Shannon Wing-Ngor Au

Publications of Shannon Wing-Ngor Au

• Structural basis for RNA-binding and homo-oligomer formation by influenza B virus nucleoprotein.

  Authors: Andy Ka-Leung Ng, Mandy Ka-Han Lam, Hongmin Zhang, Jinhuan Liu, Shannon Wing-Ngor Au, Paul Kay-Sheung Chan, Jiahuai Wang, Pang-Chui Shaw

  Journal of virology. 04/2012;

  Influenza virus nucleoprotein (NP) is the major component of the viral ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have determined theInfluenza virus nucleoprotein (NP) is the major component of the viral ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have determined the crystal structure of influenza B virus NP to a resolution of 3.2 Å. Influenza B NP contains a head, a body domain and a tail loop. The electropositive groove between the head and body domains of influenza B NP is crucial for RNA binding. This groove also contains an extended flexible charged loop (aa. 125-149) and two lysine clusters at the first half of this loop were shown to be crucial for binding RNA. Influenza B NP forms a crystallographic homo-tetramer by inserting the tail loop into the body domain of the neighboring NP molecule. A deeply buried salt bridge R472-E395 and a hydrophobic cluster at F468 are the major driving forces for the insertion. The analysis of the influenza B virus NP structure and function and comparisons with influenza A virus NP provide insights into the mechanisms of action and underpin efforts to design inhibitors for this class of proteins.

• Multiple conformations of the FliG C-terminal domain provide insight into flagellar motor switching.

  Authors: Kwok-Ho Lam, Wing-Sang Ip, Yun-Wah Lam, Sun-On Chan, Thomas Kin-Wah Ling, Shannon Wing-Ngor Au

  Structure (London, England : 1993). 02/2012; 20(2):315-25.

  Bacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliGBacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliG are closely associated with this switching mechanism. We present two crystal structures of FliG(MC) from Helicobacter pylori, each showing distinct domain orientations from previously solved structures. A 180° rotation of the charged ridge-containing C-terminal subdomain FliG(Cα1-6) that is prompted by the rotational freedom of Met245 psi and Phe246 phi at the MFXF motif was revealed. Studies on the swarming and swimming behavior of Escherichia coli mutants further identified the importance of the ₂₄₅MFXF₂₄₈ motif and a highly conserved residue, Asn216, in motor switching. Additionally, multiple conformations of FliG(Cα1-6) were demonstrated by intramolecular cysteine crosslinking. The conformational flexibility of FliGc leads us to propose a model that accounts for the symmetrical torque generation process and for the dynamics of the motor.

• Influenza Polymerase Activity Correlates with the Strength of Interaction between Nucleoprotein and PB2 through the Host-Specific Residue K/E627.

  Authors: Andy Ka-Leung Ng, Wai-Hon Chan, Sze-Ting Choi, Mandy Ka-Han Lam, Kwok-Fai Lau, Paul Kay-Sheung Chan, Shannon Wing-Ngor Au, Ervin Fodor, Pang-Chui Shaw

  PloS one. 01/2012; 7(5):e36415.

  The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) andThe ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal '627' domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2.

• Measuring rapid kinetics by a potentiometric method in droplet-based microfluidic devices.

  Authors: Zuoyan Han, Yuen Yan Chang, Shannon Wing Ngor Au, Bo Zheng

  Chemical communications (Cambridge, England). 07/2011; 48(10):1601-3.

  Droplets containing RNA and Mg(2+) were generated in microfluidic channels. By integrating a group of pneumatic valves and phase separation channels in the microfluidic system, the rapid RNA-Mg(2+)Droplets containing RNA and Mg(2+) were generated in microfluidic channels. By integrating a group of pneumatic valves and phase separation channels in the microfluidic system, the rapid RNA-Mg(2+) binding kinetics was studied by measuring the Mg(2+) ion concentration using an ion-selective electrode.

• Effects of cathelicidin and its fragments on three key enzymes of HIV-1.

