Jing Liu

University of Science and Technology of China, Hefei, Anhui Sheng, China

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Publications (48)133.48 Total impact

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    ABSTRACT: Agkisacucetin is a snake C-type lectin isolated from the venom of Agkistrodon acutus (A. acutus). It binds specifically to the platelet glycoprotein (GP) Ib and prevents the von Willebrand factor (VWF) accessing it. We determined the crystal structure of agkisacucetin to 1.9Å resolution. The structure of agkisacucetin has an (αβ) fold similar to another GPIb-binding protein, flavocetin-A, but lacks the C-terminal cysteine in the β-subunit, does not form (βα)(4) tetramers, and does not cluster GPIbs, like flavocetin-A.
    Proteins Structure Function and Bioinformatics 02/2012; 80(6):1707-11. · 3.34 Impact Factor
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    ABSTRACT: p185(her2/neu) belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185(her2/neu) monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185(her2/neu) mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185(her2/neu)-overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185(her2/neu), which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185(her2/neu). We propose a structure-based model in which chA21 cross-links two p185(her2/neu) molecules on separate homo- or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process."
    Journal of Biological Chemistry 06/2011; 286(36):31676-83. · 4.65 Impact Factor
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    ABSTRACT: p185her2/neu belongs to the erbB receptor tyrosine kinase family which has been associated with human breast, ovarian and lung cancers. Targeted therapies employing ectodomain specific p185her2/neu monoclonal antibodies (mAb) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185her2/neu mAb are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185her2/neu -over-expressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185her2/neu, which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185her2/neu. We propose a structure based model in which chA21 cross-links two p185her2/neu molecules on separate homo- or hetero- dimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAb to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process".
    Journal of Biological Chemistry 06/2011; · 4.65 Impact Factor
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    Cell Research 12/2010; 20(12):1386-9. · 10.53 Impact Factor
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    ABSTRACT: Our work is concerned with the origins and therapy of human cancers. Members of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, also known as erbB or HER receptors, are over expressed and/or activated in many types of human tumors and represent important therapeutic targets in cancer therapy. Studies from our laboratory identified targeted therapy as a way to treat cancer. Rational therapeutics targeting and disabling erbB receptors have been developed to reverse the malignant properties of tumors. Reversal of the malignant phenotype, best seen with disabling the HER2 receptors using monoclonal antibodies is a distinct process from that seen with blocking of ligand binding to cognate receptors as has been done for EGFr receptors. Here we review the mechanisms of action deduced from a number of approaches developed in our laboratory and elsewhere, including monoclonal antibodies, peptide mimetics, recombinant proteins and small molecules. The biochemical and biological principles which have been uncovered during these studies of disabling HER2 homomeric or HER2-EGFr heteromeric receptors will help the development of novel and more efficient therapeutics targeting erbB family receptors.
    Seminars in Cell and Developmental Biology 12/2010; 21(9):961-6. · 6.20 Impact Factor
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    ABSTRACT: It was well studied that ErbB2 (HER2/p185(her2/neu)) overexpression in human malignant cancers correlates with poor prognosis and chemo-resistance. Although Trastuzumab (Herceptin) has been widely used in patients with ErbB2-overexpressing metastatic breast cancer, many patients either do not respond to Trastuzumab therapy or progress within 1 year of initiating Trastuzumab treatment. Previously, we reported a novel tumor-inhibitory antibody chA21, which recognized ErbB2 extracellular domain with an epitope distinct from other tumor-inhibitory anti-ErbB2 antibodies. Here, we report that chA21 combined with Paclitaxel or Trastuzumab significantly enhances the tumor-inhibition effects on ErbB2-overexpressing breast and ovarian cancer in xenograft mice. Moreover, the study reveals that the effects by chA21 to cause an enhanced inhibition on cancer cell proliferation and angiogenesis was highly associated with the intrinsic ability of chA21 to down-regulate ErbB2 receptor, inhibit downstream MAPK and PI3K-AKT signal transduction and activate natural killer cells. Our findings show that chA21 may represent a unique anti-ErbB2 antibody with potentials as therapeutic candidate alone or combination with other anti-ErbB2 reagents in cancer therapy.
