Serum non-transferrin-bound iron and low-density lipoprotein oxidation in heterozygous hemochromatosis

Non-transferrin-bound iron (NTBI) is implicated in lipid peroxidation but the relation with oxidative modification of low-density lipoprotein (LDL) is not known. We assessed variables reflecting in vitro and in vivo LDL oxidation in two age- and sex-matched groups (n=23) of hereditary hemochromatosis heterozygotes (C282Y), characterized by a clear difference in mean serum NTBI (1.55+/-0.57 micromol/L vs 3.70+/-0.96 micromol/L). Plasma level of oxidized LDL (absolute and relative to plasma apolipoprotein B), and IgG and IgM antibodies to oxidized LDL, markers of in vivo LDL oxidation, did not differ between the groups with low and high serum NTBI. Mean lag-phase of in vitro LDL oxidation was also not significantly different between both study groups. Conclusion: these findings do not support the hypothesis that NTBI promotes oxidative modification of plasma LDL.     

Oxidative damage to surfactant protein D in pulmonary diseases

Surfactant protein D is an important innate host defence molecule that has been shown to interact with a variety of pathogens and to play a role in surfactant homeostasis. The aim of this study was to examine the influence of oxidation on surfactant protein D in different lung diseases. Bronchoalveolar lavage fluids (BALFs) from patients with different grade of protein oxidation were examined for changes in the primary chain and the quaternary structure of surfactant protein D. Significant changes of quaternary surfactant protein-D (SP-D) structure were detected under oxidative conditions in vitro and in vivo. The functional capacity of surfactant protein D to agglutinate bacteria was impaired by oxidation. We conclude that surfactant protein D is an important target of free radicals generated in the lungs. Host defence may be impaired due to the oxidation of surfactant protein D and may contribute to the suppurative lung diseases like cystic fibrosis (CF).     

Cholesterol, not polyunsaturated fatty acids, is target molecule in oxidation induced by reactive oxygen species in membrane of human erythrocytes

The oxidation of polyunsaturated fatty acids (PUFAs) by reactive oxygen species (ROS) is linked to aging and to many diseases. We herein employ initiating peroxyl radical (ROO•) derived from the decomposition of 2,2′-azobis(2-amidinopropane dihydrochloride), hydroxyl radical generated by the Fenton reaction and peroxyl radical (ROO•) and alkoxyl radical (LO•) derived from PUFAs by addition of Cu²⁺ as ROS sources to oxidize glycerides under alkaline conditions in the presence of methanol instead of being treated traditionally by diazomethane (CH₂N₂) under acidic conditions (pH=2.0), to obtain corresponding methyl esters for the combination of gas chromatography with mass spectrometry determination. It was found that all the PUFAs in the membrane are perfectly preserved after oxidation by ROS, even though sufficient time is available for the interaction between human erythrocytes and ROS. This indicates that ROS do not damage PUFAs during reaction time. However, three products (cholesta-4,6-dien-3-ol, cholesta-4,6-dien-3-one, and cholesta-3,5-dien-7-one) are produced from the oxidation of cholesterol within this time frame. This qualitative finding, suggests that the cholesterol in the membrane of human erythrocytes is more susceptible to ROS-induced oxidation than are PUFAs, and compels us to re-evaluate the physiological roles of cholesterol and PUFAs in the human erythrocyte membrane.     

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30–40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α₃β₃γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ϵ inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ϵ inhibition.     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP^C) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.     

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.     

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris

Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.     

Modification of carboxymethyl cellulose through oxidation

The periodate oxidation reaction of carboxymethyl cellulose involve the primary and secondary hydroxyl groups of the pyranose ring. The reaction is accompanied by the opening of the pyranose ring and resulting product is dialdehyde carboxymethyl cellulose along with some hydrated forms. In this process the glucosidic bond becomes weaker; the formation of carboxyl groups induces a depolymerization, thus reducing the polymerization degree and the physical and mechanical strength of the material. The reaction has been has been carried out at pH 3.5, temperature 45 °C for 0.5-4 h.