Der-Lii M. Tzou

Vaccinia Virus 4c (A26L) Protein on Intracellular Mature Virus Binds to the Extracellular Cellular Matrix Laminin

ABSTRACT Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG− sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.

Poxvirus Host Range Protein CP77 Contains an F-Box-Like Domain That Is Necessary To Suppress NF-κB Activation by Tumor Necrosis Factor Alpha but Is Independent of Its Host Range Function

ABSTRACT Tumor necrosis factor alpha (TNF-α) activates the nuclear factor κB (NF-κB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-κB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-κB activation by TNF-α. Immunofluorescence analyses revealed that nuclear translocation of NF-κB subunit p65 protein in TNF-α-treated HeLa cells was blocked by CP77. CP77 did so without blocking IκBα phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-κB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-κB activation. We thus propose a model in which poxvirus CP77 suppresses NF-κB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-κB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-κB modulation and binding to SCF complex, is not required for CP77s host range function. Consistently, knocking down the protein level of NF-κB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

Poxviral host range protein CP77 contains a F-box-like domain that is necessary to suppress NF-κB activation by TNF-α but is independent of its host range function

ABSTRACT Tumor necrosis factor alpha (TNF-α) activates the nuclear factor κB (NF-κB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-κB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-κB activation by TNF-α. Immunofluorescence analyses revealed that nuclear translocation of NF-κB subunit p65 protein in TNF-α-treated HeLa cells was blocked by CP77. CP77 did so without blocking IκBα phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-κB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-κB activation. We thus propose a model in which poxvirus CP77 suppresses NF-κB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-κB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-κB modulation and binding to SCF complex, is not required for CP77s host range function. Consistently, knocking down the protein level of NF-κB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

Stepwise Orthogonal Click Chemistry toward Fabrication of Paclitaxel/Galactose Functionalized Fluorescent Nanoparticles for HepG2 Cell Targeting and Delivery

In this report, we used stepwise orthogonal click chemistry (SOCC) involving strain-promoted azide-alkyne cycloaddition (SPAAC) and microwave-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to assemble an anticancer drug (paclitaxel, PTX) and a targeting ligand (trivalent galactoside, TGal) on a fluorescent silicon oxide nanoparticle (NP) by using dialkyne linker 8 as a bridge. The fluorescent NH2@Cy3SiO2NP was fabricated using a competition method to incorporate Cy3 without loss of the original surface amine density on the NPs. The concept of SOCC was first investigated in a solution-phase model study that showed quantitative reaction yield. In the fabrication of TGal-PTX@Cy3SiO2NP, the expensive compound azido-functionalized PTX 12 used in SPAAC can be easily recovered due to the absence of other reagents in the reaction mixture. High loading of the sugar ligand on the NP surface serves a targeting function and also overcomes the low water solubility of PTX. Confocal fluorescence microscopy and cytotoxicity assay showed that TGal-PTX@Cy3SiO2NP was taken up by HepG2 cells and was affected by the microtubule skeleton in these cells and inhibited the proliferation of these cells in a dose-dependent manner. The presence of a fluorescent probe, a targeting ligand, and an anticancer drug on the multifunctional TGal-PTX@Cy3SiO2NP allows for real-time imaging, specific cancer-cell targeting, and the cell-killing effect which is better than free PTX.

A turn-like structure "KKPE" segment mediates the specific binding of viral protein A27 to heparin and heparan sulfate on cell surfaces.

Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21–84 of A27) with Cys71 and Cys72 mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21–32; 21STKAAKKPEAKR32) and a rigid coiled-coil domain (residues 43–84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (KA = 1.25 × 108 m−1) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (KA = 1.65 × 105 m−1). Using site-directed mutagenesis of HBS and solution NMR, we identified a “KKPE” segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the KKPE segment remained intact showed an extremely high affinity for HP (KA = 1.9 × 1011 m−1). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the KKPE segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces.

Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy

This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid α-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid α-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual α-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu41, Leu45, and Leu48) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.