  Authors: Jack Ho Wong, Anna Legowska, Krzysztof Rolka, Tzi Bun Ng, Mamie Hui, Chi Hin Cho, Wendy Wai Ling Lam, Shannon Wing Ngor Au, Oscar Wangang Gu, David Chi Cheong Wan

  Peptides. 06/2011; 32(6):1117-22.

  Cathelicidins exhibit anti-HIV activity but it is not known if they reduce the activity of enzymes crucial to the life cycle of the retrovirus. It is shown in this investigation that humanCathelicidins exhibit anti-HIV activity but it is not known if they reduce the activity of enzymes crucial to the life cycle of the retrovirus. It is shown in this investigation that human cathelicidin LL37 and its fragments LL13-37 and LL17-32 inhibited HIV-1 reverse transcriptase dose-dependently with an IC50 value of 15μM, 7μM, and 70μM, respectively. The three peptides inhibited HIV-1 protease with a weak potency, achieving 20-30% inhibition at 100μM. The mechanism of inhibition was protein-protein interaction as revealed by surface plasmon resonance. The peptides were devoid of the ability to inhibit translocation of HIV-1 integrase, which has been labeled with green fluorescent protein, into the nucleus. The peptides did not exert toxicity on human peripheral blood mononuclear cells.

• The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response.

  Authors: Kin-Hang Kok, Pak-Yin Lui, Ming-Him James Ng, Kam-Leung Siu, Shannon Wing Ngor Au, Dong-Yan Jin

  Cell host & microbe. 04/2011; 9(4):299-309.

  RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. DicerRIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response.

• Molecular interaction of flagellar export chaperone FliS and cochaperone HP1076 in Helicobacter pylori.

  Authors: Wendy Wai Ling Lam, Eui Jeon Woo, Masayo Kotaka, Wai Kwan Tam, Yun Chung Leung, Thomas Kin Wah Ling, Shannon Wing Ngor Au

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 10/2010; 24(10):4020-32.

  Flagellar export chaperone FliS prevents premature polymerization of flagellins and is critical for flagellar assembly and bacterial colonization. Previously, a yeast 2-hybrid study identifiedFlagellar export chaperone FliS prevents premature polymerization of flagellins and is critical for flagellar assembly and bacterial colonization. Previously, a yeast 2-hybrid study identified various FliS-associated proteins in Helicobacter pylori, but the implications of these interactions are not known. Here we demonstrate the biophysical interaction of FliS (HP0753) and the uncharacterized protein HP1076 from H. pylori. HP1076 possesses a cochaperone activity that promotes the folding and chaperone activity of FliS. We further determined the crystal structures of FliS, HP1076, and the binary complex at 2.7, 1.8, and 2.7 Å resolution, respectively. HP1076 adopts a helix-rich bundle structure and interestingly shares a similar fold with a flagellin homologue, hook-associated protein, and FliS. The FliS-HP1076 complex revealed an extensive electrostatic and hydrophobic binding interface, which is distinct from the flagellin binding pocket in FliS. The helical stacking interaction between HP1076 and FliS suggests that HP1076 stabilizes 2 α helices of FliS and therefore the overall structure of the bundle. Our findings provide new insights into flagellar export chaperones and may have implications for other secretion chaperones in the type III secretion system.

• Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities.

  Authors: Wai-Hon Chan, Andy Ka-Leung Ng, Nicole C Robb, Mandy Ka-Han Lam, Paul Kay-Sheung Chan, Shannon Wing-Ngor Au, Jia-Huai Wang, Ervin Fodor, Pang-Chui Shaw

  Journal of virology. 07/2010; 84(14):7337-45.

  Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. TheHomo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

• Crystal structure of activated CheY1 from Helicobacter pylori.

  Authors: Kwok Ho Lam, Thomas Kin Wah Ling, Shannon Wing Ngor Au

  Journal of bacteriology. 03/2010; 192(9):2324-34.

