48) typical toxins. Additionally, a mass fingerprint obtained by MALDI-TOF MS indicated that the scorpion venom contained more than 200 different molecular mass components.Biological significanceThis work firstly gave a systematic investigation of the M. martensii venom by combined proteomics strategy coupled with genomics and transcriptomics. A large number of protein components were unambiguously identified from the venom of M. martensii, most of which were confirmed for the first time. We also contributed 7 novel-toxin sequences and 93 protein sequences previously unknown to be part of the venom, for which we assigned potential biological functions. Besides, we obtained a mass fingerprint of the M. martensii venom. Together, our study not only provides the most comprehensive catalog of the molecular diversity of the M. martensii venom at the proteomic level, but also enriches the composition information of scorpion venom.
Co-reporter:Tao Wang;Xian-guo Wang;Jun-hua Xu;Xiang-Peng Wu;Hong-ling Qiu;Hong Yi ;Wen-Xin Li
Journal of Cellular and Molecular Medicine 2012 Volume 16( Issue 5) pp:1134-1145
Publication Date(Web):
DOI:10.1111/j.1582-4934.2011.01388.x
Abstract
Zinc finger proteins (ZNF) play important roles in various physiological processes. Here we report that ZNF300, a novel zinc finger protein, identified specifically in humans, promotes tumour development by modulating the NF-κB pathway. Inflammatory factors were found to induce ZNF300 expression in HeLa cell line, and ZNF300 expression further enhanced NF-κB signalling by activating TRAF2 and physically interacting with IKKβ. Furthermore, ZNF300 overexpression increased ERK1/2 phosphorylation and the expression of c-myc, IL-6, and IL-8 but decreased the expression of p21waf-1 and p27Kip1; whose down-regulation led to the opposite effect. Most importantly, ZNF300 overexpression stimulated cancer cell proliferation in vitro and significantly enhanced tumour development and metastasis in mouse xenograft model, while knocking down ZNF300 led to the opposite effects. We have identified a novel function for ZNF300 in tumour development that may uniquely link inflammation and NF-κB to tumourigenesis in humans but not in mice.
Co-reporter:Yibao Ma, Yawen He, Ruiming Zhao, Yingliang Wu, Wenxin Li, Zhijian Cao
Journal of Proteomics 2012 Volume 75(Issue 5) pp:1563-1576
Publication Date(Web):16 February 2012
DOI:10.1016/j.jprot.2011.11.029
Venom is an important genetic development crucial to the survival of scorpions for over 400 million years. We studied the evolution of the scorpion venom arsenal by means of comparative transcriptome analysis of venom glands and phylogenetic analysis of shared types of venom peptides and proteins between buthids and euscorpiids. Fifteen types of venom peptides and proteins were sequenced during the venom gland transcriptome analyses of two Buthidae species (Lychas mucronatus and Isometrus maculatus) and one Euscorpiidae species (Scorpiops margerisonae). Great diversity has been observed in translated amino acid sequences of these transcripts for venom peptides and proteins. Seven types of venom peptides and proteins were shared between buthids and euscorpiids. Molecular phylogenetic analysis revealed that at least five of the seven common types of venom peptides and proteins were likely recruited into the scorpion venom proteome before the lineage split between Buthidae and Euscorpiidae with their corresponding genes undergoing individual or multiple gene duplication events. These are α-KTxs, βKSPNs (β-KTxs and scorpines), anionic peptides, La1-like peptides, and SPSVs (serine proteases from scorpion venom). Multiple types of venom peptides and proteins were demonstrated to be continuously recruited into the venom proteome during the evolution process of individual scorpion lineages. Our results provide an insight into the recruitment pattern of the scorpion venom arsenal for the first time.Highlights► More than 200 scorpion venom peptides and proteins were identified by transcriptome analysis, expanding venom composition. ► These toxic compositions were categorized into 15 types, including two novel types, opening new avenues for drug discovery. ► Multiple toxin types were continuously recruited into the venom proteome during the evolution process of scorpion lineages. ► Proteome evolution of the scorpion venom arsenal was firstly clarified by comparative transcriptome and molecular phylogeny.
