Van Boom Research Articles

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C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Conformational Flexibility in DNA Duplexes Containing Single G · G Mismatches

Purine-purine mismatches can base-pair in a variety of configurations depending on solution conditions. The G.G mismatch, which also occurs in the G-quartet structure, has been shown by both x-ray crystallography and NMR to adopt G(anti).G(syn) mispairs, with very different hydrogen bonding patterns [Skelly, J., Edwards, K., Jenkins, T. C. & Neidle, S. (1993) Proc. Natl Acad. Sci. USA 90, 804-808; Cognet, J. A. H., Gabarro-Arpa, J., Le Bret, M., van der Marel, G. A. van Boom, J. H. & Fazakerley, G. V. (1991) Nucleic Acids. Res. 19, 6771-6779] while we have recently suggested the presence of weakly hydrogen-bonded G(anti).G(anti) pairs in solution [Borden, K. L. B., Jenkins, T. C., Skelly, J. V., Brown, T. & Lane, A. N. (1992) Biochemistry 31, 5411-5422]. Spectral overlap and additional exchange processes have made detailed structural analysis difficult in these mismatched oligomers. We have used NMR to characterise the conformations of four duplexes containing single G.G mismatches, including a nonamer d(CATCGGATG), two undecamers d(GCATTGAATGC) and d(CATGTGACGTG) that can each form a self-complementary duplex with a single G.G mispair in the centre, and the non-self-complementary d(GTAACGACATG).d(CATGTGGTTAC). The three self-complementary duplexes have a single set of NMR resonances, and all four duplexes show evidence of conformational exchange at the mismatch site. The N1H resonances of the mismatched G residues each integrate to two protons, ruling out the enol tautomer. They resonate between 10.5-10.7 ppm, far upfield of the Watson-Crick hydrogen-bonded GN1H and exchange readily with water protons. Intraresidue GH8-H1' NOE intensities are two-threefold larger for the mismatched G residues than in G.C base pairs, indicating the presence of syn conformations. NOE time courses for the self-complementary duplexes were consistent with an equimolar mixture of G(syn).G(anti) and G (anti).G(syn) states. By symmetry, these states must be interconverting at a rate that is fast on the chemical shift timescale. In the non-self-complementary undecamer, the NOE data indicated that the distinguishable mismatched G residues also spend a significant, but different, fraction of the time in both the syn and anti conformations. The rate constant for the syn/anti transition in the non-self-complementary undecamer was determined as approximately 14,000 s-1 at 303 K from rotating frame T1 measurements, and the apparent frequency difference was > 250 Hz. Calculations based on NOEs and coupling constants showed that the duplexes are overall in the B form.(ABSTRACT TRUNCATED AT 250 WORDS)

Willem H. Van Boom and Marco Loos (eds), Collective Enforcement of Consumer Law: Securing Compliance in Europe through Private Group Action and Public Authority Intervention

Willem H. Van Boom and Marco Loos (eds), <i>Collective Enforcement of Consumer Law: Securing Compliance in Europe through Private Group Action and Public Authority Intervention</i>

An Unorthodox Bacteriophytochrome from Rhodobacter sphaeroides Involved in Turnover of the Second Messenger c-di-GMP

Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.

Inhibition of Tumor Endothelial ERK Activation, Angiogenesis, and Tumor Growth by Sorafenib (BAY43-9006)

Activation of the Raf-MEK-ERK signal transduction pathway in endothelial cells is required for angiogenesis. Raf is the kinase most efficiently inhibited by the multikinase inhibitor sorafenib, which has shown activity against certain human cancers in clinical trials. To understand the mechanisms underlying this activity, we studied how it controlled growth of K1735 murine melanomas. Therapy caused massive regional tumor cell death accompanied by severe tumor hypoxia, decreased microvessel density, increased percentage of pericyte-covered vessels, and increased caliber and decreased arborization of vessels. These signs of K1735 angiogenesis inhibition, along with its ability to inhibit Matrigel neovascularization, showed that sorafenib is an effective anti-angiogenic agent. Extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells, revealed by immunostaining for phospho-ERK and CD34, was inhibited, whereas AKT activation, revealed by phospho-AKT immunostaining, was not inhibited in K1735 and two other tumor types treated with sorafenib. Treatment decreased endothelial but not tumor cell proliferation and increased both endothelial cell and tumor cell apoptosis. These data indicate that sorafenib's anti-tumor efficacy may be primarily attributable to angiogenesis inhibition resulting from its inhibition of Raf-MEK-ERK signaling in endothelial cells. Assessing endothelial cell ERK activation in tumor bio-psies may provide mechanistic insights into and allow monitoring of sorafenib's activity in patients in clinical trials.

