Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress.

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H(2)O(2) and cDNA microarray technique was used to produce gene expression profiles. We found that H(2)O(2) treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF-kappa B, ERK, JNK, p38, PKC and INF-gamma . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.

Study of beta-amyloid peptide (Abeta40) insertion into phospholipid membranes using monolayer technique.

beta-Amyloid peptide (Abeta), a normal constituent of neuronal and non-neuronal cells, has been proved to be the major component of extracellular plaque of Alzheimer's disease (AD). The interaction of Abeta with lipid membranes may be essential for its neurotoxicity. Our previous study revealed that membrane insertion may provide a possible pathway by which Abeta prevents itself from aggregation and fibril formation. In the present work we studied the membrane insertion of Abeta and the factors that affect its insertion ability using a monolayer approach. The results show that Abeta is surface active and can insert into lipid monolayers. When a high level of cholesterol is present, Abeta40 can insert into the phospholipid mixtures simulating physiological membrane composition. Acidic pH enhances Abeta insertion, while the effect of ionic strength is rather complex. Abeta insertion ability may be ultimately relative to cholesterol-rich domains in the monolayers, which indicates strong interaction between Abeta and cholesterol.

Specific binding of integrin alphaIIbbeta3 to RGD peptide immobilized on a nitrilotriacetic acid chip: a surface plasmon resonance study.

Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni2+. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His-epsilon-aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca2+ bound on low affinity sites or adding of Mn2+ can increase the binding ability of integrin.

Study of the correlation of secondary structure of beta-amyloid peptide (Abeta40) with the hydrophobic exposure under different conditions.

Abeta is the core protein of extracellular plaque of Alzheimer's disease, and its neurotoxicity is relative to its conformation. In the current work, the effects of various factors, such as pH, ionic strength and lipid membranes, on the secondary structure of Abeta were studied by circular dichroism. In addition, we detected the exposure of hydrophobic sites of Abeta under different conditions using ANS fluorescence. The results showed that the hydrophobic exposure of the protein was correlated with the content of 3betasheet conformation in the phospholipid-containing environment. The beta-sheet content and hydrophobic exposure of Abeta both increased when reacted with pure PC vesicles, while no beta-sheet content and very low hydrophobic exposure were detected after reaction with 30% cholesterol containing PC vesicles. Since beta-sheet conformation is considered as the toxic conformation of Afbeta such correlation may be important for the pathology of AD.

Study of the spontaneous dissociation of rabbit C-reactive protein.

C-Reactive protein (CRP) is composed of five identical noncovalently linked monomers and characterized as an important acute-phase protein. The CRP subunit obtained by denaturing treatments, which is termed modified CRP, has also been widely studied. In the current work, we found that there exists some degree of natural dissociation of CRP in stock solution. This dissociation is critically dependent on the absence of Ca2+. Low pH could enhance the dissociation of CRP, while ionic strength has little effect. Anilinonaphthalenesulfonate (ANS) fluorescence detections indicate that the exposure of hydrophobic surface increases during the dissociation. Acidic pH conditions also induce an increase in ANS fluorescence. This suggests that hydrophobic interactions between CRP subunits may contribute to the study of its pentameric structure. Surface plasmon resonance experiments indicate that monomeric CRP does not specifically bind to phosphatidylcholine-containing membrane as native CRP does. Electron microscopy shows that monomeric CRP binds to negatively charged lipid through electrostatic forces, and such lipid may induce the dissociation of CRP due to the acidic pH in the diffuse double layer near the membrane.

Effect of phospholipid on trichosanthin adsorption at the air-water interface.

Trichosanthin (TCS) is a toxic protein with multiple pharmacological properties. It belongs to the type I ribosome inactivating protein (RIP) family and can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. The interaction between TCS and phospholipid membrane was thought to be essential for its physiological effect, for it must get across the cell membrane before it can enter the cytoplasm and exert its RIP function. In order to study the TCS-phospholipid interaction, the difference between spontaneous and phospholipid induced adsorption of TCS at the air-water interface was investigated, and the results were analyzed according to the diffusion-penetration-rearrangement adsorption model. The results showed that both negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and neutral 1,2-dipalmitoyl-sn-glycero-3-phosphocholine can accelerate the adsorption rate, while there exists a possible membrane induced conformational change of TCS which is specific for the negatively charged DPPG. We also proposed a revised model for the diffusion controlled initial adsorption period.

Dissociation and subunit rearrangement of membrane-bound human C-reactive proteins.