Co-reporter:Zongyun Chen;Youtian Hu;Song Han;Shijin Yin;Yawen He;Yingliang Wu;Zhijian Cao
Journal of Biochemical and Molecular Toxicology 2011 Volume 25( Issue 4) pp:244-251
Publication Date(Web):
DOI:10.1002/jbt.20382
Abstract
Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure–function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC50 of 1.70 n± 1.35 µM, whereas 10 µM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 µM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca2+ channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K+ channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel. © 2011 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:244–251, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20382
Co-reporter:Song Han, Shijin Yin, Hong Yi, Stéphanie Mouhat, Su Qiu, Zhijian Cao, Jean-Marc Sabatier, Yingliang Wu and Wenxin Li
Journal of Proteome Research 2010 Volume 9(Issue 6) pp:3118-3125
Publication Date(Web):2017-2-22
DOI:10.1021/pr100027k
Protein−protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein−protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein−protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.
Co-reporter:Lu Xue;Hongling Qiu;Jian Ma;Mingxiong Guo
Cellular & Molecular Biology Letters 2010 Volume 15( Issue 4) pp:530-540
Publication Date(Web):2010 December
DOI:10.2478/s11658-010-0025-1
ZNF300 was recently identified as a member of the human KRAB/C2H2 zinc finger protein family. Little is known about the role of ZNF300 in human gene regulation networks. In this study, the DNA-binding property of ZNF300 was further analyzed. We found that the recombinant ZNF300 could bind to the binding site 5′-GCGGGGGCG-3′ of Egr1, another member of the KRAB/C2H2 zinc finger protein family. Similarly, recombinant Egr1 also showed a similar binding affinity to the ZNF300 binding site 5′-CTGGGGGCG-3′. Bioinformatics analysis revealed that there is an overlapping ZNF300/Egr1 binding site in the human IL-2Rβ promoter region, which was previously known to be recognized by endogenous Egr1. Electrophoretic mobility shift assays showed that endogenous ZNF300 could also bind to this site. A transient transfection assay revealed that both ZNF300 and Egr1 could transactivate the IL-2Rβ promoter, and that the activation was abrogated by a mutation of residues in the overlapping ZNF300/Egr1 binding site. Co-expression of ZNF300 and Egr1 led to enhanced IL-2Rβ promoter activity. Thus, ZNF300 is likely to be another regulator of the human IL-2Rβ promoter.
Co-reporter:Su Qiu, Hong Yi, Hui Liu, Zhijian Cao, Yingliang Wu and Wenxin Li
Journal of Chemical Information and Modeling 2009 Volume 49(Issue 7) pp:1831-1838
Publication Date(Web):June 5, 2009
DOI:10.1021/ci900025n
The scorpion toxin, charybdotoxin (ChTX), is the first identified peptide inhibitor for the large-conductance Ca2+ and voltage-dependent K+ (BK) channel, and the chemical information of the interaction between ChTX and BK channel remains unclear today. Using combined computational methods, we obtained a ChTX-BK complex structure model, which correlated well with the mutagenesis data. In this complex, ChTX mainly used its β-sheet domains to associate the BK channel with a conserved pore-blocking Lys27. Another crucial Tyr36 residue of ChTX lied over the loop connecting selectivity filter and S6 helix of BK channel, forming a hydrogen bond with Gly291 of BK channel. Besides, the unique turret region of BK channel was found to be far away from bound ChTX, which could explain the fact that many BK channel blockers show less selectivity over Kv channels. Together, all these information is helpful to reveal the diverse interactions between scorpion toxins and potassium channels and can accelerate the molecular engineering of specific inhibitor design.
Co-reporter:Xin Mao, Chun Rong Yu, Wen Hua Li and Wen Xin Li
Cell Research 2008 18(8) pp:879-888
Publication Date(Web):July 29, 2008
DOI:10.1038/cr.2008.86
This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 M) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminal kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.
Co-reporter:Shi-Jin Yin, Ling Jiang, Hong Yi, Song Han, Dai-Wen Yang, Mai-Li Liu, Hui Liu, Zhi-Jian Cao, Ying-Liang Wu and Wen-Xin Li
Journal of Proteome Research 2008 Volume 7(Issue 11) pp:4890-4897
Publication Date(Web):2017-2-22
DOI:10.1021/pr800494a
The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.
