Wuyuan Lu

Publications (84)434.28 Total impact

• Article: Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: Role of the conserved salt-bridge.

  Changyou Zhan, Le Zhao, Xishan Chen, Wei-Yue Lu, Wuyuan Lu
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  ABSTRACT: The dengue capsid protein C is a highly basic alpha-helical protein of ∼100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55'. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C ((WT)DEN2C) and its salt bridge-void analog (E87A)DEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.
  Bioorganic & medicinal chemistry 04/2013; · 2.82 Impact Factor
• Article: Total Chemical Synthesis of Dengue 2 Virus Capsid Protein via Native Chemical Ligation: Role of the Conserved Salt-bridge

  Changyou Zhan, Le Zhao, Xishan Chen, Wei-Yue Lu, Wuyuan Lu
  Bioorganic & Medicinal Chemistry 04/2013; · 2.92 Impact Factor
• Article: Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53-MDM2 interaction

  Chong Li, Changyou Zhan, Le Zhao, Xishan Chen, Wei-Yue Lu, Wuyuan Lu
  Bioorganic & Medicinal Chemistry 04/2013; · 2.92 Impact Factor
• Article: Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53-MDM2 interaction.

  Chong Li, Changyou Zhan, Le Zhao, Xishan Chen, Wei-Yue Lu, Wuyuan Lu
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  ABSTRACT: Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0-3.4kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX-the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.(1.)
  Bioorganic & medicinal chemistry 04/2013; · 2.82 Impact Factor
• Article: Structural and Functional Analysis of the Pro-Domain of Human Cathelicidin, LL-37.

  Marzena Pazgier, Bryan Ericksen, Minhua Ling, Eric Toth, Jishu Shi, Xiangdong Li, Amy Galliher-Beckley, Liqiong Lan, Guozhang Zou, Changyou Zhan, Weirong Yuan, Edwin Pozharski, Wuyuan Lu
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  ABSTRACT: Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.
  Biochemistry 03/2013; 52(9):1547-58. · 3.42 Impact Factor
• Article: An ultrahigh affinity d-peptide antagonist Of MDM2.

  Changyou Zhan, Le Zhao, Xiaoli Wei, Xueji Wu, Xishan Chen, Weirong Yuan, Wei-Yue Lu, Marzena Pazgier, Wuyuan Lu
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  ABSTRACT: The oncoprotein MDM2 negatively regulates the activity and stability of the p53 tumor suppressor and is an important molecular target for anticancer therapy. Aided by mirror image phage display and native chemical ligation, we have previously discovered several proteolysis-resistant duodecimal d-peptide antagonists of MDM2, termed (D)PMI-α, β, γ. The prototypic d-peptide inhibitor (D)PMI-α binds ((25-109))MDM2 at an affinity of 220 nM and kills tumor cells in vitro and inhibits tumor growth in vivo by reactivating the p53 pathway. Herein, we report the design of a superactive d-peptide antagonist of MDM2, termed (D)PMI-δ, of which the binding affinity for ((25-109))MDM2 has been improved over (D)PMI-α by 3 orders of magnitude (K(d) = 220 pM). X-ray crystallographic studies validate (D)PMI-δ as an exceedingly potent inhibitor of the p53-MDM2 interaction, promising to be a highly attractive lead drug candidate for anticancer therapeutic development.
  Journal of Medicinal Chemistry 06/2012; 55(13):6237-41. · 4.80 Impact Factor
• Article: Functional determinants of human enteric α-defensin HD5: crucial role for hydrophobicity at dimer interface.

  Mohsen Rajabi, Bryan Ericksen, Xueji Wu, Erik de Leeuw, Le Zhao, Marzena Pazgier, Wuyuan Lu
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  ABSTRACT: Human α-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins, such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human α-defensin family: four human neutrophil peptides, including HNP1, and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However, the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine (Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel β strands. The effect of mutating Leu(29) is similar to that of a C-terminal hydrophobic residue of HNP1, Trp(26). In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by N-methylation of the Glu(21) residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the α-defensins, revealing a crucial role of hydrophobicity-mediated dimerization.
  Journal of Biological Chemistry 05/2012; 287(26):21615-27. · 4.77 Impact Factor
• Article: Invariant gly residue is important for α-defensin folding, dimerization, and function: a case study of the human neutrophil α-defensin HNP1.

