Co-reporter:Yaxin Zhang;Ke Zheng;Zhuo Chen;Jincan Chen
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 11) pp:4691-4700
Publication Date(Web):01 March 2017
DOI:10.1007/s00253-017-8133-8
Photodynamic antimicrobial chemotherapy (PACT) uses non-traditional mechanisms (free radicals) and is a highly advocated method with promise of inactivating drug-resistance bacteria for local infections. However, there is no related drug used in clinical practice yet. Therefore, new photosensitizers for PACT are under active development. Here, we report the synthesis of a series of photosensitizers with variable positive charges (ZnPc(TAP)4n+, n = 0, 4, 8, 12) and their inactivation against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The binding kinetics of ZnPc(TAP)4n+ to bacteria were measured by flow cytometer. Reactive oxygen species (ROS) generation mechanism of the photosensitizers was studied. The toxicity of these compounds to human blood cells was also evaluated. These compounds showed negligible toxicity against human erythocytes but potent bactericidal effects. The compound with 8 positive charges, ZnPc(TAP)48+, turned out to have the strongest antibacterial effect among this series of compounds, giving IC50 value of 59 nM at a light dosage of 5 J/cm2 toward E. coli. For a multi-resistant E. coli strain, ZnPc(TAP)48+ decreased the bacteria load by 1000-fold at a concentration of 1 μM. Interestingly, ZnPc(TAP)412+, instead of ZnPc(TAP)48+, exhibited the highest amount of binding to bacteria. Flow cytometry studies showed that all PSs have fast binding onto bacteria, reaching saturated binding within 5 min. Mechanistically, ZnPc(TAP)412+ generated ROS primarily via Type I mechanism, while ZnPc(TAP)44+ or ZnPc(TAP)48+ created ROS by both type I and type II mechanisms. ZnPc(TAP)4n+ are highly potent, rapid-acting and non-toxic photosensitizers capable of inactivating bacteria.
Co-reporter:Xiaolei Zhou, Ke Zheng, Rui Li, Zhuo Chen, Cai Yuan, Ping Hu, Jincan Chen, Jinping Xue, Mingdong Huang
Acta Biomaterialia 2015 Volume 23() pp:116-126
Publication Date(Web):1 September 2015
DOI:10.1016/j.actbio.2015.05.017
Abstract
Photodynamic therapy (PDT) has been used as an effective therapeutical modality for tumors. In PDT, a photosensitizer was used to capture the light of specific wavelength, leading to the generation of reactive oxygen species and cytotoxicity surrounding the photosensitizer. Modifications of photosensitizers to enhance tumor specificity are common approaches to increase the efficacy and reduce the side effects of PDT. Previously, we developed a human serum albumin (HSA)-based drug carrier fused with the human amino-terminal fragment (hATF), which binds to a tumor surface marker (urokinase receptor, uPAR). However, hATF-HSA binds to murine uPAR much weaker (79-fold) than to human uPAR, and is not optimal for applications on murine tumor models. In this study, we developed a murine version of the drug carrier (mATF-HSA). A photosensitizer (mono-substituted β-carboxy phthalocyanine zinc, CPZ) was loaded into this carrier, giving a rather stable macromolecule (mATF-HSA:CPZ) that was shown to bind to murine uPAR in vitro. In addition, we evaluated both the photodynamic therapy efficacy and tumor retention capability of the macromolecule (at a dose of 0.05 mg CPZ/kg mouse body weight) on murine hepatoma-22 (H22) tumor bearing mouse model. mATF-HSA:CPZ showed more accumulation in tumors compared to its human counterpart (hATF-HSA:CPZ) measured by quantitative fluorescence molecular tomography (FMT). Besides, mATF-HSA:CPZ exhibited a higher tumor killing efficacy than hATF-HSA:CPZ. Together, the macromolecule mATF-HSA is a promising tumor-specific drug carrier on murine tumor models and is an useful tool to study tumor biology on murine tumor models.