Interaction of Human Lactoferrin with Cell Adhesion Molecules through RGD Motif Elucidated by Lactoferrin-binding Epitopes

Lactoferrin (LF) is an iron-binding secretory protein, which is distributed in the secondary granules of polynuclear lymphocytes as well as in the milk produced by female mammals. Although it has multiple functions, for example antimicrobial, immunomodulatory, antiviral, and anti-tumor metastasis activities, the receptors responsible for these activities are not fully understood. In this study, the binding epitopes for human LF were first isolated from a hexameric random peptide library displayed on T7 phage. Interestingly, two of the four isolated peptides had a representative cell adhesion motif, Arg-Gly-Asp (RGD), implying that human LF interacts with proteins with the RGD motif. We found that human LF bound to the RGD-containing human extracellular matrix proteins, fibronectin and vitronectin. Furthermore, human LF inhibited cell adhesion to these matrix proteins in a concentration-dependent manner but not to the RGD-independent cell adhesion molecule like laminin or collagen. These results indicate that a function of human LF is to block the various interactions between the cell surface and adhesion molecules. This may explain the multifunctionality of LF.

Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

Some Aspects of Selectivity in the Reaction of Glycosyl Donors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

1,6‐C—H and 1,5‐O—Si Insertion Reactions of Alkylidenecarbene Derivatives of Monosaccharides.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of an α‐Gal epitope α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐β‐D‐Glcp NAc–lipid conjugate*

The synthesis of a neoglycoconjugate containing the Galili epitope trisaccharide connected to a spacer‐lipid entity is described. The α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐β‐D‐GlcpNAc trisaccharide, equipped with a 3‐aminopropyl spacer, is efficiently assembled from easily accessible building blocks in a one‐pot procedure. Global deprotection of the trisaccharide and ensuing introduction of a bis(palmitamido)‐ propanamido moiety afforded title compound 1 as depicted in Scheme 1. *Dedicated to the memory of Prof. Dr. Jacques H. van Boom, our teacher, colleague, and friend.

2‐[Dimethyl(2‐naphthylmethyl)silyl]ethoxy Carbonate (NSEC) as a New Mode of Hydroxyl Group Protection*

2‐[Dimethyl(2‐naphthylmethyl)silyl]ethoxy carbonate (NSEC) is a novel protecting group to mask hydroxyl groups. NSECCl is available in three steps from chlorodimethylvinylsilane and 2‐(bromomethyl)naphthalene. Introduction and removal of the NSEC group are performed easily and rapidly in the presence of most protecting groups currently used in carbohydrate chemistry. The removal of NSEC can be carried out under mild conditions in the presence of various ether and ester protecting groups. Additionally, the NSEC group is stable to glycosylation conditions using glycosyl phosphates. The synthesis of disaccharide 18 containing NSEC has been accomplished. * In memory of Professor Jacques H. van Boom

Preparation of S‐ and N‐Linked Glycosylated Amino Acid Building Blocks for Solid‐phase Glycopeptide Library Synthesis*

A general route for the preparation of 1,2‐trans‐linked S‐glycosylated amino acid building blocks by a Lewis‐acid‐promoted condensation of peracetylated glycosyl donors and N α‐Fmoc‐Cys‐OH, in good overall yield, is described. In addition, a short and time‐efficient route was applied for the synthesis of N‐glycosylated amino acid building blocks in good overall yields by coupling unprotected glycosylamines and N α‐Fmoc‐Asp(OH)‐O t Bu using TBTU activation. * In memory of Professor Jacques H. van Boom.