  Chemotaxis is an important virulence factor for Helicobacter pylori colonization and infection. The chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1,Chemotaxis is an important virulence factor for Helicobacter pylori colonization and infection. The chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1, one CheY-like-containing CheA protein (CheAY2), and three CheV proteins. Recent studies have demonstrated that these molecules play unique roles in the chemotactic signal transduction mechanisms of H. pylori. Here we report the crystal structures of BeF(3(-)-activated CheY1 from H. pylori resolved to 2.4 A. Structural comparison of CheY1 with active-site residues of BeF3(-)-bound CheY from Escherichia coli and fluorescence quenching experiments revealed the importance of Thr84 in the phosphotransfer reaction. Complementation assays using various nonchemotactic E. coli mutants and pull-down experiments demonstrated that CheY1 displays differential association with the flagellar motor in E. coli. The structural rearrangement of helix 5 and the C-terminal loop in CheY1 provide a different interaction surface for FliM. On the other hand, interaction of the CheA-P2 domain with CheY1, but not with CheY2/CheV proteins, underlines the preferential recognition of CheY1 by CheA in the phosphotransfer reaction. Our results provide the first structural insight into the features of the H. pylori chemotactic system as a model for Epsilonproteobacteria.

• SUMO proteases: Redox regulation and biological consequences.

  Authors: Shannon Wing-Ngor Au, Kwok Ho Lam, Wai Ling Lam, Levina Suk Mi Lam, Ho Yin Chan, Zheng Xu, Tzi Bun Ng

  Antioxidants & redox signaling. 02/2009;

  Small-ubiquitin modifier (SUMO) has emerged as a novel modification system that governs the activities of a wide spectrum of protein substrates. SUMO specific proteases (SENP) are of particularSmall-ubiquitin modifier (SUMO) has emerged as a novel modification system that governs the activities of a wide spectrum of protein substrates. SUMO specific proteases (SENP) are of particular interest, as they are responsible for both the maturation of SUMO precursors and for their deconjugation. The interruption of SENPs has been implicated in embryonic defects and carcinoma cells, indicating that a proper balance of SUMO conjugation and deconjugation is crucial. Recent advances in molecular and cellular biology have highlighted the distinct subcellular localization, endopeptidase and isopeptidase activities of SENPs, suggesting that they are non-redundant. A better understanding of the molecular basis of SUMO recognition and hydrolytic cleavage has been obtained from the crystal structures of SENP-substrate complexes. While a number of proteomic studies have shown an up-regulation of sumoylation, attention is now increasingly being directed towards the regulatory mechanism of sumoylation, in particular the oxidative effect. Findings on the oxidation-induced intermolecular disulfide of E1-E2 ligases and SENP1/2 have improved our understanding of the mechanism by which modification is switched up or down. More intriguingly, a growing body of evidence suggests that sumoylation cross-talks with other modifications, and that the upstream and downstream signaling pathway is co-regulated by more than one modifier.

• The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome.

  Authors: Priscilla Hiu-Mei Too, Meiji Kit-Wan Ma, Amanda Nga-Sze Mak, Yuen-Ting Wong, Christine Kit-Ching Tung, Guang Zhu, Shannon Wing-Ngor Au, Kam-Bo Wong, Pang-Chui Shaw

  Nucleic acids research. 01/2009;

  Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the bindingRibosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 A crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs.

• Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design.

  Authors: Andy Ka-Leung Ng, Hongmin Zhang, Kemin Tan, Zongli Li, Jin-huan Liu, Paul Kay-Sheung Chan, Sui-Mui Li, Wood-Yee Chan, Shannon Wing-Ngor Au, Andrzej Joachimiak, Thomas Walz, Jia-huai Wang, Pang-Chui Shaw

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 08/2008;

  The threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virusThe threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virus and the host cell. It, therefore, plays an important structural and functional role and represents an attractive drug target. Here, we report the 3.3-A crystal structure of H5N1 NP, which is composed of a head domain, a body domain, and a tail loop. Our structure resolves the important linker segments (residues 397-401, 429-437) that connect the tail loop with the remainder of the molecule and a flexible, basic loop (residues 73-91) located in an arginine-rich groove surrounding Arg150. Using surface plasmon resonance, we found the basic loop and arginine-rich groove, but mostly a protruding element containing Arg174 and Arg175, to be important in RNA binding by NP. We also used our crystal structure to build a ring-shaped assembly of nine NP subunits to model the miniribonucleoprotein particle previously visualized by electron microscopy. Our study of H5N1 NP provides insight into the oligomerization interface and the RNA-binding groove, which are attractive drug targets, and it identifies the epitopes that might be used for universal vaccine development.-Ng, A. K.-L., Zhang, H., Tan, K., Li, Z., Liu, J.-h., Chan, P. K.-S., Li, S.-M., Chan, W.-Y., Au, S. W.-N., Joachimiak, A., Walz, T., Wang, J.-H., Shaw, P.-C. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design.