Co-reporter:Cai Yuan;Joy He Huang;Min Liu
Acta Crystallographica Section F 2015 Volume 71( Issue 11) pp:1442-1447
Publication Date(Web):
DOI:10.1107/S2053230X15018944
Urokinase plasminogen activator receptor-associated protein (uPARAP) is an endocytic receptor that internalizes collagen for lysosomal degradation and plays an important role in matrix remodelling. Previous recombinant protein production of uPARAP in Pichia pastoris generated protein with highly heterogeneous glycans that was prone to proteolytic degradation, resulting in highly twinned crystals. In this study, the uPARAP ligand-binding region was expressed in stably transfected Drosophila S2 insect cells. The recombinant protein was homogeneous after purification by metal-affinity and anion-exchange chromatography. Crystals were obtained at two different pH values (5.3 and 7.4) and diffracted to 2.44 and 3.13 Å resolution, respectively. A model of the ligand-binding region of uPARAP was obtained by molecular replacement combined with autobuilding. As the first multidomain crystal structure of the mannose receptor family, structural characterization of the uPARAP ligand-binding region will provide insight into the pH-induced conformational rearrangements of the mannose receptor family.
Co-reporter:Zhuo Chen, Peng Xu, Jincan Chen, Hongwei Chen, Ping Hu, Xueyuan Chen, Lin Lin, Yunmei Huang, Ke Zheng, Shanyong Zhou, Rui Li, Song Chen, Jianyong Liu, Jinping Xue, Mingdong Huang
Acta Biomaterialia 2014 Volume 10(Issue 10) pp:4257-4268
Publication Date(Web):October 2014
DOI:10.1016/j.actbio.2014.06.026
Abstract
Photodynamic therapy (PDT) has attracted much interest for the treatment of cancer due to the increased incidence of multidrug resistance and systemic toxicity in conventional chemotherapy. Phthalocyanine (Pc) is one of main classes of photosensitizers for PDT and possesses optimal photophysical and photochemical properties. A higher specificity can ideally be achieved when Pcs are targeted towards tumor-specific receptors, which may also facilitate specific drug delivery. Herein, we develop a simple and unique strategy to prepare a hydrophilic tumor-targeting photosensitizer ATF-ZnPc by covalently coupling zinc phthalocyanine (ZnPc) to the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA), a fragment responsible for uPA receptor (uPAR, a biomarker overexpressed in cancer cells), through the carboxyl groups of ATF. We demonstrate the high efficacy of this tumor-targeting PDT agent for the inhibition of tumor growth both in vitro and in vivo. Our in vivo optical imaging results using H22 tumor-bearing mice show clearly the selective accumulation of ATF-ZnPc in tumor region, thereby revealing the great potential of ATF-ZnPc for clinical applications such as cancer detection and guidance of tumor resection in addition to photodynamic treatment.
Co-reporter:Fanglei Chen, Guiping Zhang, Zebin Hong, Zhonghui Lin, Min Lei, Mingdong Huang, Lihong Hu
Bioorganic & Medicinal Chemistry Letters 2014 Volume 24(Issue 10) pp:2379-2382
Publication Date(Web):15 May 2014
DOI:10.1016/j.bmcl.2014.03.045
The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of 4.94 μM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC50 value of 0.18 μM.
Co-reporter:Zhuo Chen, Shanyong Zhou, Jincan Chen, Linsen Li, Ping Hu, Song Chen, Mingdong Huang
Journal of Luminescence 2014 152() pp: 103-107
Publication Date(Web):
DOI:10.1016/j.jlumin.2013.10.067
Co-reporter:Shanyong Zhou;Dr. Wei Zheng;Dr. Zhuo Chen;Dr. Datao Tu;Dr. Yongsheng Liu;En Ma;Renfu Li;Dr. Haomiao Zhu; Mingdong Huang; Xueyuan Chen
Angewandte Chemie International Edition 2014 Volume 53( Issue 46) pp:12498-12502
Publication Date(Web):
DOI:10.1002/anie.201405937
Abstract
Conventional dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) using molecular probes suffer from a low labeling ratio of lanthanide ions (Ln3+) per biomolecule. Herein, we develop a unique bioassay based on the dissolution-enhanced luminescence of inorganic lanthanide nanoparticles (NPs). As a result of the highly concentrated Ln3+ ions in a single Ln3+ NP, an extremely high Ln3+ labeling ratio can be achieved, which amplifies significantly the luminescence signal and thus improves the detection sensitivity compared to DELFIA. Utilizing sub-10 nm NaEuF4 NPs as dissolution-enhanced luminescent nanoprobes, we demonstrate the successful in vitro detection of carcinoembryonic antigen (CEA, an important tumor marker) in human serum samples with a record-low detection limit of 0.1 pg mL−1 (0.5 fM). This value is an improvement of approximately 3 orders of magnitude relative to that of DELFIA. The dissolution-enhanced luminescent bioassay shows great promise in versatile bioapplications, such as ultrasensitive and multiplexed in vitro detection of disease markers in clinical diagnosis.