As one of the most important acute-phase reactants in human serum, C-reactive protein plays its physiological roles mainly on membranes. Here we show that the human C-reactive protein is two-dimensionally crystallized upon specific adsorption on the phosphorylcholine ligand containing membranes by monolayer approach. The 2.0-nm resolution projection structure of the two-dimensional crystals analyzed by electron microscopy and image reconstruction reveals open-ring-like pentamers in the crystals. The electron microscope graphs also show that the dissociated pentamers with open-ring-like structure occur in a closed packing region (not two-dimensionally crystallized). These results indicate a membrane-induced dissociation and rearrangement of hCRP, which may relate to the variety of hCRP's physiological functions.

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

The membrane insertion of trichosanthin is membrane-surface-pH dependent.

Trichosanthin (TCS) is the active component extracted from Tianhuafen, a traditional herbal medicine that has been used for abortion in China for centuries. It belongs to the type-I ribosome-inactivating protein (RIP) family and can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. Recent studies have shown TCS to be multifunctional, its pharmacological properties including immunomodulatory, anti-tumour and anti-HIV activities. The membrane-insertion property of TCS is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In this paper, the membrane-insertion mechanism of TCS was studied. The monolayer experiment revealed that TCS's membrane-insertion ability was dependent on low pH. Fluorescence spectroscopy using 1-anilinonaphthalene-8-sulphonic acid as a probe showed that low pH may induce the conformational change of TCS that leads to the hydrophobic-site exposure, and the CD result showed that this conformational change did not alter its secondary structure. Such conformational change leads to an intermediate state, called the 'molten globular state' by previous investigators. The pH-dependent membrane insertion and conformational change were related by the fact that the optimal membrane-surface pH needed was the same for the two events. From these and other results, a membrane-insertion model was proposed.

Membrane-induced conformational change in human apolipoprotein H.

The interaction of apolipoprotein H (Apo H) with lipid membrane has been considered to be a basic mechanism for the biological function of the protein. Previous reports have demonstrated that Apo H can interact only with membranes containing anionic phospholipids. Here we study the membrane-induced conformational change of Apo H by CD spectroscopy with two different model systems: anionic-phospholipid-containing liposomes [such as 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and cardiolipin], and the water/methanol mixtures at moderately low pH, which mimic the micro-physicochemical environment near the membrane surface. It is found that Apo H undergoes a remarkable conformational change on interaction with liposomes containing anionic phospholipid. To interact with liposomes containing DMPG, there is a 6.8% increase in alpha-helix in the secondary structures; in liposomes containing cardiolipin, however, there is a 12.6% increase in alpha-helix and a 9% decrease in beta-sheet. The similar conformation change in Apo H can be induced by treatment with an appropriate mixture of water/methanol. The results indicate that the association of Apo H with membrane is correlated with a certain conformational change in the secondary structure of the protein.

Membrane-induced conformational change of proteins.

Many proteins exhibit both a water-soluble and a membrane-bound state. The proteins in the membrane-bound state obtain a distinct structure from that in the bulk, which exists in many important biological processes. In the present paper we would stress that the variation of the physical chemistry properties of the microenvironment adjacent to the membrane-surface region play an important role in the process of the membrane-induced conformational changes of the proteins.

Orientation of membrane-bound melittin studied by a combination of HPLC and liquid secondary ion mass spectrometry (LSIMS).

Combining two analytical techniques, HPLC and liquid secondary ion mass spectrometry, the orientation of liposomal membrane-bound melittin was analyzed through its trypsin-digested products. We found that trypsin can access all proteolytic sites of the membrane-bound melittin when the liposomes have no transmembrane potential, whereas the proteolytic site near the N terminus of melittin is blocked when the liposomes have a negative transmembrane potential. The results suggest that the negative transmembrane potential may induce the melittin molecules to insert into the membrane perpendicularly, whereas melittin lies flat on the membrane surface in the absence of a negative potential.

The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond.

A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.

Antibodies against human IFN-alpha and -beta recognized the immunosuppressive domain of HIV-1 gp41 and inhibit gp41-binding to the putative cellular receptor protein p45.

Sequence-comparison indicates existing sequence-similarity between receptor-binding regions of human type 1 IFNs (IFN-alpha, -beta and -omega) and HIV-1 gp41. Previous findings had suggested that the increased levels of antibodies against human IFN-alpha and -beta in HIV-1-infected individuals are associated with a common epitope on gp41, IFN-alpha and -beta. To clarify the relationship between human type I interferon and HIV-1 gp41 and the protective mechanism of an IFN-alpha-vaccine, we prepared antisera against human IFN-alpha, -beta and HIV-1 gp41, and examined crossreaction of these antisera and their inhibition of gp41 binding to its binding protein p45. Mouse antisera against IFN-alpha and -beta could recognize HIV-1 recombinant soluble (aa539-684) and gp41 immunosuppressive peptide (ISP, aa583-599), while normal mouse sera (pre-immune sera) did not. Mouse antisera to rsgp41 crossreacted with IFN-alpha and -beta. Besides, mouse antisera to IFN-alpha and beta, like mouse anti-rsgp41 antiserum, could inhibit gp41-binding to its putative cellular receptor protein p45, while normal mouse serum did not. These results indicate that antibodies crossreacting with gp41 ISP, IFN-alpha and -beta, could be induced by this common immunological epitope in vivo.