Co-reporter:Hongling Qiu;Lu Xue;Li Gao;Huanjie Shao;Di Wang
Cellular & Molecular Biology Letters 2008 Volume 13( Issue 3) pp:391-403
Publication Date(Web):2008 September
DOI:10.2478/s11658-008-0005-x
The human ZNF300 gene is a member of the KRAB/C2H2 zinc finger gene family, the members of which are known to be involved in various developmental and pathological processes. Here, we show that the ZNF300 gene encodes a 68-kDa nuclear protein that binds DNA in a sequence-specific manner. The ZNF300 DNA binding site, C(t/a)GGGGG(c/g)G, was defined via a random oligonucleotide selection assay, and the DNA binding site was further confirmed by electrophoretic mobility shift assays. A potential ZNF300 binding site was found in the promoter region of the human IL-2Rβ gene. The results of electrophoretic mobility shift assays indicated that ZNF300 bound to the ZNF300 binding site in the IL-2Rβ promoter in vitro. Transient co-transfection assays showed that ZNF300 could activate the IL-2Rβ promoter, and that the activation was abrogated by the mutation of residues in the ZNF300 binding site. Identifying the DNA binding site and characterizing the transcriptional regulation property of ZNF300 would provide critical insights into its potential as a transcriptional regulator.
Co-reporter:Li Xu;Zhouzhou Zhao;Jiqun Sheng;Chengang Zhu;Hui Liu
Biochemistry (Moscow) 2008 Volume 73( Issue 9) pp:1025-1030
Publication Date(Web):2008 September
DOI:10.1134/S0006297908090101
C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.
Co-reporter:Chengang Zhu;Zhouzhou Zhao;Mingxiong Guo;Huanjie Shao
Biochemistry (Moscow) 2008 Volume 73( Issue 8) pp:881-885
Publication Date(Web):2008 August
DOI:10.1134/S0006297908080051
Nonsense-mediated mRNA decay (NMD), also called RNA surveillance, is a process that degrades mRNAs with premature translation termination codons. In Saccharomyces cerevisiae, it has also been shown that NMD can regulate gene expression at the transcriptional level. To date, there has been no example where promoters are regulated by the NMD path-way in higher eukaryotes. Taking advantage of our previous research on ZNF268 transcription control, we studied the relationship between the ZNF268 promoter and the NMD pathway. We showed by transient transfection that the ZNF268 promoter activity was influenced by hUpf1, not hSmg6, in HeLa cells. This result was confirmed by the analysis of the steady state mRNA of ZNF268 after depletion of endogenous hUpf1 or hSmg6 in HeLa cells. Direct mutational analysis revealed that the C/EBP site in the promoter region is important for hUpf1 function on ZNF268 promoter. Together our results demonstrated that the mammalian gene ZNF268 is regulated by hUpf1 via its promoter.
Co-reporter:Chao Dai;Zhijian Cao;Yingliang Wu;Hong Yi
Cellular & Molecular Biology Letters 2007 Volume 12( Issue 3) pp:362-369
Publication Date(Web):2007 September
DOI:10.2478/s11658-007-0008-z
Since its discovery, green fluorescence protein (GFP) has been used as a reporter in a broad range of applications, including the determination of gene expresion in diverse organisms, and subcellular protein localization. pEGFP-N1 is a eukayotic expression vector encoding EGFP, the MCS of which locates at the N terminus of EGFP. In this study, the cDNA sequence of scorpion toxin BmKK2 was inserted into the XhoI-HindIII cut of pEGFP-N1 to construct a toxin-EGFP fusion gene (named pEGFP-BmKK2). Fluorescence imaging revealed that HEK 293T cells that were transfected by pEGFP-BmKK2 emitted green fluorescence. Transcription of pEGFP-BmKK2 was confirmed by RT-PCR. However, western blotting analysis showed that the transfected HEK 293T cells expressed mostly EGFP, but little toxin-EGFP fusion protein, implying that pEGFP-N1 cannot be used as a fusion expression vector for subcellular protein localization for the BmKK2 gene. Consequently, two modified recombinant vectors (pEGFP-BmKK2-M1 and pEGFP-BmKK2-M2) were constructed based on pEGFP-BmKK2. This greatly improved the expression of toxin-EGFP fusion protein from pEGFP-BmKK2-M2.