    Cancer Immunology and Immunotherapy 11/2010; 60(3):339-48. · 3.64 Impact Factor
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    ABSTRACT: Anti-HER-2 antibodies targeting distinct epitopes have different biological functions on cancer cells. In a previous study, we demonstrated that anti-HER-2 engineering antibody ChA21 was able to bind to subdomain I of HER-2 extracellular domain. In this study, The effects of ChA21 on growth and apoptosis against ovarian carcinoma cell SK-OV-3 over-expressing HER-2 in vitro and in vivo were investigated. Cell growth inhibition was evaluated by MTT assay. Apoptosis was detected by TUNEL stain, transmission electron microscopy and flow cytometry on cultured cells and tissue sections from nude mice xenografts. The apoptosis-related proteins Bax and Bcl-2 were assessed by immunohistochemistry. We found that treatment of ChA21 caused a dose-dependent decrease of cell proliferation in vitro and a significant inhibition of tumor growth in vivo. ChA21 therapy led to a significant increase in the induction of apoptosis, and up-regulated the expression of Bax, while the expression of Bcl-2 was down-regulated. These data suggest that ChA21 inhibits the growth and induces apoptosis of SK-OV-3 via regulating the balance between Bax and Bcl-2.
    Journal of Experimental & Clinical Cancer Research 03/2010; 29:23. · 3.07 Impact Factor
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    ABSTRACT: chA21 is a novel tumor-inhibitory antibody which recognized subdomain I of HER2 extracellular domain with an epitope distinct from other HER2 antibodies. Previously, we demonstrated that chA21 inhibits human ovarian carcinoma cell line SKOV-3 growth in vitro and in vivo study. In this study, we further investigated the anti-angiogenic efficacy combination of chA21 with trastuzumab in SKOV-3 xenograft model. Nude mice were s.c. challenged with SKOV-3 cells and received treatment of chA21 alone, trastuzumab alone or both antibodies together twice a week for 21 days. Tumor volume and microvessel density (MVD) were evaluated. The effect of chA21 plus trastuzumab treament on vascular endothelial growth factor (VEGF) secretion, endothelial cells proliferation and migration, and the status of HER2 downstream pathway AKT/phosphorylated AKT (pAKT) were evaluated in vitro. In vivo study combination of chA21 with trastuzumab resulted in reduce tumor growth and angiogenesis than each monotherapy. In vitro study, the combination of chA21 with trastuzumab inhibits VEGF secretion, endothelial cells proliferation and migration. Furthermore, the combination treatment inhibits pAKT expression. Our findings suggested that the combination of chA21 with trastuzumab can cause augmented inhibition of angiogenesis in SKOV-3 xenograft model. Inhibition of agniogenesis may through suppression of AKT pathway. The therapeutic benefits of combination chA21 with trastuzumab warrant further study in an attempt to make the translation into the clinic.
    Journal of Ovarian Research 01/2010; 3:20. · 2.43 Impact Factor
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    ABSTRACT: Anti-ErbB2 antibodies are well researched for the therapy of ErbB2-overexpressing tumors. The therapeutic potential and efficacy of these antibodies are closely related to their affinities to ErbB2. Previously we reported that an anti-ErbB2 antibody A21 targeting a conformational epitope comprising several loops in ErbB2 extracellular subdomain I and II could inhibit the proliferation of ErbB2-overexpressing cancer cells in vitro and in vivo. Here we found that another structureless and non-conserved loop in subdomain I of ErbB2 extracellular domain (ECD) was important for binding to A21, and then the antigen-contact sites on A21 were determined by site-directed mutation. The loop was constructed by molecular modeling, and a new model of A21-ErbB2 complex was generated by docking using the crystal structure of the scfv A21 and the model of ErbB2 ECD with the loop built. Based on the complex model, computational design for A21 affinity improvement was performed to enhance its affinity to ErbB2. Two mutants with about 1.7-fold improvement in affinity were obtained. Our study provided a rational molecular basis for affinity improvement and mechanism investigation of A21.
    Journal of Computer-Aided Molecular Design 12/2009; 24(1):37-47. · 3.17 Impact Factor
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    ABSTRACT: Centrosome cohesion and segregation are accurately regulated to prevent an aberrant separation of duplicated centrosomes and to ensure the correct formation of bipolar spindles by a tight coupling with cell cycle machinery. CPAP is a centrosome protein with five coiled-coil domains and plays an important role in the control of brain size in autosomal recessive primary microcephaly. Previous studies showed that CPAP interacts with tubulin and controls centriole length. Here, we reported that CPAP forms a homodimer during interphase, and the fifth coiled-coil domain of CPAP is required for its dimerization. Moreover, this self-interaction is required for maintaining centrosome cohesion and preventing the centrosome from splitting before the G(2)/M phase. Our biochemical studies show that CPAP forms homodimers in vivo. In addition, both monomeric and dimeric CPAP are required for accurate cell division, suggesting that the temporal dynamics of CPAP homodimerization is tightly regulated during the cell cycle. Significantly, our results provide evidence that CPAP is phosphorylated during mitosis, and this phosphorylation releases its intermolecular interaction. Taken together, these results suggest that cell cycle-regulated phosphorylation orchestrates the dynamics of CPAP molecular interaction and centrosome splitting to ensure genomic stability in cell division.
