Claudia S. Maier

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

Acrolein (2-propenal) is ubiquitously present in (cooked) foods and in the environment. It is formed from carbohydrates, vegetable oils and animal fats, amino acids during heating of foods, and by combustion of petroleum fuels and biodiesel. Chemical reactions responsible for release of acrolein include heat-induced dehydration of glycerol, retro-aldol cleavage of dehydrated carbohydrates, lipid peroxidation of polyunsaturated fatty acids, and Strecker degradation of methionine and threonine. Smoking of tobacco products equals or exceeds the total human exposure to acrolein from all other sources. The main endogenous sources of acrolein are myeloperoxidase-mediated degradation of threonine and amine oxidase-mediated degradation of spermine and spermidine, which may constitute a significant source of acrolein in situations of oxidative stress and inflammation. Acrolein is metabolized by conjugation with glutathione and excreted in the urine as mercapturic acid metabolites. Acrolein forms Michael adducts with ascorbic acid in vitro, but the biological relevance of this reaction is not clear. The biological effects of acrolein are a consequence of its reactivity towards biological nucleophiles such as guanine in DNA and cysteine, lysine, histidine, and arginine residues in critical regions of nuclear factors, proteases, and other proteins. Acrolein adduction disrupts the function of these biomacromolecules which may result in mutations, altered gene transcription, and modulation of apoptosis.

Pyrolysis of cellulose under ammonia leads to nitrogen-doped nanoporous carbon generated through methane formation.

Here, we present a simple one-step fabrication methodology for nitrogen-doped (N-doped) nanoporous carbon membranes via annealing cellulose filter paper under NH3. We found that nitrogen doping (up to 10.3 at %) occurs during cellulose pyrolysis under NH3 at as low as 550 °C. At 700 °C or above, N-doped carbon further reacts with NH3, resulting in a large surface area (up to 1973.3 m(2)/g). We discovered that the doped nitrogen, in fact, plays an important role in the reaction, leading to carbon gasification. CH4 was experimentally detected by mass spectrometry as a product in the reaction between N-doped carbon and NH3. When compared to conventional activated carbon (1533.6 m(2)/g), the N-doped nanoporous carbon (1326.5 m(2)/g) exhibits more than double the unit area capacitance (90 vs 41 mF/m(2)).

Tricking the guard: exploiting plant defense for disease susceptibility.

Necrophilic Bandit Fungi The immune systems that plants use to defend against pathogens normally deflect attack. However, Lorang et al. (p. 659, published online 18 October) have identified a rearguard susceptibility that a necrotrophic fungus exploits. The fungal toxin victorin interacts with the defense protein LOV1 in Arabidopsis and activates it, but the result, counterintuitively, is that the plant succumbs to the disease rather than fighting it off. What seems to be happening is that victorin targets a thioredoxin that regulates the systemic acquired resistance pathway. That interaction triggers activity of LOV1, which in turn incites cell death. The invading fungus then benefits from the ready access to dead cells. Cochliobolus victoriae, a necrotrophic fungal pathogen with devastating effects on oat crops reveals its strategies. Typically, pathogens deploy virulence effectors to disable defense. Plants defeat effectors with resistance proteins that guard effector targets. We found that a pathogen exploits a resistance protein by activating it to confer susceptibility in Arabidopsis. The guard mechanism of plant defense is recapitulated by interactions among victorin (an effector produced by the necrotrophic fungus Cochliobolus victoriae), TRX-h5 (a defense-associated thioredoxin), and LOV1 (an Arabidopsis susceptibility protein). In LOV1’s absence, victorin inhibits TRX-h5, resulting in compromised defense but not disease by C. victoriae. In LOV1’s presence, victorin binding to TRX-h5 activates LOV1 and elicits a resistance-like response that confers disease susceptibility. We propose that victorin is, or mimics, a conventional pathogen virulence effector that was defeated by LOV1 and confers virulence to C. victoriae solely because it incites defense.

Pox proteomics: mass spectrometry analysis and identification of Vaccinia virion proteins.

BackgroundAlthough many vaccinia virus proteins have been identified and studied in detail, only a few studies have attempted a comprehensive survey of the protein composition of the vaccinia virion. These projects have identified the major proteins of the vaccinia virion, but little has been accomplished to identify the unknown or less abundant proteins. Obtaining a detailed knowledge of the viral proteome of vaccinia virus will be important for advancing our understanding of orthopoxvirus biology, and should facilitate the development of effective antiviral drugs and formulation of vaccines.ResultsIn order to accomplish this task, purified vaccinia virions were fractionated into a soluble protein enriched fraction (membrane proteins and lateral bodies) and an insoluble protein enriched fraction (virion cores). Each of these fractions was subjected to further fractionation by either sodium dodecyl sulfate-polyacrylamide gel electophoresis, or by reverse phase high performance liquid chromatography. The soluble and insoluble fractions were also analyzed directly with no further separation. The samples were prepared for mass spectrometry analysis by digestion with trypsin. Tryptic digests were analyzed by using either a matrix assisted laser desorption ionization time of flight tandem mass spectrometer, a quadrupole ion trap mass spectrometer, or a quadrupole-time of flight mass spectrometer (the latter two instruments were equipped with electrospray ionization sources). Proteins were identified by searching uninterpreted tandem mass spectra against a vaccinia virus protein database created by our lab and a non-redundant protein database.ConclusionSixty three vaccinia proteins were identified in the virion particle. The total number of peptides found for each protein ranged from 1 to 62, and the sequence coverage of the proteins ranged from 8.2% to 94.9%. Interestingly, two vaccinia open reading frames were confirmed as being expressed as novel proteins: E6R and L3L.