Co-reporter:Cao Zhijian;Dai Chao;Li Wenxin;Jiang Dahe
Journal of Biochemical and Molecular Toxicology 2006 Volume 20(Issue 3) pp:127-132
Publication Date(Web):20 JUN 2006
DOI:10.1002/jbt.20127
Previously reported results showed that the BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found in the second exon. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression level of the toxin-GFP fusion protein (Cao et al., J Biochem Mol Toxicol 2006;20:1–6). In this investigation, the BmKK2's intron with 79 nucleotides length was artificially shifted from the 49th nt (the 17th Gly codon between the first base and the second base) to the 100th nt (the 34th Gly codon between the first base and the second base). Based on the constructed intron-splicing system, the results of RT-PCR and the western blotting analysis showed that the BmKK2's shifted-intron (named BmKK2-s) was not recognized and spliced correctly, but the cryptic splicing site of BmKK2 gene was still spliced in the second exon, which possibly indicated that locations of introns were very important to the recognition and splicing of introns, and splicing of introns was very much associated with the corresponding upstream and downstream exons. This result possibly provides evidence for splice-site recognition across the exons. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:127–132, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20127
Co-reporter:Yin Shijin;Wu Yingliang;Dai Chao;Li Wenxin;Cao Zhijian;Sha Yonggang;Sheng Jiqun
Journal of Biochemical and Molecular Toxicology 2006 Volume 20(Issue 1) pp:1-6
Publication Date(Web):23 FEB 2006
DOI:10.1002/jbt.20111
Using GFP as a reporter gene, splicing of scorpion toxin gene BmKK2 was investigated in cultured HEK 293T cells. The results of RT-PCR and western blotting showed that BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found at the 91st nucleotide site of the second exon, which is a typical form of alternative splicing. For the first time, alternative splicing would partially explain the diversity of scorpion toxins at the gene level. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression of the toxin-GFP fusion protein by fluorescence imaging, which indicated that both introns may regulate the expression of toxin-GFP fusion protein. The artificial BmKK2-BmP03 mosaic gene was also spliced into two kinds of mRNA molecules, which showed that sequence of intron was not absolutely conserved. The results suggested that introns of scorpion toxin genes BmKK2 and BmP03 increase the diversity of scorpion toxins and regulate the expression of their genes. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:1–6, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20111
Co-reporter:Sheng Jiqun;Xu Xiuling;Cao Zhijian;Zhu Shunyi;Jiang Dahe;Wu Yingliang;Liu Hui;Zeng Xianchun;Mao Xin;Li Wenxin;Wang Teng;Liu Wanhong
Journal of Biochemical and Molecular Toxicology 2004 Volume 18(Issue 4) pp:187-195
Publication Date(Web):27 SEP 2004
DOI:10.1002/jbt.20026
Scorpion venom contains many small polypeptide toxins, which can modulate Na+, K+, Cl−, and Ca2+ ion–channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K+-channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K+-channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K+-channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0μM rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K+ current (Ito), delayed inward rectifier K+ current (Ik1), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K+-channel scorpion toxin. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:187–195, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20026
Co-reporter:Cao Zhijian;Wu Yingliang;Sheng Jiqun;Liu Wanhong;Xiao Fan;Mao Xin;Liu Hui;Jiang Dahe;Li Wenxin
Journal of Biochemical and Molecular Toxicology 2003 Volume 17(Issue 4) pp:235-238
Publication Date(Web):29 JUL 2003
DOI:10.1002/jbt.10083
