Paul Wentworth

Investigating antibody-catalyzed ozone generation by human neutrophils.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H2O3) and ozone (O3) from singlet oxygen (1O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document}) and water. Given that neutrophils have the potential both to produce 1O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document} and to bind antibodies, we considered that these cells could be a biological source of O3. We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O3. This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated 1O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document}. Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O3 to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation.

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy

Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific Fab fragments. The density map at a resolution of ≈16 Å reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins.

Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen (1O*2) induced antibody-catalyzed water-oxidation pathways proceeds via the formation of dihydrogen trioxide (H2O3), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{_{3}}^{{\bullet}}}}\end{equation*}\end{document}, and we point to the remarkable potential of this either H2O3- or O3-derivable species to act as a masked hydroxyl radical (HO•) in a biological environment.

A chemical approach for cell-specific targeting of nanomaterials: small-molecule-initiated misfolding of nanoparticle corona proteins.

A major challenge in nanomaterial science is to develop approaches that ensure that when administered in vivo, nanoparticles can be targeted to their requisite site of action. Herein we report the first approach that allows for cell-specific uptake of nanomaterials by a process involving reprogramming of the behavior of the ubiquitous protein corona of nanomaterials. Specifically, judicious surface modification of quantum dots with a small molecule that induces a protein-misfolding event in a component of the nanoparticle-associated protein corona renders the associated nanomaterials susceptible to cell-specific, receptor-mediated endocytosis. We see this chemical approach as a new and general method for exploiting the inescapable protein corona to target nanomaterials to specific cells.

Catalytic antibodies: structure and function.

More than 10 years have now elapsed since the first reports confirmed that antibodies could be programmed as catalysts for chemical processes. Much of the initial research focussed on exploring the scope and utility of these new biocatalysts. Recently however, increasing information gleaned from X-ray analyses is allowing an exciting insight into the structural basis of antibody catalyzed reactions. This review details the evolving knowledge of the structure-function relationship for catalytic antibodies that accelerate a range of different reaction classes.

Generating and analyzing combinatorial chemistry libraries

Abstract The past year has seen developments in key areas of combinatorial chemistry, including new encoding and deconvolution strategies, techniques for analyzing library components and alternatives to the obligatory solid-supports utilized for library construction. Highlights include the utility of radiofrequency encoding strategies and the expansion of solution-phase combinatorial synthesis.

Lipid-derived aldehydes accelerate light chain amyloid and amorphous aggregation.

Antibody light chain (LC) aggregation in vivo leads to the systemic deposition of Ig light chain domains in the form of either amyloid fibrils (AL-amyloidosis) or amorphous deposits, light-chain deposition disease (LCDD), in mainly cardiac or renal tissue and is a pathological condition that is often fatal. Molecular factors that may contribute to the propensity of LCs to aggregate in vivo, such as the protein primary structure or local environment, are intensive areas of study. Herein, we show that the aggregation of a human antibody kappa-(kappa-MJM) and lambda-(lambda-L155) light chain (1 mg/mL) can be accelerated in vitro when they are incubated under physiologically relevant conditions, PBS, pH 7.4 and 37 degrees C, in the presence of a panel of biologically relevant lipid-derived aldehydes, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), glyoxal (GLY), atheronal-A (KA), and atheronal-B (ALD). Thioflavin-T (ThT) and Congo Red (CR) binding assays coupled with turbidity studies reveal that this aldehyde-induced aggregation can be associated with alteration of protein secondary structure to an increased beta-sheet conformation. We observed that the nature of the conformational change is primarily dependent upon the lipidic aldehyde studied, not the protein sequence. Thus, the cholesterol 5,6-secosterols, KA and ALD, cause an amorphous-type aggregation which is ThT and CR negative for both the kappa-MJM and lambda-L155 light chains, whereas 4-HNE, MDA, and GLY induce aggregates that bind both ThT and CR. TEM analysis revealed that amyloid fibrils were formed during the 4-HNE-mediated aggregation of kappa-MJM and lambda-L155 light chains, whereas ALD-induced aggregates of these LCs where amorphous in nature. Kinetic profiles of LC aggregation reveal clear differences between the aldehydes, KA and ALD, causing a classic nucleated polymerization-type aggregation, with a lag phase (of approximately 150 h) followed by a growth phase that plateaus, whereas 4-HNE, MDA, and GLY trigger a seeded-type aggregation process that has no lag phase. In-depth studies of the 4-HNE-accelerated aggregation of kappa-MJM and lambda-L155 reveal a clear aldehyde concentration dependence and a process that can be inhibited by the naturally occurring osmolyte trimethylamine N-oxide (TMAO). Given these data, we feel our recently discovered paradigm of inflammatory aldehyde-induced protein misfolding may now extend to LC aggregation.

Adduction of cholesterol 5,6-secosterol aldehyde to membrane-bound myelin basic protein exposes an immunodominant epitope.

Myelin degradation in the central nervous system (CNS) is a clinical hallmark of multiple sclerosis (MS). A reduction in the net positive charge of myelin basic protein (MBP) via deimination of arginine to citrulline has been shown to correlate strongly with disease severity and has been linked to myelin instability and a defect that precedes neurodegeneration and leads to autoimmune attack. Recently, we have shown that lipid-derived aldehydes, such as cholesterol 5,6-secosterols atheronal A (1a) and atheronal B (1b), modulate the misfolding of certain proteins such as apolipoprotein B(100), β-amyloid, α-synuclein, and κ- and λ-antibody light chains in a process involving adduction of the hydrophobic aldehyde to lysine side chains, resulting in a decrease in the net positive charge of the protein. In this study, we show that the presence of either atheronal A (1a) or atheronal B (1b) in large unilamellar vesicles (cyt-LUVs) with the lipid composition found in the cytosolic myelin sheath and bovine MBP (bMBP) leads to an atheronal concentration-dependent increase in the surface exposure of the immunodominant epitope (V86-T98) as determined by antibody binding. Other structural changes in bMBP were also observed; specifically, 1a and 1b induce a decrease in the surface exposure of L36-P50 relative to control cyt-LUVs as measured both by antibody binding and by a reduction in the level of cathepsin D proteolysis of F42 and F43. Structure-activity relationship studies with analogues of 1a and 1b point to the aldehyde moiety of both compounds being critical to their effects on bMBP structure. The atheronals also cause a reduction in the size of the bMBP-cyt-LUV aggregates, as determined by fluorescence microscopy and dynamic light scattering. These results suggest that formation of an imine between inflammatory-derived aldehydes, which effectively reduces the cationic nature of MBP, can lead to structural changes in MBP and a decrease in myelin stability akin to deimination and as such may make a hitherto unknown contribution to the onset and progression of MS.

A cofactor approach to copper-dependent catalytic antibodies

A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, LysH93, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [kcat(11) = 2.3 min−1 and Km(11) 2.2 mM] with a rate enhancement [kcat(11)kuncat(11)] of 2.1 × 105. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 × 104 in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2.

Cholesterol Secosterol Aldehydes Induce Amyloidogenesis and Dysfunction of Wild Type Tumor Protein p53

Epidemiologic and clinical evidence points to an increased risk for cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and -B (ALD), but not the polyunsaturated fatty acid (PUFA)-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by the analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction and cancer risk.