Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.

Oxidative stress, inflammation and alpha-synuclein overexpression confer risk for development of alpha-synucleinopathies-neurodegenerative diseases that include Parkinson disease and Lewy body dementia. Dopaminergic neurons undergo degeneration in these diseases and are particularly susceptible to oxidative stress because dopamine metabolism itself creates reactive oxygen species. Intraneuronal deposition of alpha-synuclein as amyloid fibrils or Lewy bodies is the hallmark of these diseases. Herein, we demonstrate that concentrations of oxidative cholesterol metabolites derived from reactive oxygen species are elevated in the cortices of individuals with Lewy body dementia relative to those of age-matched controls, and we show that these metabolites accelerate alpha-synuclein aggregation in vitro. The increase in the production of these cytotoxic cholesterol metabolites is also observed in a dopaminergic cell line that overexpresses alpha-synuclein. By extension, these data lead to the hypothesis that oxidative stress produces cholesterol aldehydes that enable alpha-synuclein aggregation, leading to a pathologic cycle.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify A␤, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD. Anfinsen's classic experiments demonstrated that a protein's amino acid sequence specifies its conformation (1). These ideas were extended to explain the misfolding susceptibility of mutant proteins associated with a growing number of familial amyloid diseases (2-5). Although it is thus clear why mutant proteins might be more susceptible to misfolding, it is harder to understand why a wild-type protein or peptide adopts a native conformation in some individuals but it misfolds in others in what should be a common extracellular environment, leading to sporadic amyloid diseases. This discrepancy suggests that other events likely trigger misfolding in sporadic amyloid disease, but their nature remains elusive.The misfolding of secreted amyloid ␤ peptides (A␤) 39-43 residues in length is linked by a plethora of evidence to the pathology of Alzheimer's disease (AD) (6, 7). A␤ misfolding occurs when the soluble, monomeric, extracellular ensemble of extended conformations and low M r oligomers is transformed first into spherical assemblies, then into a number of intermediates, and lastly into fibrillar cross ␤-sheet quaternary structures known as amyloid (8)(9)(10)(11)(12). Amyloid fibrils and related structures recruit soluble A␤ to the aggregate by a seeded polymerization mechanism (10). The direct neurotoxicity of A␤ aggregates (8, 13) combined with their role in mediating chronic inflammation by microglia (14) and complement cascade activation (15) suggests that aggregation then mediates inflammation (16), which in turn promotes aggregation, in a vicious cycle of AD pathology.It is known that atherosclerosis and AD share many risk factors, including hypercholesterolemia and inflammation. The apoE-4 allele, which exacerbates hypercholesterolemia, has been linked to AD by data from both epidemiological and transgenic mouse studies (17)(18)(19)(20). It has also recently been shown t...

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody-antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.

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 F ab fragments. The density map at a resolution of Ϸ16 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. membrane protein ͉ viroporin ͉ single particle analysis ͉ random conical tilt reconstruction T he hepatitis C virus (HCV) poses a major global health problem. It puts more than 170 million people worldwide at risk of developing liver cirrhosis and hepatocellular carcinoma. HCV comprises 6 different genotypes and is one of the fastest mutating viruses known to man. There is no vaccine available, and treatment options are genotype-specific, prone to viral escape mutations, and inadequate.The HCV p7 ion channel is a more recent addition to the growing list of potential drug targets encoded by HCV, reflecting the urgent need for a therapeutic approach. p7 is critical for the release of infectious virions in vitro (1, 2) and in vivo (3). It is not involved in HCV RNA replication (4, 5), but is required for late steps of viral particle assembly (2) and potentially cell entry (6). However, the prerequisite incorporation of p7 into budding virions has not been demonstrated. p7 belongs to the viroporins, small virally encoded proteins with at least 1 membrane-spanning helix that oligomerize to form channels or pores that modify the permeability of the cell membrane to ions and other small molecules (7). In planar lipid bilayers, p7 monomers oligomerize to form cation-selective ion channels that can be specifically inhibited by long alkylchain iminosugars, amiloride, and amantadine derivatives, with varying reported efficacies (6,(8)(9)(10)(11)(12)(13)(14)(15). Each HCV p7 monomer consists of 63 aa, most of which are hydrophobic and possibly contain endoplasmic reticulum (ER) retention signals (16-18). Computational secondary structure predictions suggest that the monomers contain 2 transmembrane spanning helices connected by a short basic loop (19,20). The loop is assumed to face the cytoplasm, with the N and C termini facing...

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H 2O3) and ozone (O3) from singlet oxygen ( 1 O 2 * ) and water. Given that neutrophils have the potential both to produce 1 O 2 * and to bind antibodies, we considered that these cells could be a biological source of O 3. We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O 3. 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 1 O 2 * . 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 O 3 to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation. N eutrophils (PMNs) are the most abundant leukocytes in the bloodstream. Their function is the killing of bacteria and fungi, in part by the triggering of an oxidative burst that is composed of a set of enzymatic and chemical reactions ultimately leading to the formation of hypohalous acid, 1 O 2 * , and hydroxyl radical (HO • ) (1, 2). The first step in this cascade, the reduction of dioxygen, is initiated by the enzyme NAD(P)H oxidase. This oxidase is a complex enzyme composed of five components: gp91 phox (with phox being phagocyte oxidase), a heavily glycosylated 56-kDa protein that contains the electroncarrying components of the oxidase; p67 phox , p47 phox , and p22 phox , which are proteins named according to their approximate molecular weights; and rac2, a low molecular weight GTPase. In the resting cell, p47 phox and p67 phox form a complex in the cytosol (which also contains p40 phox , a protein whose effect on oxidase activity is unclear), whereas gp91 phox and p22 phox are in the membrane. When the PMN is activated by antibody-coated bacteria, p47 phox is phosphorylated on particular serines and moves to the membrane to assemble the active oxidase, carrying with it its cargo of p67 phox and the enigmatic p40 phox . Rac 2, also necessary for oxidase activity, picks up a GTP and moves into the oxidase assembly. The NAD(P)H oxidase then produces superoxide anion (O 2•Ϫ ) (Eq.