All scorpion toxins from different 30 species are simply reviewed. A new classification system of scorpion toxins is first proposed: scorpion toxins are classified into three families (long-chain scorpion toxins with 4 disulfide bridges, short-chain scorpion toxins with 3 disulfide bridges, and intermediate-type scorpion toxins with 3 or 4 disulfide bridges). Intermediate-type scorpion toxins provide a strong proof for the conclusion that channel toxins from scorpion venoms evolve from a common ancestor. Common organization of precursor nucleotides and genomic sequence, similar 3-dimensional structure, and the existence of intermediate type scorpion toxins and functionally intercrossing scorpion toxins show that all scorpion toxins affecting ion channels evolve from the common ancestor, which produce millions of scorpion toxins with function-diversity. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:235–238, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10083
Co-reporter:Qiaoli Li, Zhenhuan Zhao, Dihan Zhou, Yaoqing Chen, Wei Hong, Luyang Cao, Jingyi Yang, Yan Zhang, Wei Shi, Zhijian Cao, Yingliang Wu, Huimin Yan, Wenxin Li
Peptides (July 2011) Volume 32(Issue 7) pp:1518-1525
Publication Date(Web):1 July 2011
DOI:10.1016/j.peptides.2011.05.015
Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC50 of 7.15 μg/ml (3.52 μM) and a CC50 of 70.46 μg/ml (34.70 μM) against measles virus, an EC50 of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC50 of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.Highlights► We investigated the antiviral activities of mucroporin and optimized mucroporin-M1. ► Mucroporin and mucroporin-M1 were scorpion venom-derived host defense peptides. ► Mucroporin-M1 but not mucroporin had antiviral activities against tested viruses. ► The tested viruses were measles, SARS-CoV and influenza H5N1 viruses. ► Mucroporin-M1 exerted its effects on viruses by directly binding to virus membranes.
Co-reporter:Zongyun Chen, Song Han, Zhijian Cao, Yingliang Wu, Renxi Zhuo, Wenxin Li
Peptides (January 2013) Volume 39() pp:145-151
Publication Date(Web):1 January 2013
DOI:10.1016/j.peptides.2012.11.013
Animal toxins are powerful tools for testing the pharmacological, physiological, and structural characteristics of ion channels, proteases, and other receptors. However, most animal toxins are disulfide-rich peptides that are difficult to produce functionally. Here, a glutathione S-transferase (GST) fusion expression strategy was used to produce four recombinant animal toxin peptides, ChTX, StKTx23, BmP01, and ImKTx1, with different isoelectric points from 4.7 to 9.2. GST tags were removed by enterokinase, a widely used and effective commercial protease that cleaves after lysine at the cleavage site DDDDK. Using this strategy, two disulfide-rich animal toxins ChTX and StKTx23 were obtained successfully with a yield of approximately 1–2 mg/l culture. Electrophysiological experiments further showed that these two recombinant toxins showed good bioactivities, indicating that our method was effective in producing large amounts of functional disulfide-rich animal toxins. Interestingly, by analyzing the separated fractions of BmP01, StKTx23, and ImKTx1 using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, four new enterokinase secondary cleavage sites were found, consisting of the sequences “WEYR,” “EDK,” “QNAR,” and “DNDK.” To our knowledge, this is the first report of the presence of secondary cleavage sites for commercial enterokinase in animal toxins. These findings will help us use commercial enterokinase appropriately as a cleavage tool in the production of animal toxins.Highlights► A glutathione S-transferase (GST) fusion expression strategy was used to produce four animal toxins. ► GST tags were removed by enterokinase that cleaves after lysine at the cleavage site DDDDK. ► Four enterokinase secondary cleavage sites “WEYR”, “EDK”, “QNAR”, and “DNDK” were found. ► It is the first report of the presence of secondary cleavage sites for commercial enterokinase in animal toxins.