Co-reporter:Shanyong Zhou;Dr. Wei Zheng;Dr. Zhuo Chen;Dr. Datao Tu;Dr. Yongsheng Liu;En Ma;Renfu Li;Dr. Haomiao Zhu; Mingdong Huang; Xueyuan Chen
Angewandte Chemie 2014 Volume 126( Issue 46) pp:12706-12710
Publication Date(Web):
DOI:10.1002/ange.201405937
Abstract
Conventional dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) using molecular probes suffer from a low labeling ratio of lanthanide ions (Ln3+) per biomolecule. Herein, we develop a unique bioassay based on the dissolution-enhanced luminescence of inorganic lanthanide nanoparticles (NPs). As a result of the highly concentrated Ln3+ ions in a single Ln3+ NP, an extremely high Ln3+ labeling ratio can be achieved, which amplifies significantly the luminescence signal and thus improves the detection sensitivity compared to DELFIA. Utilizing sub-10 nm NaEuF4 NPs as dissolution-enhanced luminescent nanoprobes, we demonstrate the successful in vitro detection of carcinoembryonic antigen (CEA, an important tumor marker) in human serum samples with a record-low detection limit of 0.1 pg mL−1 (0.5 fM). This value is an improvement of approximately 3 orders of magnitude relative to that of DELFIA. The dissolution-enhanced luminescent bioassay shows great promise in versatile bioapplications, such as ultrasensitive and multiplexed in vitro detection of disease markers in clinical diagnosis.
Co-reporter:Zhonghui Lin, Jan K. Jensen, Zebin Hong, Xiaoli Shi, Lihong Hu, Peter A. Andreasen, Mingdong Huang
Chemistry & Biology 2013 Volume 20(Issue 2) pp:253-261
Publication Date(Web):21 February 2013
DOI:10.1016/j.chembiol.2013.01.002
Plasminogen activator inhibitor-1 (PAI-1), a serpin, is the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and thus also an inhibitor of fibrinolysis and tissue remodeling. It is a potential therapeutic target in many pathological conditions, including thrombosis and cancer. Several types of PAI-1 antagonist have been developed, but the structural basis for their action has remained largely unknown. Here we report X-ray crystal structure analysis of PAI-1 in complex with a small-molecule antagonist, embelin. We propose a mechanism for embelin-induced rapid conversion of PAI-1 into a substrate for its target proteases and the subsequent slow conversion of PAI-1 into an irreversibly inactivated form. Our work provides structural clues to an understanding of PAI-1 inactivation by small-molecule antagonists and an important step toward the design of drugs targeting PAI-1.Highlights► Identification of a small molecular PAI-1 antagonist, embelin ► Crystal structure of PAI-1 in complex with embelin ► Structural insight into the mechanism of PAI-1 inactivation by embelin
Co-reporter:Linsen Li, Zhipu Luo, Zhuo Chen, Jincan Chen, Shanyong Zhou, Peng Xu, Ping Hu, Jundong Wang, Naisheng Chen, Jinling Huang, and Mingdong Huang
Bioconjugate Chemistry 2012 Volume 23(Issue 11) pp:2168
Publication Date(Web):October 12, 2012
DOI:10.1021/bc3002997
Zinc phthalocyanine (ZnPc) is a promising photosensitizer for photodynamic therapy, but faces some challenges: ZnPc is insoluble in water and thus requires either special formulation of ZnPc by, e.g., liposome or Cremophor EL, or chemical modification of Pc ring to enhance its bioavailability and photodynamic efficacy. Here, we conjugated monosubstituted ZnPc-COOH with a series of oligolysine moieties with different numbers of lysine residues (ZnPc-(Lys)n (n = 1, 3, 5, 7, 9) to improve the water solubility of the ZnPc conjugates. We measured the photosensitizing efficacies and the cellular uptakes of this series of conjugates on a normal and a cancerous cell line. In addition, we developed a sensitive in situ method to distinguish the difference in photodynamic efficacy among conjugates. Our results showed that ZnPc-(Lys)7 has the highest photodynamic efficacy compared to the other conjugates investigated.