  Le Zhao, Bryan Ericksen, Xueji Wu, Changyou Zhan, Weirong Yuan, Xu Li, Marzena Pazgier, Wuyuan Lu
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  ABSTRACT: The human α-defensins (HNP) are synthesized in vivo as inactive prodefensins, and contain a conserved glycine, Gly(17), which is part of a β-bulge structure. It had previously been shown that the glycine main chain torsion angles are in a D-configuration, and that d-amino acids but not L-alanine could be substituted at that position to yield correctly folded peptides without the help of a prodomain. In this study, the glycine to L-alanine mutant defensin was synthesized in the form of a prodefensin using native chemical ligation. The ligation product folded correctly and yielded an active peptide upon CNBr cleavage. The L-Ala(17)-HNP1 crystal structure depicted a β-bulge identical to wild-type HNP1. However, dimerization was perturbed, causing one monomer to tilt with respect to the other in a dimerization model. Inhibitory activity against the anthrax lethal factor showed a 2-fold reduction relative to wild-type HNP1 as measured by the inhibitory concentration IC(50). Self-association was slightly reduced, as detected by surface plasmon resonance measurements. According to the results of the virtual colony count assay, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus exhibited a less than 2-fold reduction in virtual lethal dose values. Prodefensins with two other L-amino acid substitutions, Arg and Phe, at the same position did not fold, indicating that only small side chains are tolerable. These results further elucidate the factors governing the region of the β-bulge structure that includes Gly(17), illuminating why glycine is conserved in all mammalian α-defensins.
  Journal of Biological Chemistry 04/2012; 287(23):18900-12. · 4.77 Impact Factor
• Article: Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined.

  Changyou Zhan, Kristen Varney, Weirong Yuan, Le Zhao, Wuyuan Lu
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  ABSTRACT: The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.
  Journal of the American Chemical Society 03/2012; 134(15):6855-64. · 9.91 Impact Factor
• Article: Soluble factors from T cells inhibiting X4 strains of HIV are a mixture of β chemokines and RNases.

  Fiorenza Cocchi, Anthony L DeVico, Wuyuan Lu, Mikulas Popovic, Olga Latinovic, Mohammad M Sajadi, Robert R Redfield, Mark K Lafferty, Massimo Galli, Alfredo Garzino-Demo, Robert C Gallo
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  ABSTRACT: T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three β chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three β chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4(+) and CD8(+) T cells (chemokines, angiogenin) or only by CD8(+) T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive β chemokines.
  Proceedings of the National Academy of Sciences 03/2012; 109(14):5411-6. · 9.68 Impact Factor
• Article: Sometimes it takes two to tango: contributions of dimerization to functions of human α-defensin HNP1 peptide.

  Marzena Pazgier, Gang Wei, Bryan Ericksen, Grace Jung, Zhibin Wu, Erik de Leeuw, Weirong Yuan, Henryk Szmacinski, Wei-Yue Lu, Jacek Lubkowski, Robert I Lehrer, Wuyuan Lu
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  ABSTRACT: Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.
  Journal of Biological Chemistry 01/2012; 287(12):8944-53. · 4.77 Impact Factor
• Article: α-Defensins in human innate immunity.

  Robert I Lehrer, Wuyuan Lu
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  ABSTRACT: Defensins are small, multifunctional cationic peptides. They typically contain six conserved cysteines whose three intramolecular disulfides stabilize a largely β-sheet structure. This review of human α-defensins begins by describing their evolution, including their likely relationship to the Big Defensins of invertebrates, and their kinship to the β-defensin peptides of many if not all vertebrates, and the θ-defensins found in certain non-human primates. We provide a short history of the search for leukocyte-derived microbicidal molecules, emphasizing the roles played by luck (good), preconceived notions (mostly bad), and proper timing (essential). The antimicrobial, antiviral, antitoxic, and binding properties of human α-defensins are summarized. The structural features of α-defensins are described extensively and their functional contributions are assessed. The properties of HD6, an enigmatic Paneth cell α-defensin, are contrasted with those of the four myeloid α-defensins (HNP1-4) and of HD5, the other α-defensin of human Paneth cells. The review ends with a decalogue that may assist researchers or students interested in α-defensins and related aspects of neutrophil function.
  Immunological Reviews 01/2012; 245(1):84-112. · 11.15 Impact Factor
• Source

  Available from: PubMed Central

  Article: Human defensins 5 and 6 enhance HIV-1 infectivity through promoting HIV attachment.

  Aprille Rapista, Jian Ding, Bernadette Benito, Yung-Tai Lo, Matthew B Neiditch, Wuyuan Lu, Theresa L Chang
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  ABSTRACT: Concurrent sexually transmitted infections (STIs) increase the likelihood of HIV transmission. The levels of defensins are frequently elevated in genital fluids from individuals with STIs. We have previously shown that human defensins 5 and 6 (HD5 and HD6) promote HIV entry and contribute to Neisseria gonorrhoeae-mediated enhancement of HIV infectivity in vitro. In this study, we dissect the molecular mechanism of the HIV enhancing effect of defensins. HD5 and HD6 primarily acted on the virion to promote HIV infection. Both HD5 and HD6 antagonized the anti-HIV activities of inhibitors of HIV entry (TAK 779) and fusion (T-20) when the inhibitors were present only during viral attachment; however, when these inhibitors were added back during viral infection they overrode the HIV enhancing effect of defensins. HD5 and HD6 enhanced HIV infectivity by promoting HIV attachment to target cells. Studies using fluorescent HIV containing Vpr-GFP indicated that these defensins enhanced HIV attachment by concentrating virus particles on the target cells. HD5 and HD6 blocked anti-HIV activities of soluble glycosaminoglycans including heparin, chondroitin sulfate, and dextran sulfate. However, heparin, at a high concentration, diminished the HIV enhancing effect of HD5, but not HD6. Additionally, the degree of the HIV enhancing effect of HD5, but not HD6, was increased in heparinase-treated cells. These results suggest that HD5 and haparin/heparan sulfate compete for binding to HIV. HD5 and HD6 increased HIV infectivity by concentrating virus on the target cells. These defensins may have a negative effect on the efficacy of microbicides, especially in the setting of STIs.
  Retrovirology 06/2011; 8:45. · 6.47 Impact Factor
• Article: Peptide activators of the p53 tumor suppressor.