Protein conformations, interactions, and H/D exchange.

Modern mass spectrometry (MS) is well known for its exquisite sensitivity in probing the covalent structure of macromolecules, and for that reason, it has become the major tool used to identify individual proteins in proteomics studies. This use of MS is now widespread and routine. In addition to this application of MS, a handful of laboratories are developing and using a methodology by which MS can be used to probe protein conformation and dynamics. This application involves using MS to analyze amide hydrogen/deuterium (H/D) content from exchange experiments. Introduced by Linderstøm-Lang in the 1950s, H/D exchange involves using (2)H labeling to probe the rate at which protein backbone amide protons undergo chemical exchange with the protons of water. With the advent of highly sensitive electrospray ionization (ESI)-MS, a powerful new technique for measuring H/D exchange in proteins at unprecedented sensitivity levels also became available. Although it is still not routine, over the past decade the methodology has been developed and successfully applied to study various proteins and it has contributed to an understanding of the functional dynamics of those proteins.

Epigallocatechin gallate (EGCG) potentiates the cytotoxicity of rotenone in neuroblastoma SH-SY5Y cells

Exposure to rotenone, a widely used pesticide, has been suggested to increase the risk of developing Parkinsons disease. Studies indicate that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species. The present work was conducted to determine to what extent (-)-epigallocatechin-3-gallate (EGCG), a widely used dietary supplement, modulates the cytotoxicity of rotenone in human neuroblastoma SH-SY5Y cells. Our results indicate that EGCG shows concentration-dependent effects on ROS production and cytotoxicity in SH-SY5Y cells. Treatment of these dopaminergic cells with rotenone (1-50 microM) alone or EGCG (25 or 50 microM) alone caused a significant decrease in cell viability. Pretreatment of SH-SY5Y cells with 25 or 50 microM EGCG potentiated the cytotoxicity of rotenone. The exacerbating effect of EGCG on rotenone toxicity may involve an apoptotic mechanism as shown by the enhancement of caspase-3 activity and activation of other caspases in rotenone-treated SH-SY5Y cells. The potentiating effect of EGCG on rotenone toxicity may be attributed to the enhanced production of intracellular superoxide in SH-SY5Y cells. The enhanced intracellular production of ROS by rotenone-EGCG combination may also account for the increased formation of protein carbonyls in 10,000xg fraction of SH-SY5Y cells detected by anti-HNE antibody. For instance, core histones and nuclear ribonuclear proteins were identified as major putative in vivo targets of HNE. Our present findings indicate that more detailed mechanistic studies are necessary to fully understand the chemistry of EGCG and to justify its use as potentially health-promoting dietary supplement, e.g. in the prevention of neurodegenerative diseases associated with oxidative stress.

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.

Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

Nano‐electrospray ionization time‐of‐flight mass spectrometry (ESI‐MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI‐MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far‐ and near‐UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI‐MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid‐induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI‐MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn2+. Gas‐phase dissociation experiments of the deuterium‐labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas‐phase dissociation experiments of the ternary E9 DNase‐Zn2+‐Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.

Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein.

Khao Dawk Mali 105 rice bran protein (RBP) was fractionated into albumin (12.5%), globulin (13.9%), glutelin (70.8%) and prolamine (2.9%). The native and denatured RBP fractions were hydrolyzed with papain and trypsin for 3h at optimum conditions. The RBP fractions and their hydrolysates were evaluated for their antioxidant activity by the Oxygen Radical Absorbance Capacity (ORAC) assay. The trypsin-hydrolyzed denatured albumin exhibited the highest antioxidant activity with an ORAC value of 4.07 μmol of Trolox equivalent (TE)/mg protein. This hydrolysate was separated by using RP-HPLC and three fractions with high antioxidant activity were examined by LTQ-FTICR ESI mass spectrometry. The MW of the peptides from these fractions were 800-2100 Da. and consisted of 6-21 amino acid residues. Most of the peptides from the fractions demonstrated typical characteristics of well-known antioxidant peptides. The results suggest that trypsin-hydrolyzed denatured rice bran albumin might be useful as a natural food antioxidant.

Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction, and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. This study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin alpha-1B chain, Cys-351 and Cys-499 in alpha-actinin-4, Cys-328 in vimentin, Cys-369 in D-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.