蛋白质中二硫键附近肽段识别规律研究

本文对蛋白质中二硫键附近的残基进行了计算机统计分析,结果发现平行和反平行残基间存在着特异的配对规律。这种残基间的相互作用或识别,可能与蛋白质折叠过程中正确地形成二硫键有关。该结果有助于蛋白质工程设计。     

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.     

Evidence for Disulfide Bonds in Membrane-Bound and Solubilized Opioid Receptors

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane-bound and digitonin-solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D-Ala2-D-Leu5-enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin-solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2. [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.     

Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

Optimizing the MALDI-TOF-MS Observation of Peptides Containing Disulfide Bonds

The observation of peaks corresponding to both disulfide-bonded and reduced peptides in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of disulfides could suggest that the samples are either mixtures prior to analysis or that the measurement process has converted single compounds into mixtures. This is an important distinction when characterizing potentially disulfide-bonded peptides obtained from proteolyzed proteins or from oxidized synthetic peptides. It is well documented that disulfides can undergo in-source decay (ISD) when using a 337-nm laser. However, the mixed matrix 2-(4-hydroxyphenylazo)benzoic acid:α-cyano-4-hydroxycinnamic acid (1:10) not only suppresses the ISD reduction of disulfides to thiols but allows the same low threshold laser power generally used with α-cyano-4-hydroxycinnamic acid to be applied.     

One-Way Allosteric Communication between the Two Disulfide Bonds in Tissue Factor

Tissue factor (TF) is a transmembrane glycoprotein that plays distinct roles in the initiation of extrinsic coagulation cascade and thrombosis. TF contains two disulfide bonds, one each in the N-terminal and C-terminal extracellular domains. The C-domain disulfide, Cys186-Cys209, has a ?RHStaple configuration in crystal structures, suggesting that this disulfide carries high pre-stress. The redox state of this disulfide has been proposed to regulate TF encryption/decryption. Ablating the N-domain Cys49-Cys57 disulfide bond was found to increase the redox potential of the Cys186-Cys209 bond, implying an allosteric communication between the domains. Using molecular dynamics simulations, we observed that the Cys186-Cys209 disulfide bond retained the ?RHStaple configuration, whereas the Cys49-Cys57 disulfide bond fluctuated widely. The Cys186-Cys209 bond featured the typical ?RHStaple disulfide properties, such as a longer S-S bond length, larger C-S-S angles, and higher bonded prestress, in comparison to the Cys49-Cys57 bond. Force distribution analysis was used to sense the subtle structural changes upon ablating the disulfide bonds, and allowed us to identify a one-way allosteric communication mechanism from the N-terminal to the C-terminal domain. We propose a force propagation pathway using a shortest-pathway algorithm, which we suggest is a useful method for searching allosteric signal transduction pathways in proteins. As a possible explanation for the pathway being one-way, we identified a pronounced lower degree of conformational fluctuation, or effectively higher stiffness, in the N-terminal domain. Thus, the changes of the rigid domain (N-terminal domain) can induce mechanical force propagation to the soft domain (C-terminal domain), but not vice versa.     

An Unconventional Role for Cytoplasmic Disulfide Bonds in Vaccinia Virus Proteins

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mercapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within 1 h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).     

Contribution of Disulfide Bonds to the Characteristics of Casein Micelles

Drainage in foam was analyzed by the capillary model proposed by Haas and Johnson. Foam was produced by introducing air into an ovalbumin solution through a spinneret. The electric conductivity at selected positions in the foam and the drained liquid height below the foam were measured at constant intervals. The measured electric conductivity was converted to the liquid volume fraction by Prager’s equation. The capillary model described well not only the liquid leakage rate from foam but also the liquid volume fraction in foam. The ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border was much larger than the value estimated from Haas and Johnson’s result. It was confirmed that a time constant, T, involved in the model was suitable for estimating foam stability only by one parameter. However, T is affected not only by material characteristics but also by the foam height. On the other hand, since the ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border reflects the material characteristics, it may be used for comparing the foam stability between materials with the same viscosity and density.     

Location of the Disulfide Bonds of the Sweetness-suppressing Polypeptide Gurmarin

The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gymnema sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing pep tides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at Cys3–Cys18, Cys10–Cys23, and Cys17–Cys33.     

Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds

Abstract: Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys⁷⁶⁴, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys⁷⁶⁴ is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.     

Influence of Disulfide Bonds on the Induction of Helical Conformation in Proteins

The effect(s) of TFE (2,2,2-trifluoroethanol) on three different conformational states (native, denatured, and carboxymethylated) of CTX III and RNase A has been examined. Contrary to the general belief, the results of the present study reveal that TFE can induce helical conformation in a protein which has no sequence propensity to form a helix. It is found that the helix induction in TFE is intricately related to the destabilization of the tertiary structural conformation in proteins. More importantly, the disulfide bonds in proteins are found to have significant influence on the TFE-mediated helix induction. The results obtained in this study strongly suggest that information pertaining to the influence of disulfide bonds on helix induction need to be considered to improve the accuracy of secondary structure prediction algorithms.     

The Role of Intermolecular Disulfide Bonds in Stabilizing the Structure of Peroxiredoxins

The comparative characterization of thermal stability of human peroxiredoxins 1–6 (Prx1–Prx6) has been performed by physicochemical and biochemical methods and the role of disulfide bonds in stabilizing their structure has been shown. Prx1 and Prx2 among the tested peroxiredoxins exhibit the highest peroxidase activity and thermal stability. Prx1 and Prx2 are more than 2 times more active on average with H₂O₂ and tert-butyl hydroperoxide as substrates compared to other peroxiredoxins and retain at least 50% activity after 30 min heating at a temperature of 64°C, which is more than 10°C higher compared to Prx3–Prx6. The reduction of the disulfide bonds between Prx1 and Prx2 leads to a decrease of their thermal stability, comparable to the thermal stability of Prx3–Prx6, which confirms the important role of the intermolecular S–S bonds in stabilizing the structure of these proteins.     

Force Generation by Microtubule Assembly/Disassembly in Mitosis and Related Movements

In this article, we review the dynamic nature of the filaments (microtubules) that make up the labile fibers of the mitotic spindle and asters, we discuss the roles that assembly and disassembly of microtubules play in mitosis, and we consider how such assembling and disassembling polymer filaments can generate forces that are utilized by the living cell in mitosis and related movements.     

A Thermodynamic Model of Microtubule Assembly and Disassembly

Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.     

Protein Disulfide Bonds Detected by Tagging with High Molecular Weight Maleimide Derivative

Molecular Biology - Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity...     

Reduction of Disulfide Bonds by Streptomyces pactum during Growth on Chicken Feathers

For disintegration of chicken feathers by Streptomyces pactum, keratinolytic proteinases and extracellular reduction of disulfide bonds were necessary. Conditions for disulfide reduction were examined with oxidized glutathione as model substrate. The reduction of glutathione depended on the presence of metabolically active cells. The mycelium also reduced tetrazolium dyes and cystine.