    Journal of Biological Chemistry 11/2009; 285(4):2488-97. · 4.65 Impact Factor
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    ABSTRACT: ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody-antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1-192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 A resolution from a single flash-cooled crystal; the crystal belonged to space group P2(1)2(1)2(1).
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2009; 65(Pt 7):692-4. · 0.55 Impact Factor
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    ABSTRACT: The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins-called plus-end tracking proteins (+TIPs)-bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150-MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.
    EMBO Reports 07/2009; 10(8):857-65. · 7.19 Impact Factor
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    ABSTRACT: During cell division, chromosome segregation is governed by the interaction of spindle microtubules with the kinetochore. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, microtubule-bundling protein PRC1, and Aurora B kinase complex. However, the precise role of PRC1 in central spindle organization has remained elusive. Here we show that PRC1 recruits CLASP1 to the central spindle at early anaphase onset. CLASP1 belongs to a conserved microtubule-binding protein family that mediates the stabilization of overlapping microtubules of the central spindle. PRC1 physically interacts with CLASP1 and specifies its localization to the central spindle. Repression of CLASP1 leads to sister-chromatid bridges and depolymerization of spindle midzone microtubules. Disruption of PRC1-CLASP1 interaction by a membrane-permeable peptide abrogates accurate chromosome segregation, resulting in sister chromatid bridges. These findings reveal a key role for the PRC1-CLASP1 interaction in achieving a stable anti-parallel microtubule organization essential for faithful chromosome segregation. We propose that PRC1 forms a link between stabilization of CLASP1 association with central spindle microtubules and anti-parallel microtubule elongation.
    Journal of Biological Chemistry 07/2009; 284(34):23059-71. · 4.65 Impact Factor
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    ABSTRACT: Cardiotoxin III (CTX III), a 60-amino acid basic polypeptide isolated from Naja venom, showed potential therapeutic activity toward cancer cells. Here we report that CTX III inhibited proliferation of human leukemia K562 cells by G2/M phase arresting and apoptosis which was associated with the activation of caspase-8 and cytochrome c release as well as the p38 and c-Jun N-terminal protein kinase (JNK) phosphorylation signaling pathway. We further demonstrated that daily administration of CTX III for 2 d to chicken chorioallantoic membrane (CAM) bearing tumours derived from the CAM at E10 administration of K562 cells resulted in inhibition of the tumours in vivo. Importantly, this in vivo inhibition was also associated with caspase-8 activation and cytochrome c release. Our results suggest that CTX III-induced apoptosis is mediated via the p38 and JNK pathway as well as the caspase-8-dependent Bid-Bax pathway in human K562 cells.
    Biological & Pharmaceutical Bulletin 05/2009; 32(4):583-8. · 1.85 Impact Factor
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    ABSTRACT: HRG-beta1 stimulation of breast cancer cell line SKBR3 resulted in not only increased cell migration and invasion, upregulation of some mesenchymal markers, and downregulation of epithelial marker, but also upregulation of transcription factor Snail and its nuclear translocation. Similar results were acquired for cells transfected with Snail cDNA. Furthermore, downregulation of Snail by siRNA attenuated HRG-beta1 induced EMT-like phenotype. Inhibition of Akt kinase activation by a PI3K inhibitor LY294002, or exogenous expression of a kinase-dead mutant of Akt abrogated the increase of Snail expression induced by HRG-beta1. Conversely, expression of a constitutively active Akt resulted in increase of Snail expression. These results indicated that Snail upregulation by HRG-beta1 is mediated via the PI3K/Akt signaling pathway and that Snail plays a key role in HRG-beta1 induced breast cancer cell metastasis through induction of EMT.
    Cancer letters 04/2009; 280(1):50-60. · 5.02 Impact Factor
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    ABSTRACT: We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2008; 24(11):1918-23.