Co-reporter:Zhipu Luo, Xiaoli Shi, Qin Hu, Bin Zhao, and Mingdong Huang
Chemical Research in Toxicology 2012 Volume 25(Issue 5) pp:990
Publication Date(Web):April 6, 2012
DOI:10.1021/tx300112p
Perfluorooctane sulfonate (PFOS) is a man-made fluorosurfactant and globally persistent organic pollutant. PFOS is mainly distributed in blood with a long half-life for elimination. PFOS was found mainly bound to human serum albumin (HSA) in plasma, the most abundant protein in human blood plasma, which transports a variety of endogenous and exogenous ligands. However, the structural basis of such binding remains unclear. Here, we report the crystal structure of the HSA–PFOS complex and show that PFOS binds to HSA at a molar ratio of 2:1. In addition, PFOS binding renders the HSA structure more compact. Our results provide a structural mechanism to understand the retention of surfactants in human serum.
Co-reporter:Zhuo Chen;Shanyong Zhou;Jincan Chen;Yicai Deng Dr.;Zhipu Luo;Hongwei Chen;MichaelR. Hamblin
ChemMedChem 2010 Volume 5( Issue 6) pp:890-898
Publication Date(Web):
DOI:10.1002/cmdc.201000042
Abstract
Unsymmetrical phthalocyanine derivatives have been widely studied as photosensitizers for photodynamic therapy (PDT), targeting various tumor types. However, the preparation of unsymmetrical phthalocyanines is always a challenge due to the presence of many possible structural isomers. Herein we report a new unsymmetrical zinc phthalocyanine, pentalysine β-carbonylphthalocyanine zinc (ZnPc-(Lys)5), that was prepared in large quantity and high purity. This is a water-soluble cationic photosensitizer and maintains a high quantum yield of singlet oxygen generation similar to that of unsubstituted zinc phthalocyanine (ZnPc). Compared with anionic ZnPc counterparts, ZnPc-(Lys)5 shows a higher level cellular uptake and 20-fold higher phototoxicity toward tumor cells. Pharmacokinetics and PDT studies of ZnPc-(Lys)5 in S180 tumor-bearing mice showed a high ratio of tumor versus skin retention and significant tumor inhibition. This new molecular framework will allow synthetic diversity in the number of lysine residues incorporated and will facilitate future QSAR studies.
Co-reporter:C. Yuan;M. Huang
Cellular and Molecular Life Sciences 2007 Volume 64( Issue 9) pp:
Publication Date(Web):2007 May
DOI:10.1007/s00018-007-6498-z
Multiple cellular functions of urokinase and its receptor are associated with the receptor’s capability to interact with a number of ligands at the molecular level. The presence of urokinase is generally needed for the urokinase receptor to acquire this capability. Recent X-ray studies of the structure of the urokinase receptor in complex with either its ligand or peptide inhibitors demonstrate the flexibility of the domain organization of the receptor, suggesting that unliganded urokinase receptor may exist in a latent form that has a conformation different from its ligand-binding form.
Co-reporter:Qing Huai;Andrew P. Mazar;Alice Kuo;Graham C. Parry;David E. Shaw;Jennifer Callahan;Yongdong Li;Cai Yuan;Chuanbing Bian;Liqing Chen;Bruce Furie;Barbara C. Furie;Douglas B. Cines
Science 2006 Vol 311(5761) pp:656-659
Publication Date(Web):03 Feb 2006
DOI:10.1126/science.1121143
Abstract
The urokinase plasminogen activator binds to its cellular receptor with high affinity and initiates signaling cascades that are implicated in pathological processes including tumor growth, metastasis, and inflammation. We report the crystal structure at 1.9 angstroms of the urokinase receptor complexed with the urokinase amino-terminal fragment and an antibody against the receptor. The three domains of urokinase receptor form a concave shape with a central cone-shaped cavity where the urokinase fragment inserts. The structure provides insight into the flexibility of the urokinase receptor that enables its interaction with a wide variety of ligands and a basis for the design of urokinase-urokinase receptor antagonists.