  Changyou Zhan, Wuyuan Lu
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  ABSTRACT: The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the p53 tumor suppressor, directly contributing to the development and progression of many tumors harboring wild type p53. Antagonizing MDM2 and MDMX to activate the p53 pathway has thus become an attractive new strategy for anticancer drug design. Several different classes of MDM2 and MDMX antagonists have been reported, including low molecular weight compounds, small peptides, miniature proteins, and peptidomimetics. This review aims to summarize the latest progress in the design of peptide activators of the p53 tumor suppressor.
  Current pharmaceutical design 03/2011; 17(6):603-9. · 4.41 Impact Factor
• Article: Mucosal human defensins 5 and 6 antagonize the anti-HIV activity of candidate polyanion microbicides.

  Jian Ding, Aprille Rapista, Natalia Teleshova, Wuyuan Lu, Mary E Klotman, Theresa L Chang
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  ABSTRACT: Defensins are highly abundant antimicrobial peptides in the female genital mucosa. We have previously shown that human defensins 5 and 6 (HD5 and HD6), produced by cervicovaginal epithelial cells, significantly enhance HIV infectivity in vitro. Candidate polyanion microbicides, including PRO 2000, cellulose sulfate and carrageenan, failed to protect women against HIV infection in large-scale clinical trials, but the molecular basis of ineffectiveness was not clear. We hypothesized that mucosal host factors such as HD5 an HD6 may alter the activity of polyanion microbicides against HIV. Our results demonstrated that HD5 and HD6 but not their linear analogs antagonized the anti-HIV activity of PRO 2000, cellulose sulfate and carrageenan in vitro. Polyanion microbicides also reduced the HIV-enhancing effect of these defensins. We conclude that mucosal host factors could negatively impact the efficacy of topical microbicides against HIV, and their impact on the activity of candidate microbicides needs to be considered during the preclinical evaluation.
  Journal of Innate Immunity 01/2011; 3(2):208-12. · 4.21 Impact Factor
• Article: Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes.

  Eusondia Arnett, Robert I Lehrer, Pratikhya Pratikhya, Wuyuan Lu, Stephanie Seveau
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  ABSTRACT: Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.
  Cellular Microbiology 12/2010; 13(4):635-51. · 5.46 Impact Factor
• Article: Topology of the disulfide bonds in the antiviral lectin scytovirin.

  Tinoush Moulaei, Olga Stuchlik, Matthew Reed, Weirong Yuan, Jan Pohl, Wuyuan Lu, Lauren Haugh-Krumpe, Barry R O'Keefe, Alexander Wlodawer
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  ABSTRACT: The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the "cystine clusters" [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment.
  Protein Science 09/2010; 19(9):1649-61. · 2.80 Impact Factor
• Source

  Available from: Changyou Zhan

  Article: D-peptide inhibitors of the p53-MDM2 interaction for targeted molecular therapy of malignant neoplasms.

  Min Liu, Chong Li, Marzena Pazgier, Changqing Li, Yubin Mao, Yifan Lv, Bing Gu, Gang Wei, Weirong Yuan, Changyou Zhan, Wei-Yue Lu, Wuyuan Lu
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  ABSTRACT: The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed (D)PMI-alpha (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed (D)PMI-gamma (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both (D)PMI-alpha and (D)PMI-gamma failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, (D)PMI-alpha exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.
  Proceedings of the National Academy of Sciences 08/2010; 107(32):14321-6. · 9.68 Impact Factor
• Article: A left-handed solution to peptide inhibition of the p53-MDM2 interaction.

  Min Liu, Marzena Pazgier, Changqing Li, Weirong Yuan, Chong Li, Wuyuan Lu
  Angewandte Chemie International Edition 05/2010; 49(21):3649-52. · 13.45 Impact Factor
• Article: Limitations of peptide retro-inverso isomerization in molecular mimicry.

  Chong Li, Marzena Pazgier, Jing Li, Changqing Li, Min Liu, Guozhang Zou, Zhenyu Li, Jiandong Chen, Sergey G Tarasov, Wei-Yue Lu, Wuyuan Lu
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  ABSTRACT: A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an alpha-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288-16289) made a surprising finding that the retro-inverso isomer of p53(15-29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the D-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15-29) binding to MDM2 or MDMX by 3.2-3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that D-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level.
  Journal of Biological Chemistry 04/2010; 285(25):19572-81. · 4.77 Impact Factor