A 13C solid-state NMR analysis of vitamin D compounds

(13)C cross-polarization/magic-angle spinning (CP/MAS) solid-state NMR spectroscopy has been employed to analyze four vitamin D compounds, namely vitamin D3 (D3), vitamin D2 (D2), and the precursors ergosterol (Erg) and 7-dehydrocholesterol (7DHC). The (13)C NMR spectrum of D3 displays a doublet pattern for each of the carbon atoms, while that of Erg contains both singlet and doublet patterns. In the cases of 7DHC and D2, the (13)C spectra display various multiplet patterns, viz. singlets, doublets, triplets, and quartets. To overcome the signal overlap between the (13)C resonances of protonated and unprotonated carbons, we have subjected these vitamin D compounds to 1D (1)H-filtered (13)C CP/MAS and {(1)H}/(13)C heteronuclear correlation (Hetcor) NMR experiments. As a result, assisted by solution NMR data, all of the (13)C resonances have been successfully assigned to the respective carbon atoms of these vitamin D compounds. The (13)C multiplets are interpreted due to the presence of s-cis and s-trans configurations in the alpha- and beta-molecular conformers, consistent with computer molecular modeling determined by molecular dynamics and energy minimization calculations. To further characterize the ring conformations in D3, we have successfully extracted chemical shift tensor elements for the (13)C doublets. It is demonstrated that (13)C solid-state NMR spectroscopy provides a robust and high sensitive means of characterizing molecular conformations in vitamin D compounds.

Multiple conformations of the metal-bound pyoverdine PvdI, a siderophore of Pseudomonas aeruginosa: a nuclear magnetic resonance study.

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdine PvdI, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, d-Ser- l-Arg- d-Ser- l-FoOHOrn-( l-Lys- l-FoOHOrn- l-Thr- l-Thr), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the l-Lys side chain. In aqueous solution at room temperature, the (1)H NMR spectrum of pyoverdine PvdI-Ga(III) showed clear evidence of exchange broadening. At 253 K, two distinct conformations were observed and the measurement of structural constraints was possible. The three-dimensional structures of the two PvdI-Ga(III) conformers were determined, and analysis of the structures indicates that the observed conformational exchange involves a stereoisomerization of the metal binding coordination accompanied by a change in the global shape of the siderophore. This conformational transition was further characterized by heteronuclear relaxation experiments. The possible implications of this dynamic behavior for siderophore recognition by the receptor FpvAI are discussed.

A 13C solid‐state NMR analysis of steroid compounds

13C CP/MAS solid‐state NMR spectroscopy has been utilized to analyze six steroid compounds, namely testosterone (Tes), hydrocortisone (Cor), trans‐dehydroandrosterone (Adr), prednisolone (Prd), prednisone (Pre) and estradiol (Est). Among them, Tes displays a doublet pattern for all residues, whereas Prd, Pre and Est, exhibit exclusively singlets. For Cor and Adr, the 13C spectra contain both doublet and singlet patterns. The 13C doublet signal, with splittings of 0.2–1.5 ppm, are ascribed to local differences in the ring conformations associated with polymorphism. We have assigned all of the 13C resonances to the different residues in these steroid compounds on the basis of solution NMR data. The C‐7, C‐8, C‐10, C‐15 and C‐16 residues of Tes, Cor and Adr consistently give rise to singlets or doublets with splittings of less than 0.5 ppm, indicating similar local conformations. Accompanying hydration and dehydration processes, a reversible phase transformation between δ‐ and α‐crystal forms has been observed in Tes, corresponding to singlet and doublet 13C patterns, respectively. To further characterize the ring conformations in the α‐form, we have successfully extracted chemical shift tensor elements for the 13C doublets. It is demonstrated that 13C solid‐state NMR spectroscopy provides a reliable and sensitive means of characterizing polymorphism in steroids. Copyright

Immobilization of Rh(PPh3)3Cl on phosphinated MCM-41 for catalytic hydrogenation of olefins

The complex Rh(PPh3)3Cl immobilized on MCM-41 modified with (OEt)3Si(CH2)3PPh2 results in a stable hydrogenation catalyst with turnover frequency (TOF) three times higher than that of Rh(PPh3)3Cl in the hydrogenation of cyclohexene.