Co-reporter:ChuanBing Bian, Cai Yuan, Lin Lin, JunHan Lin, XiaoLi Shi, XiaoMing Ye, ZiXiang Huang, MingDong Huang
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2005 Volume 1752(Issue 1) pp:99-102
Publication Date(Web):31 August 2005
DOI:10.1016/j.bbapap.2005.07.014
PF898 is a strain of Penicillium expansum optimized for the high level production of Penicillium expansum lipase (PEL). This PEL is unique compared with other lipases in several aspects, For example, the PEL shows low sequence identities (<30%) to all other known lipases, and high percentage of hydrophobic residues in the N-terminal region. The PEL was purified to homogeneity and shown to be 28 kDa by SDS-PAGE. Crystals suitable for X-ray diffraction analysis were obtained by the sitting-drop method of vapor diffusion with ammonia sulfate as the precipitating agent at 298 K. The crystals have tetragonal lattice and unit-cell parameters of a = b = 88.09 Å, c = 126.54 Å. Diffraction data were collected to a resolution of 2.08 Å on an in-house rotating-anode generator.
Co-reporter:Gengxiang Zhao, Cai Yuan, Troels Wind, Zixiang Huang, Peter A. Andreasen, Mingdong Huang
Journal of Structural Biology (October 2007) Volume 160(Issue 1) pp:1-10
Publication Date(Web):1 October 2007
DOI:10.1016/j.jsb.2007.06.003
Urokinase-type plasminogen activator (uPA) plays a crucial role in the regulation of plasminogen activation, tumor cell adhesion and migration. The inhibition of uPA activity is a promising mechanism for anti-cancer therapy. A cyclic peptidyl inhibitor, upain-1, CSWRGLENHRMC, was identified recently as a competitive and highly specific uPA inhibitor. We determined the crystal structure of uPA in complex with upain-1 at 2.15 Å. The structure reveals that the cyclic peptide adopts a rigid conformation stabilized by a disulfide bond (residues 1–12) and three tight beta turns (residues 3–6, 6–9, 9–12). The Glu7 residue of upain-1 forms hydrogen bonds with the main chain nitrogen atoms of residues 4, 5, and 6 of upain-1, and is also critical for maintaining the active conformation of upain-1. The Arg4 of upain-1 is inserted into the uPA’s specific S1 pocket. The Ser2 residue of upain-1 locates close to the S1β pocket of uPA. The Gly5 and Glu7 residues of upain-1 occupy the S2 pocket and the oxyanion hole of uPA, respectively. Furthermore, the Asn8 residue of upain-1 binds to the 37- and 60-loops of uPA and renders the specificity of upain-1 for uPA. Based on this structure, a new pharmacophore for the design of highly specific uPA inhibitors was proposed.
Co-reporter:Longguang Jiang, Lisbeth Moreau Andersen, Peter A. Andreasen, Liqing Chen, Mingdong Huang
Biochimica et Biophysica Acta (BBA) - General Subjects (March 2016) Volume 1860(Issue 3) pp:599-606
Publication Date(Web):March 2016
DOI:10.1016/j.bbagen.2015.12.009
Co-reporter:Lihu Gong, Min Liu, Tu Zeng, Xiaoli Shi, Cai Yuan, Peter A. Andreasen, Mingdong Huang
Data in Brief (March 2016) Volume 6() pp:550-555
Publication Date(Web):1 March 2016
DOI:10.1016/j.dib.2015.12.050
Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7–18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19,20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].
Co-reporter:Lili Zhu, Cai Yuan, Zhuo Chen, Wanyu Wang, Mingdong Huang
Toxicon (February–March 2010) Volume 55(Issues 2–3) pp:375-380
Publication Date(Web):1 February 2010
DOI:10.1016/j.toxicon.2009.08.016
Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin.