Co-reporter:Ran Yan, Zhenhuan Zhao, Yawen He, Lin Wu, Dawei Cai, Wei Hong, Yingliang Wu, Zhijian Cao, Congyi Zheng, Wenxin Li
Peptides (January 2011) Volume 32(Issue 1) pp:11-19
Publication Date(Web):1 January 2011
DOI:10.1016/j.peptides.2010.10.008
Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. There is no vaccine available for HCV, and almost half of patients cannot be cured using standard combination therapy. Thus, new anti-HCV strategies and drugs are urgently needed. Here, the gene encoding a new α-helical peptide, Hp1090, was screened from the venomous gland cDNA library of the scorpion Heterometrus petersii. Structural analysis showed that Hp1090 is an amphipathic α-helical peptide. In vitro HCV RNA inhibitory assays indicated that Hp1090 peptide inhibited HCV infection with an IC50 of 7.62 μg/ml (5.0 μM), whereas Hp1035 peptide, showing high homology to Hp1090, exhibited no anti-HCV activity. Hp1090 acted as a viricide against HCV particles in vitro and prevented the initiation of HCV infection. Furthermore, this peptide interacted with HCV particles directly and rapidly permeabilized phospholipid membranes. Collectively, it seems that Hp1090 is virocidal for HCV in vitro, directly interacting with the viral membrane and decreasing the virus infectivity. These results suggest that Hp1090 could be considered an anti-HCV lead compound with virocidal mechanism that offers a potential therapeutic approach to HCV infection. Our work opens a new avenue for antiviral drug discovery in natural scorpion venom.Research highlights▶ a novel α-helical peptide Hp1090 was high-throughput screened from the scorpion venom;▶ the peptide Hp1090 can inhibit HCV replication and prevents the initiation of HCV infection;▶ the peptide Hp1090 kills HCV by the mechanism of breaking viral membrane; ▶ Hp1090 peptide is the first natural anti-HCV peptide from animal venoms.
Co-reporter:Wenying Yuan, Luyang Cao, Yibao Ma, Panyong Mao, Weipeng Wang, Ruiming Zhao, Yingliang Wu, Zhijian Cao, Wenxin Li
Peptides (January 2010) Volume 31(Issue 1) pp:22-26
Publication Date(Web):1 January 2010
DOI:10.1016/j.peptides.2009.10.008
Scorpion has an innovative venom gland, which is an important determinant in contributing to its successful survival for more than 400 million years. Scorpion venom contains a diversity of bioactive peptides, which represent a tremendous hitherto unexplored resource for use in drug design and development. Here, StCT1, a new antimicrobial peptide gene, was screened and isolated from the venomous gland cDNA library of the scorpion Scorpiops tibetanus. The full-length cDNA of StCT1 is 369 nucleotides encoding the precursor that contains a putative 24-residue signal peptide, a presumed 14-residue mature peptide, and an uncommon 37-residue acidic propeptide at the C-terminus. The minimal inhibitory concentrations (MICs) of the synthetic StCT1 peptide against Staphylococcus aureus and Micrococcus luteus were 12.5 μg/ml and 100 μg/ml, respectively. The MICs of StCT1 against clinical antibiotics-resistant bacterial strains, were 50–250 μg/ml, 2–40 folds lower than those of penicillin. These results show that the antimicrobial peptide encoded by StCT1 gene from the venom of the scorpion S. tibetanus is a potential anti-infective polypeptide or lead compound, especially for treating antibiotics-resistant pathogens.
Co-reporter:Yibao Ma, Ruiming Zhao, Songryong Li, Shaozhong Fan, Yingliang Wu, Hui Liu, Zhijian Cao, Wenxin Li
Toxicon (January 2009) Volume 53(Issue 1) pp:129-134
Publication Date(Web):1 January 2009
DOI:10.1016/j.toxicon.2008.10.025
Scorpion venoms are rich resources of bioactive peptides with extreme variability. Multiple molecular mechanisms are involved in the diversity of scorpion venom peptides. However, alternative splicing, which plays a major role in the generation of proteomic and functional diversity in metazoan organisms, hasn't been reported in genes coding for scorpion venom peptides. In the EST analysis of venom peptide transcripts from scorpion Lychas mucronatus, we reported an alternative splicing event. Transcripts of LmTxLP11 and LmVP1.1 share identical 5′ region. LmVP1.1 is a novel type of scorpion venom peptides constrained by one disulfide bridge, whereas LmTxLP11 is an extended version of LmVP1.1. By transcript alignment with its genomic sequence, it is found that both transcripts are generated from a single gene by alternative poly A site and terminal exon. The gene encoding LmTxLP11 and LmVP1.1 is the first one harboring three introns ever reported from scorpion venoms. This work demonstrates for the first time that alternative splicing is involved in regulating the diversity of scorpion venom peptides.