Block Synthesis of Streptococcus pneumoniae Type 14 Capsular Polysaccharide Structures*

Synthesis of a thioglycoside tetrasaccharide block, β‐D‐Galp‐(1→4)‐β‐DGlcp‐(1→6)‐[β‐D‐Galp‐(1→4)]‐β‐D‐GlcNPhthp‐(1→SEt, corresponding to the repeating unit of Streptococcus pneumoniae serotype 14 CPS is described. Coupling of this block with a spacer followed by removal of an isopropylidene acetal yielded an acceptor, which was elongated with the donor block to give a protected dimer of the repeating unit. Iteration of this methodology yielded the trimer. Deprotection then produced an octa and a dodecasaccharide derivative ready for conjugation to proteins to afford immunoactive glycoconjugates. * In memory of Professor Jacques H. van Boom

Inositolphosphoglycan Mediators: An Effective Synthesis of the Conserved Linear GPI Anchor Structure*

An effective new preparative synthesis of the conserved linear pseudopentasaccharide structure of the GPI anchors and of the full GPI structure has been carried out that has permitted obtaining both molecules in sufficient quantities as to perform further structural and biologic studies. The synthesis involves a 3+2 block synthesis strategy in which a conveniently protected Man α(1→4) GlcN3 α(1→6) myo‐Ins building block, previously used in the synthesis of inositolphosphoglycan (IPG) mediators, is glycosylated with a protected Man α(1→2) Man trichloroacetimidate. * Dedicated to the memory of Professor Jacques H. van Boom.

Synthesis of Ganglioside Mimics for Binding Studies with Myelin‐Associated Glycoprotein (MAG)*

Glycosylation of 3‐O‐unprotected 2‐azido‐2‐deoxy‐galactopyranoside (compound 5) with O‐(2,3‐di‐O‐acyl‐4,6‐O‐benzylidene‐D‐galactopyranosyl) trichloroacetimidates (compounds 4A, B) as glycosyl donors afforded β (1–3)‐linked disaccharides (9A, B) in high yield. Removal of the 2,3‐O‐acyl groups and selective 3‐O‐alkylation with α‐benzyloxycarbonyl‐alkyl triflates furnished the protected target molecules, which could be readily transformed into the desired ganglioside mimics. * Dedicated to the memory of late Professor Jacques H. van Boom.

Improved Synthesis of 1‐Benzenesulfinyl Piperidine and Analogs for the Activation of Thioglycosides in Conjunction with Trifluoromethanesulfonic Anhydride*

An improved protocol for the large‐scale production of 1‐benzenesulfinyl piperidine and other sulfinamides is described. It is demonstrated that 1‐benzenesulfinyl pyrrolidine and N,N‐diethyl benzenesulfinamide function analogously to 1‐benzenesulfinyl piperidine in the trifluoromethanesulfonic anhydride‐mediated activation of thioglycosides, and that their less crystalline nature enables them to be used at −78°C as opposed to the −60°C required to keep 1‐benzenesulfinyl piperidine in solution. N,N-Dicyclohexyl benzenesulfinamide does not activate thioglycosides in combination with trifluoromethanesulfonic anhydride, which is attributed to its greater steric bulk. * Dedicated with respect to the memory of Professor Jacques H. Van Boom.

Mapping the Active Site of the Bacterial Enzyme LpxC Using Novel Carbohydrate‐Based Hydroxamic Acid Inhibitors*

LpxC (UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐GlcNAc deacetylase), an enzyme involved in the biosynthesis of lipid A, is crucial for the growth of Gram‐negative bacteria. This enzyme has accordingly been identified as a potential target for the development of novel antibiotics against Gram‐negative bacteria. The carbohydrate‐derived hydroxamic acid 1 (1,5‐anhydro‐2‐C‐(carboxymethyl N‐hydroxyamide)‐2‐deoxy‐3‐O‐myristoyl‐ D‐glucitol) was previously shown to exhibit a wide spectrum of inhibitory activity against LpxC enzymes. Here we describe the preparation of seven analogs of 1 and their enzymatic evaluation. Two of the hydroxyl groups (OH‐3 and 6) of the GlcNAc residue were found to be involved in the binding interaction, and there is an important hydrophobic interaction in the vicinity O‐3 position with the enzyme that recognizes aromatic as well as aliphatic substituents. * In memory of Jacques H. van Boom.