Co-reporter:Feng Yang, Chuanbing Bian, Lili Zhu, Gengxiang Zhao, Zixiang Huang, Mingdong Huang
Journal of Structural Biology (February 2007) Volume 157(Issue 2) pp:348-355
Publication Date(Web):1 February 2007
DOI:10.1016/j.jsb.2006.08.015
Human serum albumin (HSA) is the most abundant plasma protein in the human body with a plasma concentration of 0.6 mM. HSA plays an important role in drug transport and metabolism. Enzymatic activity of HSA on different substrates or drugs has been studied and documented. The structural mechanism of this activity, however, is unknown. In this study, we have determined the crystal structures of HSA–myristate in a complex of aspirin and of salicylic acid, respectively. The crystal structure of HSA–myristate–aspirin illustrates that aspirin transfers acetyl group to Lys199 and is hydrolyzed into salicylic acid by HSA. The hydrolysis product, salicylic acid, remains bound to HSA at a similar location, but it shows a very different orientation when compared with the salicylic acid in the HSA–myristate–salicylic acid ternary complex. These results not only provide the structural evidence of esterase activity of HSA, and demonstrate the conformational plasticity of HSA on drug binding, but also may provide structural information for the modulation of HSA–drug interaction by computational approach based on HSA–drug structure.
Co-reporter:Baoyu Zhao, Sonu Gandhi, Cai Yuan, Zhipu Luo, Rui Li, Henrik Gårdsvoll, Valentina de Lorenzi, Nicolai Sidenius, Mingdong Huang, Michael Ploug
Data in Brief (December 2015) Volume 5() pp:107-113
Publication Date(Web):1 December 2015
DOI:10.1016/j.dib.2015.08.027
The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2–4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].
Co-reporter:Lili Zhu, Feng Yang, Liqing Chen, Edward J. Meehan, Mingdong Huang
Journal of Structural Biology (April 2008) Volume 162(Issue 1) pp:40-49
Publication Date(Web):1 April 2008
DOI:10.1016/j.jsb.2007.12.004
3′-Azido-3′-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug–drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA–Myr–AZT complex and a quaternary HSA–Myr–AZT–SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA–Myr–AZT–SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA–drug interaction and drug–drug interaction on HSA.
Co-reporter:Baoyu Zhao, Sonu Gandhi, Cai Yuan, Zhipu Luo, ... Michael Ploug
Journal of Molecular Biology (27 March 2015) Volume 427(Issue 6) pp:1389-1403
Publication Date(Web):27 March 2015
DOI:10.1016/j.jmb.2015.01.022
•The urokinase receptor (uPAR) is a flexible modular receptor with dual roles in cell adhesion and pericellular proteolysis.•Functional and structural epitopes for new anti-uPAR mAbs are defined by surface plasmon resonance and X-ray crystallography.•These mAbs trap uPAR in its globally closed conformation by stabilizing the vitronectin binding interface.•Bidirectional conformational cooperativity exists between uPA and vitronectin binding sites in uPAR.The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0 Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments.Download high-res image (234KB)Download full-size image
Co-reporter:Yongdong Li, Graham Parry, Liqing Chen, Jennifer A. Callahan, ... Mingdong Huang
Journal of Molecular Biology (26 January 2007) Volume 365(Issue 4) pp:1117-1129
Publication Date(Web):26 January 2007
DOI:10.1016/j.jmb.2006.10.059
Human urokinase-type plasminogen activator receptor (uPAR/CD87) is expressed at the invasive interface of the tumor-stromal microenvironment in many human cancers and interacts with a wide array of extracellular molecules. An anti-uPAR antibody (ATN615) was prepared using hybridoma technology. This antibody binds to uPAR in vitro with high affinity (Kd ∼ 1 nM) and does not interfere with uPA binding to uPAR. Here we report the crystal structure of the Fab fragment of ATN615 at 1.77 Å and the analysis of ATN615-suPAR-ATF structure that was previously determined, emphasizing the ATN615–suPAR interaction. The complementarity determining regions (CDRs) of ATN615 consist of a high percentage of aromatic residues, and form a relatively flat and undulating surface. The ATN615 Fab fragment recognizes domain 3 of suPAR. The antibody–antigen recognition involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are the key residues for the antibody recognition, while Pro189 and Arg192 render specificity of ATN615 for human uPAR. Interestingly, this antibody–antigen interface has a small contact area, mainly polar interaction with little hydrophobic character, yet has high binding strength. Furthermore, several solvent molecules (assigned as polyethylene glycols) were clearly visible in the binding interface between antibody and antigen, suggesting that solvent molecules may be important for the maximal binding between suPAR and ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR region upon antigen binding.
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