Co-reporter:Jun-Hua Xu, Tao Wang, Xian-Guo Wang, Xiang-Peng Wu, Zhou-Zhou Zhao, Chen-Gang Zhu, Hong-Ling Qiu, Lu Xue, Huan-Jie Shao, Ming-Xiong Guo, Wen-Xin Li
Leukemia Research (December 2010) Volume 34(Issue 12) pp:1636-1646
Publication Date(Web):1 December 2010
DOI:10.1016/j.leukres.2010.04.009
ZNF300, which plays the role in human embryonic development and some diseases, is a typical KRAB/C2H2 zinc finger gene expressed only in higher mammalians. Our data showed that expression of ZNF300 changed significantly in various leukemia blasts in the bone marrow aspirates of newly diagnosed leukemia patients. To investigate the potential relationship between expression of ZNF300 and the progression of leukemia development and hematopoietic differentiation, we cloned and characterized the putative human ZNF300 gene promoter and identified its transcription start sites (TSSs). Deletion and mutagenesis analysis demonstrated that a myeloid-specific transcription factor PU.1 binding site was responsible for myeloid-specific regulation of ZNF300 promoter activity. Furthermore, electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that PU.1 bound to the PU.1 binding site within ZNF300 promoter region in vitro and in vivo. Overexpression of PU.1 elevated ZNF300 promoter activity, whereas silencing of PU.1 expression significantly reduced the activity in myeloid-derived HL-60 cell but not in T-cell Jurkat. In vitro induced HL-60 cells into CD11b expressing cells by DMSO demonstrated that ZNF300 was upregulated along with upregulation of PU.1 expression. These results demonstrated that ZNF300 was activated by PU.1 and suggested that the regulation may be involved in the progression of leukemia development and hematopoietic differentiation.
Co-reporter:Luyang Cao, Zhongjie Li, Ruhong Zhang, Yingliang Wu, Wenxin Li, Zhijian Cao
Peptides (August 2012) Volume 36(Issue 2) pp:213-220
Publication Date(Web):1 August 2012
DOI:10.1016/j.peptides.2012.04.010
Bacterial infection poses an increasing threat to global public health and new types of antibacterial agents are urgently needed to respond to the threat. Scorpion venom contains series of bioactive peptides, among which antibacterial peptide is an important part. Herein, a new antimicrobial peptide StCT2 was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. The full-length cDNA of StCT2 is 369 nucleotides encoding the precursor that contains a putative 24 residues signal peptide, a presumed 14 residues mature peptide, and a putative 37 residues acidic propeptide at the C-terminus. The minimal inhibition concentrations (MICs) of StCT2 for Staphylococcus aureus were 6.25–25 μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus (MRSA). StCT2 was further found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism. Taken together, these results demonstrate the efficacy and general mechanism of StCT2 antimicrobial action and the therapeutic potential of StCT2 as a new antimicrobial peptide.Highlights► StCT2, a new antimicrobial peptide, was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. ► The MICs of StCT2 for Staphylococcus aureus were 6.25–25 μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus. ► StCT2 was found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. ► StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism.
Co-reporter:Hui Dai, Shijin Yin, Tian Li, Zhijian Cao, Yonghua Ji, Yingliang Wu, Wenxin Li
Protein Expression and Purification (April 2012) Volume 82(Issue 2) pp:325-331
Publication Date(Web):1 April 2012
DOI:10.1016/j.pep.2012.02.001
Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure–function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4 mg from 1 L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNav1.4 (EC50 = 82.3 ± 15.7 nM) rather than that of rNav1.2 (EC50 > 30 μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.Highlights► Toxin rBmαTX14 was produced by the recombinant expression in Escherichia coli. ► BmαTX14’s structure is stable with or without His6-tag. ► BmαTX14 selectively inhibits the fast inactivation of mNav1.4 channel. ► The activity of BmαTX14 inhibiting channel inactivation is less affected by His6-tag.