• Immunomodulatory and anti-SARS activities of Houttuynia cordata.

  Authors: Kit-Man Lau, Kin-Ming Lee, Chi Man Koon, Crystal Sao-Fong Cheung, Ching-Po Lau, Hei-Ming Ho, Mavis Yuk Ha Lee, Shannon Wing-Ngor Au, Christopher Hon-Ki Cheng, Clara Bik-San Lau, Stephen Kwok-Wing Tsui, David Chi-Cheong Wan, Mary Miu-Yee Waye, Kam-Bo Wong, Chun Kwok Wong, Christopher Wai-Kei Lam, Ping Chung Leung, Kwok-Pui Fung

  Journal of ethnopharmacology. 07/2008; 118(1):79-85.

  BACKGROUND: Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by SARS coronavirus (SARS-CoV). From late 2002 to mid 2003, it infected more than 8000 peopleBACKGROUND: Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by SARS coronavirus (SARS-CoV). From late 2002 to mid 2003, it infected more than 8000 people worldwide, of which a majority of cases were found in China. Owing to the absence of definitive therapeutic Western medicines, Houttuynia cordata Thunb. (Saururaceae) (HC) was shortlisted by Chinese scientists to tackle SARS problem as it is conventionally used to treat pneumonia. AIM OF THE STUDY: The present study aimed to explore the SARS-preventing mechanisms of HC in the immunological and anti-viral aspects. RESULTS: Results showed that HC water extract could stimulate the proliferation of mouse splenic lymphocytes significantly and dose-dependently. By flow cytometry, it was revealed that HC increased the proportion of CD4(+) and CD8(+) T cells. Moreover, it caused a significant increase in the secretion of IL-2 and IL-10 by mouse splenic lymphocytes. In the anti-viral aspect, HC exhibited significant inhibitory effects on SARS-CoV 3C-like protease (3CL(pro)) and RNA-dependent RNA polymerase (RdRp). On the other hand, oral acute toxicity test demonstrated that HC was non-toxic to laboratory animals following oral administration at 16g/kg. CONCLUSION: The results of this study provided scientific data to support the efficient and safe use of HC to combat SARS.

• Purification and detailed study of two clinically different human glucose 6-phosphate dehydrogenase variants, G6PD(Plymouth) and G6PD(Mahidol): Evidence for defective protein folding as the basis of disease.

  Authors: Yuxiang Huang, Mei Yee Choi, Shannon Wing Ngor Au, Deborah Man Yee Au, Veronica Min Sien Lam, Paul C Engel

  Molecular genetics and metabolism. 02/2008; 93(1):44-53.

  In an attempt to investigate the molecular mechanism underlying human glucose-6-phosphate dehydrogenase (G6PD) deficiency caused by two mutations, G6PD(Plymouth) (G163D) and G6PD(Mahidol) (G163S),In an attempt to investigate the molecular mechanism underlying human glucose-6-phosphate dehydrogenase (G6PD) deficiency caused by two mutations, G6PD(Plymouth) (G163D) and G6PD(Mahidol) (G163S), the two variants were constructed by site-directed mutagenesis and expressed in G6PD-deficient E. coli DF 213 cells. A first indication of impaired folding came from problems in expressing these clinical mutants, which were only overcome by lowering the growth temperature or co-expressing with molecular chaperones (GroEL and GroES). Both strategies significantly increased soluble expression of recombinant G6PD(Plymouth) and G6PD(Mahidol), judged by both G6PD activity in extracts and the amount of immunoreactive protein. Using a modified 3-step protocol, the two mutant enzymes were successfully purified for the first time. Steady-state kinetic parameters (K(m) for NADP(+), K(m) for G6P and k(cat)) of the two mutants are very similar to the wild-type values, indicating that the catalytic efficiency of the two mutants remains unchanged. The two mutants are, however, markedly less stable than wild-type G6PD in both thermostability and urea-induced inactivation tests. In a typical experiment at 37 degrees C and pH 7.2 after 24h G6PD WT, G6PD(Mahidol) and G6PD(Plymouth) retained 58.3%, 27.0% and 3.9%, respectively, of their corresponding initial activity. The stability of all three enzymes is enhanced by addition of NADP(+). According to unfolding and refolding experiments, the two mutants are impaired in their folding properties. Thus structural instability appears to be the molecular basis of the clinical phenotype in G6PD(Plymouth) and G6PD(Mahidol) and in particular of the differing clinical severity of the two mutations. The 3-D structure solved for G6PD(Canton) allows an interpretation of these effects in terms of steric hindrance.