Conformational changes of urea-denatured colicin E1 induced by phospholipid membranes.

The membrane insertion of urea-denatured colicin E1 was studied by using fluorescence spectroscopy, circular dichroism and monolayer techniques. The results showed that the denatured colicin E1 taking mainly the 'random coil' conformation may recover its orderliness to a certain extent under the induction of the phospholipid membrane and insert spontaneously into phospholipid membrane, indicating that unfolding of colicin E1 does not inhibit its membrane insertion. Among the four tryptophan residues of the membrane-bound colicin E1 molecules, at least two were accessible by the quenchers, i.e. not inserted into the membranes. Although urea-denatured colicin E1 interacted preferentially with negatively charged phospholipids, it seems less dependent on the negatively charged lipid than colicin A. The addition of urea increased the speed of the adsorption of colicin E1 to the membrane, but did not affect obviously its membrane insertion ability.

Numerical simulations of surface plasmon resonance system for monitoring DNA hybridization and detecting protein-lipid film interactions.

This paper presents a simple method to extract information about thin organic films from surface plasmon resonance (SPR) spectra. From numerical simulations it was found that a shift (delta theta SPR) of an absorption peak in the SPR spectrum was directly proportional to the product of the thin organic film thickness and the refractive index difference between the thin organic film and a buffer soaking the sample. It was also found that delta theta SPR was not sensitive to the thin organic film support of a gold film and a glass cover slip. Relationships between delta theta SPR and distributions of macromolecule structures, in the thin organic films were theoretically established. Formulae were derived for a homemade SPR system to calculate length, transverse area, density and surface concentration of macromolecules in the thin organic film. The validity of these treatments was checked by precisely measuring the size of a single distearoylphosphatidylcholine molecule on a gold-supported phospholipid film; by quantitatively monitoring hybridization of synthesized oligonucleotides strands based on a biotin/avidin system; and by quantitatively detecting the steric hindrance of rabbit C-reactive protein specifically bound to phospholipid monolayers composed of synthesized lipids.

Calcium-dependent binding of rabbit C-reactive protein to supported lipid monolayers containing exposed phosphorylcholine group.

The interaction of rabbit C-reactive protein (rCRP) with a supported monolayer containing a phosphorylcholine moiety was studied. Three types of phospholipids were synthesized, each containing a insertion spacer of eight, six, or three atoms between the phosphorylcholine group and hydrophobic tail. By varying the length of the insertion spacer, we can vary the extension of the phosphorylcholine group from the membrane surface. By varying the monolayer composition, we can control the lateral distance between the exposed phosphorylcholine groups. Using the surface plasmon resonance technique (SPR), we demonstrated that the calcium-dependent binding of rCRP to the model membrane is governed not only by the ability of the ligand to access the binding pocket fully (spacer length), but also by lateral hindrance within the two-dimensional plane of the membrane. The value of the apparent binding constant was estimated by theoretical analysis, which is obviously dependent on the composition of the lipid mixture, and a maximum of (9.9 +/- 1.5) x 10(6) M-1 was obtained.

Detection of the genotype of apolipoprotein H purified from the serum of a Chinese subject by capillary isoelectric focusing electrophoresis.

Capillary isoelectric focusing electrophoresis (cIEF) has been developed to detect the genotype of apolipoprotein H, which was purified from the serum of a Chinese subject. Depending upon the observation of splitting peaks in cIEF, it is possible to determine if the protein was expressed from a heterozygote gene.

The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study.

Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (pic) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the 'immune cleaning' of plasma liposomes is discussed.

Interaction of rabbit C-reactive protein with phospholipid monolayers studied by microfluorescence film balance with an externally applied electric field.

C-reactive protein (CRP) is one of the most characteristic acute-phase proteins in humans and many other animals. It binds to phosphorylcholine in a calcium-dependent manner. In addition, CRP activates the complement systems via the classical pathway. The interaction between rabbit CRP (rCRP) and model biological membrane is studied using dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine monolayers. Observations with fluorescence microscopy indicate that rCRP is more likely to be incorporated in the liquid phase of monolayers. Such incorporation does not depend on the presence of calcium and is not inhibited by phosphocholine. The area occupied by the protein when incorporated into the monolayer was estimated. The dipole moment density of the protein crossing the air/water interface was measured by applying an external electric field. Our results indicate that calcium binding leads to a conformational change in CPR, which might modify the orientation of CRP in the monolayer. In addition, a negative charge or negative difference in dipole moment density facilitates the incorporation of CPR into the monolayer.