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    ABSTRACT: Cytotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, have potential therapeutic activity in tumor therapy. However, the therapeutic effect in solid tumor treatment with CTX III are still largely unknown. In the present study, we investigated whether CTX III affects cell growth and cell cycle progression of hepatocellular carcinoma cell (HepG2). We found that the proliferation of HepG2 cell was inhibited by CTX III, to some extent, in a time- and dose-dependent manner (IC50 2.58microg/ml at 24h). Flow cytometric analysis and annexin V labeling also demonstrated that CTX III increased the percentage of apoptotic cells being associated with cell cycle arrest at S-phase. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot revealed that cyclin D1, cyclin A and cyclin E, which involved in cell apopotosis and cell cycle progression, were down regulated both at transcription and translation levels. CTX III-induced caspase-8, -9 and caspase-3 activation, generation of truncated Bid, releasing of cytochrome c and the change of Bcl-2/Bax ratio on protein and mRNA levels. These findings demonstrated that cyclin D1, cyclin B and cyclin A down-regulation, change of Bcl-2/Bax ratio and caspase-8 and -9 activation contribute to CTX III-induced HepG2 cell apoptosis.
    Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 12/2008; 61(4):307-15. · 1.43 Impact Factor
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    ABSTRACT: Anti-ErbB2 antibodies targeting distinct epitopes can have different biological functions on cancer cells. A21 prepared by surface epitope masking (SEM) method is a tumor-inhibitory anti-ErbB2 monoclonal antibody. Previously we engineered a single chain chimeric antibody chA21 with potential for therapy of ErbB2-overexpressing tumors. Here, we mapped the A21 epitope on ErbB2 extracellular domain (ECD) by screening a combinatorial phage display peptide library, serial subdomain deletion, and mutagenesis scanning. X-ray crystal structure of the A21 scFv fragment at 2.1 A resolution was also determined. A molecular model of Ag-Ab complex was then constructed based on the crystal structures of the A21 scFv and ErbB2 ECD. Some of biological functions of the A21 mAb and its derivative antibodies including their tumor cell growth inhibition and effects on the expression, internalization, and phosphorylation of ErbB2 receptor were also investigated. The results showed that A21 recognized a conformational epitope comprising a large region mostly from ErbB2 extracellular subdomain I with several surface-exposed residues important for the binding affinity. These data provide unique functional properties of A21 that are quite different from two broadly used anti-ErbB2 mAbs, Herceptin and 2C4. It suggested that the A21 epitope may be another valuable target for designing new anti-ErbB2 therapeutics.
    Proteins Structure Function and Bioinformatics 03/2008; 70(3):938-49. · 3.34 Impact Factor
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    ABSTRACT: To prepare and identify the antibodies against snake venom C-type lectin like protein Agkisacutacin. A BALB/c mouse was immunized with Agkisacutacin and the cells were fused by standard hybridoma technique to prepare mAbs. The stability of the hybridomas secreting mAbs was detected by indirect ELISA. The nuclear type of the hybridomas was analyzed by fluorescent staining and the specificity of mAbs was detected by Western blot. Three cell strains of hybridomas that could steadily secrete anti-Agkisacutacin mAb were obtained. The successful preparation of anti-Agkisacutacin mAbs provides an important tool for studying the in vivo metabolism of new anti-thrombus drugs by detecting Agkisacutacin and investigating the mechanism of anti-thrombus of the drugs.
    Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 12/2007; 23(11):1031-3.
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    ABSTRACT: To explore the possibility of RNA interference (RNAi)-based gene therapy against HER2-overexpressing tumors using adenovirus-mediated vector. A plasmid named pHER2-GFP containing HER2 and green fluorescent protein (GFP) fusion was constructed and cotransfected into CHO-K1 cells respectively with nine small interference RNA (siRNA)-expressing plasmids targeting different regions of HER2. The siRNA-expressing plasmids with best interference effect were screened out and then used to identify the gene silence effect in HER2-overexpressing SKBR3 breast cancer cells. Subsequently, the siRNA-expressing cassettes were subcloned into adenoviral vectors. Downregulation of HER2 by adenovirus-mediated RNAi and its effect on SKBR3 cell proliferation were identified again. Two siRNA-expressing plasmids with best interference effect were screened out and HER2 was also efficiently downregulated in SKBR3 cells infected with the adenovirus containing these siRNA-expressing cassettes. Downregulation of HER2 resulted in the increase of cells in G1 phase and the induction of apoptosis. Furthermore, infection of adenovirus inhibited SKBR3 cell growth, which was confirmed by MTT and cell long-term proliferation assays. The adenovirus-mediated RNAi could downregulate the HER2 expression efficiently and exert an inhibitory effect on growth of HER2-overexpressing breast cancer cell.
    Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 09/2007; 23(8):691-5.