• Molecular basis of the redox regulation of SUMO proteases: a protective mechanism of intermolecular disulfide linkage against irreversible sulfhydryl oxidation.

  Authors: Zheng Xu, Levina Suk Mi Lam, Lok Hei Lam, So Fun Chau, Tzi Bun Ng, Shannon Wing-Ngor Au

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 02/2008; 22(1):127-37.

  Sumoylation has emerged as an indispensable post-translational modification that modulates the functions of a broad spectrum of proteins. Recent studies have demonstrated that reactive oxygen speciesSumoylation has emerged as an indispensable post-translational modification that modulates the functions of a broad spectrum of proteins. Recent studies have demonstrated that reactive oxygen species influence the equilibrium of sumoylation-desumoylation. We show herein that H2O2 induces formation of an intermolecular disulfide linkage of human SUMO protease SENP1 via the active-site Cys 603 and a unique residue Cys 613. Such reversible modification confers a higher recovery of enzyme activity, which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. In vivo formation of a disulfide-linked dimer of SENP1 is also detected in cultured cells in response to oxidative stress. The modifications are further elucidated by the crystal structures of Ulp1 with the catalytic cysteine oxidized to sulfenic, sulfinic, and sulfonic acids. Our findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors modulating the desumoylation pathway and specific cellular responses to oxidative stress.

• Proteomic characterization of Sophoraflavone J-induced apoptosis in HepG2 cells.

  Authors: Crystal Sao-Fong Cheung, Po-Ming Hon, Kwok-Pui Fung, Shannon Wing-Ngor Au

  Proteomics. Clinical applications. 12/2007; 1(12):1532-44.

  Chinese herb Radix sophorae is widely applied as an anticarcinogenic/antimetastatic agent against liver cancers. In the current study, Sophoraflavone J, a flavonoid constituent enriched in the rootChinese herb Radix sophorae is widely applied as an anticarcinogenic/antimetastatic agent against liver cancers. In the current study, Sophoraflavone J, a flavonoid constituent enriched in the root of Radix sophorae, induced apoptosis in human hepatoma HepG2 cells via the intrinsic mitochondrial death pathway. The molecular mechanism of the cytotoxic effect was further investigated by a comparative proteomic approach. Differentially expressed proteins identified included membrane proteins/antigens, structural proteins, transcriptional factors, glycolytic enzymes, heat-shock chaperon proteins, ROS-related proteins and proteosomes, etc. These findings were further validated by Western blot analysis and real-time PCR. Preliminary experiments to characterize the roles of these proteins were conducted. Our data suggested that Sophoraflavone J treatment triggered nutrient depletion and generation of ROS in cells, which subsequently led to mitochondrial dysfunction and apoptosis.

• Leachianone A as a potential anti-cancer drug by induction of apoptosis in human hepatoma HepG2 cells.

  Authors: Crystal Sao-Fong Cheung, Karen Ka-Wing Chung, Julian Chun-Kin Lui, Ching-Po Lau, Po-Ming Hon, Judy Yuet-Wa Chan, Kwok-Pui Fung, Shannon Wing-Ngor Au

  Cancer letters. 09/2007; 253(2):224-35.

  The Chinese herbal medicine Radix Sophorae is widely applied as an anti-carcinogenic/ anti-metastatic agent against liver cancer. In this study, Leachianone A, isolated from Radix Sophorae, possessedThe Chinese herbal medicine Radix Sophorae is widely applied as an anti-carcinogenic/ anti-metastatic agent against liver cancer. In this study, Leachianone A, isolated from Radix Sophorae, possessed a profound cytotoxic activity against human hepatoma cell line HepG2 in vitro, with an IC(50) value of 3.4microg/ml post-48-h treatment. Its action mechanism via induction of apoptosis involved both extrinsic and intrinsic pathways. Its anti-tumor effect was further demonstrated in vivo by 17-54% reduction of tumor size in HepG2-bearing nude mice, in which no toxicity to the heart and liver tissues was observed. In conclusion, this is the first report describing the isolation of Leachianone A from Radix Sophorae and the molecular mechanism of its anti-proliferative effect on HepG2 cells.

• Nanoliter dispensing method by degassed poly(dimethylsiloxane) microchannels and its application in protein crystallization.

  Authors: Xuechang Zhou, Lana Lau, Wendy Wai Ling Lam, Shannon Wing-Ngor Au, Bo Zheng

  Analytical chemistry. 08/2007; 79(13):4924-30.

  This paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannelsThis paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannels were reversibly bound to arrays of microwells. The PDMS elastomer was predegassed and served as an internal vacuum pumping source. Various aqueous solutions were infused into arrays of microwells through the reversibly sealed PDMS microchannels. Microwells fabricated in PDMS, poly(methyl methacrylate) (PMMA), and glass were all compatible with this dispensing method. By removing the PDMS microchannels, arrays of droplets confined in the microwells were obtained. Multiplex reaction and screening at the nanoliter scale were carried out by binding two such arrays of microwells to form microchambers. We applied this method to screening the crystallization conditions of four known proteins. Long-term incubation of over 2 months was achieved by employing glass microwells. An unknown protein was then crystallized using the screening method in microwells. The crystals with sufficient size were harvested from the reversibly bound microwells. X-ray diffraction with a resolution of 3.1 Angstrom was obtained.

• Pheophorbide a, an active component in Scutellaria barbata, reverses P-glycoprotein-mediated multidrug resistance on a human hepatoma cell line R-HepG2.

  Authors: Patrick Ming-Kuen Tang, Judy Yuet-Wa Chan, Dong-Mei Zhang, Shannon Wing-Ngor Au, Wing-Ping Fong, Siu-Kai Kong, Stephen Kwok-Wing Tsui, Mary Mui-Yee Waye, Thomas Chung-Wai Mak, Kwok-Pui Fung

  Cancer biology & therapy. 05/2007; 6(4):504-9.

  Scutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), anScutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), an active component from S. barbata, was examined on a multi-drug resistant (MDR) human hepatoma cell line R-HepG2. Our study showed that Pa could significantly inhibit the growth of R-HepG2 cells with an IC50 value at 25.0 microM after 48 hours treatment. When compared with the parental HepG2 cells, Pa showed weak resistance to R-HepG2. Efflux of Pa out of R-HepG2 cells was not observed as its cellular uptake level showed no significant difference comparing with HepG2 cells. Interestingly, significant reduction of P-glycoprotein expression on Pa-treated R-HepG2 cells was found at both transcriptional and translational levels, leading to reduction of P-glycoprotein activity. In addition, mechanistic study elucidated that Pa induced cell cycle arrest at G2/M phase and inhibited the expressions of G2/M phase cell cycle regulatory proteins, cyclin-A1 and cdc2 in a dose-dependent manner.

• Structure-function study of maize ribosome-inactivating protein: implications for the internal inactivation region and the sole glutamate in the active site.

  Authors: Amanda Nga-Sze Mak, Yuen-Ting Wong, Young Jun An, Sun-Shin Cha, Kong-Hung Sze, Shannon Wing-Ngor Au, Kam-Bo Wong, Pang-Chui Shaw

  Nucleic acids research. 02/2007; 35(18):6259-67.

  Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, whichMaize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 A, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long alpha-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins.