Disulfide-reducing Agent Research Articles

The Reducing Agent Dithiothreitol Modulates the Ventilatory Responses That Occur in Freely Moving Rats during and following a Hypoxic-Hypercapnic Challenge.

The present study examined the hypothesis that changes in the oxidation-reduction state of thiol residues in functional proteins play a major role in the expression of the ventilatory responses in conscious rats that occur during a hypoxic-hypercapnic (HH) gas challenge and upon return to room air. A HH gas challenge in vehicle-treated rats elicited robust and sustained increases in minute volume (via increases in frequency of breathing and tidal volume), peak inspiratory and expiratory flows, and inspiratory and expiratory drives while minimally affecting the non-eupneic breathing index (NEBI). The HH-induced increases in these parameters, except for frequency of breathing, were substantially diminished in rats pre-treated with the potent and lipophilic disulfide-reducing agent, L,D-dithiothreitol (100 µmol/kg, IV). The ventilatory responses that occurred upon return to room air were also substantially different in dithiothreitol-treated rats. In contrast, pre-treatment with a substantially higher dose (500 µmol/kg, IV) of the lipophilic congener of the monosulfide, N-acetyl-L-cysteine methyl ester (L-NACme), only minimally affected the expression of the above-mentioned ventilatory responses that occurred during the HH gas challenge or upon return to room air. The effectiveness of dithiothreitol suggests that the oxidation of thiol residues occurs during exposure to a HH gas challenge and that this process plays an essential role in allowing for the expression of the post-HH excitatory phase in breathing. However, this interpretation is contradicted by the lack of effects of L-NACme. This apparent conundrum may be explained by the disulfide structure affording unique functional properties to dithiothreitol in comparison to monosulfides. More specifically, the disulfide structure may give dithiothreitol the ability to alter the conformational state of functional proteins while transferring electrons. It is also possible that dithiothreitol is simply a more efficient reducing agent following systemic injection, although one interpretation of the data is that the effects of dithiothreitol are not due to its reducing ability.

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Mucolytic treatment of chronic rhinosinusitis in a murine model of primary ciliary dyskinesia.

Background: Genetic defects in motile cilia cause primary ciliary dyskinesia (PCD), a rare disease with no specific therapeutics. Individuals with PCD often have impaired fertility and laterality defects and universally suffer from upper and lower airway diseases. Chronic rhinosinusitis is a universal feature of PCD, and mucus accumulation and subsequent infections of the sinonasal cavity cause significant morbidity in individuals with PCD. Despite this, there are no approved treatments that specifically target mucus. Objective: The goals of this study were to determine whether computed tomography (CT) imaging could be used to quantify mucus accumulation and whether the use of a mucolytic agent to reduce disulfide cross-links present in mucins would improve the effectiveness of nasal lavage at removing mucus in a murine model of PCD. Methods: Adult mice with a deletion of the axonemal dynein Dnaic1 were imaged using CT scanning to characterize mucus accumulation. The animals were then treated by nasal lavage with saline, with/without the disulfide-reducing agent tris(2-carboxyethyl)phosphine. Post-treatment CT scans were used to quantify improvement in the sinonasal cavity. Results: Mucus accumulation in the nasal cavity was readily quantified by CT. Compared to sham-treated control animals, nasal lavage with/without a mucolytic agent resulted in a significant reduction of accumulated mucus (p < 0.01). Treatment with the mucolytic agent showed a greater reduction of accumulated mucus than treatment with saline alone. Conclusion: The results suggest that inclusion of a mucolytic agent may increase the effectiveness of nasal lavage at reducing mucus burden in PCD.

Serinol-Based Versatile Disulfide-Reducing Reagent.

Here, we report the synthesis of disulfide-reducing agents 2-(dibenzylamino) propane-1,3-dithiol (DPDT) and 2-(dibenzylamino)-2-methylpropane-1,3-dithiol (DMPDT) from serinol and methyl serinol, respectively. DPDT was found to show greater stability than DMPDT. Hence, the effectiveness of DPDT as a reducing agent was evaluated in both liquid and solid phases. The reducing capacity of this agent was comparable to that of DTT.

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Dithiol Based on l-Cysteine and Cysteamine as a Disulfide-Reducing Agent.

We report the synthesis, chemical properties, and disulfide bond-reducing performance of a dithiol called NACMEAA, conceived as a hybrid of two biologically relevant thiols: cysteine and cysteamine. NACMEAA is conveniently prepared from inexpensive l-cystine in an efficient manner. As a nonvolatile, highly soluble, and neutral compound at physiological pH with the first thiol pKa value of 8.0, NACMEAA is reactive and user-friendly. We also demonstrate that NACMEAA reduces disulfide bonds in GSSG and lysozyme.

Brine-Releasable Hydrogen Sulfide in Wine: Mechanism of Release from Copper Complexes and Effects of Glutathione.

Copper-sulfhydryl complexes in wine can be disrupted by addition of brine to release free hydrogen sulfide (H2S), and the resulting "brine-releasable H2S" is reported to correlate with formation of H2S during bottle storage. However, both the mechanism of the brine-release assay and factors affecting the stability of copper sulfhydryls under brine-release conditions are not well understood. By varying brine composition and concentration, it is shown that release of copper-complexed H2S requires the presence of a halide (Cl- and Br-) and is not due to a general "salting-out" effect. Release of copper-complexed H2S by the brine dilution assay is highly temperature-dependent. When H2S and Cu(II) are added to a model wine, brine-releasable H2S decreases markedly (∼10-fold) after a 20 min incubation period prior to performing the brine-release assay. In commercial wines, the fraction of added H2S recovered through the brine-release assay was correlated with the initial glutathione (GSH) concentration (r2 = 0.58) but not with initial Cu. Negligible additional release of H2S from organopolysulfanes was observed following addition of a disulfide-reducing agent (tris(2-carboxyethyl)phosphine). As previous studies have reported a correlation between H2S formed under brine-release conditions and normal storage, these results suggest that the susceptibility of a wine to form latent copper-sulfhydryl precursors of H2S following copper addition is dependent on the concentration of sulfhydryls like GSH.

Comparison of Hemoglobin Köln Erythrocyte Membranes With Malondialdehyde-Reacted Normal Erythrocyte Membranes

AbstractSplenectomized patients with hemoglobin (Hb) Köln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Köln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Köln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Köln RBC membranes and MDA-reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Köln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Köln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Köln RBCs. We conclude that Hb Köln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.

Breaking a Couple: Disulfide Reducing Agents.

Cysteine is present in a large number of natural and synthetic (bio)molecules. Although the thiol side chain of Cys can be in a free form, in most cases it forms a disulfide bond either with a second Cys (bridge) or with another thiol, as in the case of protecting groups. Efficient reduction of these disulfide bridges is a requirement for many applications of Cys-containing molecules in the fields of chemistry and biochemistry. Here we review reducing methods for disulfide bonds, taking into consideration the solubility of the substrates when selecting the appropriate reducing reagent.

2-(Dibenzylamino)butane-1,4-dithiol (DABDT), a Friendly Disulfide-Reducing Reagent Compatible with a Broad Range of Solvents.

Disulfide-reducing agents play a crucial role in bioconjugate chemistry. Herein, we describe the synthesis of a new disulfide-reducing agent [2-(dibenzylamino)butane-1,4-dithiol (DABDT)] that is conveniently prepared from inexpensive aspartic acid. Being nonmalodorous and highly soluble in a broad range of solvents, DABDT is user-friendly. We have demonstrated its performance both in solution and in the solid phase.

Beyond amino acid sequence: disulfide bonds and the origins of the extreme amyloidogenic properties of insulin's H-fragment.

The presence of disulfide bonds affects the protein stability and therefore tendency to misfold and form amyloid-like fibrils. Insulin's three disulfide bridges stabilize the native state and prevent aggregation. Partial proteolysis of insulin releases highly amyloidogenic and inherently disordered two-chain 'H-fragment' retaining insulin's Cys7A-Cys7B and Cys6A-Cys11A disulfide bonds. The abrupt self-association of H-fragment monomers into fibrils is suppressed in the presence of disulfide-reducing agent. These circumstances make the H-fragment an interesting model to study the impact of disulfide bonds on amyloidogenesis beyond the 'stabilization-of-the-native-state' paradigm. Here, we investigate fibrillization of various synthetic peptides derived from the H-fragment through modifications of Cys7A-Cys7B/Cys6A-Cys11A bonds. In comparison to H-fragment, aggregation of a two-chain 'AB' analog lacking Cys6A-Cys11A bond is decelerated, while the alternative removal of Cys7A-Cys7B bond releases a non-aggregating B-chain and a highly amyloidogenic 'ACC' fragment containing the intrachain Cys6A-Cys11A bond. Our analysis, supported by calculations of configurational entropy, suggests that Cys6A-Cys11A bond is a key factor behind the explosive self-association of H-fragment. The bond restricts the conformational space probed by nucleating monomers which is reflected by an approximately 2.4 kJ·mol-1 K-1 decrease in entropy. The fact that the intact Cys6A-Cys11A bond promotes fibrillization of the H-fragment is remarkable in light of the previously established role of the same disulfide bond in preventing formation of insulin fibrils. Our results imply that a single disulfide bond within a folded protein and its fragment may play entirely different roles in aggregation and that this role may evolve with progressing phases of misfolding.

Cytosolic increased labile Zn2+ contributes to arrhythmogenic action potentials in left ventricular cardiomyocytes through protein thiol oxidation and cellular ATP depletion

Intracellular labile (free) Zn2+-level ([Zn2+]i) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn2+]i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn2+]i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn2+]i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn2+]i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn2+]i induced inhibitions in voltage-dependent K+-channel currents, such as transient outward K+-currents, Ito, steady-state currents, Iss and inward-rectifier K+-currents, IK1, reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both Iss and IK1, while no effect on Ito. However, the increased-[Zn2+]i could induce marked activation in ATP-sensitive K+-channel currents, IKATP, depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn2+]i with no change in mRNA level of Kv4.2, while the mRNA level of IKATP subunit, SUR2A was increased significantly with increased-[Zn2+]i, being reversible with DTT. Overall we demonstrated that high-[Zn2+]i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K+-currents, although activation of IKATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn2+]i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress.

Facile Fabrication of a Modular "Catch and Release" Hydrogel Interface: Harnessing Thiol-Disulfide Exchange for Reversible Protein Capture and Cell Attachment.

Surfaces engineered to "specifically capture" and "release on demand" analytes ranging from biomolecules to cells find niche applications in areas such as diagnostics and detection. Utilization of a disulfide-based linker as a building block allows fabrication of a novel hydrogel-based platform that incorporates a "catch and release" attribute. Hydrogels incorporating pyridyl disulfide groups as thiol-reactive handles were prepared by photopolymerization in the presence of a poly(ethylene glycol) (PEG)-based cross-linker. A range of bulk and micropatterned hydrogels with varying amounts of the reactive group were prepared using PEG-based monomers with different chain lengths. Thiol-containing molecules were conjugated to these hydrogels through the thiol-disulfide exchange reaction under ambient conditions with high efficiencies, as determined by UV-vis spectroscopy. Facile conjugation of a thiol-containing fluorescent dye, namely 4,4-difluoro-1,3,5,7-tetramethyl-8-[(10-mercapto)]-4-bora-3 a,4 a-diaza- s-indacene, was demonstrated, followed by its effective cleavage in the presence of dithiothreitol (DTT), a thiol-containing disulfide-reducing agent. Conjugation of a biotin-containing ligand onto the hydrogels allowed specific binding of protein extravidin when exposed to a mixture of extravidin and bovine serum albumin. The bound protein could be released from the hydrogel by simple exposure to a DTT solution. Likewise, hydrogels modified with a cell-adhesive peptide unit containing the RGD sequence acted as favorable substrates for cellular attachment. Incubation of these cell-attached hydrogel surfaces in a DTT-containing solution leads to facile detachment of cells from the surfaces, while retaining a high level of cell viability. It can be envisioned that the benign nature of these hydrogels, their facile fabrication, and modular functionalization will make them attractive platforms for many applications.

Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography.

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using β2-microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule).

Inactivation of human DGAT2 by oxidative stress on cysteine residues.

Diacylglycerol acyltransferases (DGATs) have a crucial role in the biosynthesis of triacylglycerol (TG), the major storage form of metabolic energy in eukaryotic organisms. Even though DGAT2, one of two distinct DGATs, has a vital role in TG biosynthesis, little is known about the regulation of DGAT2 activity. In this study, we examined the role of cysteine and its oxidation in the enzymatic activity of human DGAT2 in vitro. Human DGAT2 activity was considerably inhibited not only by thiol-modifying reagents (NEM and IA) but also by ROS-related chemicals (H2O2 and β-lapachone), while human DGAT1 and GPAT1 were little affected. Particularly, ROS-related chemicals concomitantly induced intermolecular disulfide crosslinking of human DGAT2. Both the oxidative inactivation and disulfide crosslinking were almost completely reversed by the treatment with DTT, a disulfide-reducing agent. These results clearly demonstrated the significant role of ROS-induced intermolecular crosslinking in the inactivation of human DGAT2 and also suggested DGAT2 as a redox-sensitive regulator in TG biosynthesis.

Fourier transform infrared spectroscopic analysis of sperm chromatin structure and DNA stability.

Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and packing. Spectroscopic studies were corroborated with flow cytometric analyses using the DNA-intercalating fluorescent dye acridine orange. Decreased fertility of individuals correlated with increased abnormal sperm morphology and decreased stability toward induced DNA damage. Treatment with the disulfide reducing agent dithiothreitol resulted in increased sperm chromatin decondensation and DNA accessibility, similar as found for less mature epididymal spermatozoa. In situ infrared spectroscopic analysis revealed that characteristic bands arising from the DNA backbone (ν1230, ν1086, ν1051cm(-1) ) changed in response to induced oxidative damage, water removal, and decondensation. This coincided with changes in the amide-I region (intensity at ν1620 vs. ν1640cm(-1) ) denoting concomitant changes in protein secondary structure. Reduction in protein disulfide bonds resulted in a decreased value of the asymmetric to symmetric phosphate band intensity (ν1230/ν1086cm(-1) ), suggesting that this band ratio is sensitive for the degree of chromatin condensation. Moreover, when analyzing spermatozoa from different individuals, it was found that the asymmetric/symmetric phosphate band ratio negatively correlated with the percentage of morphologically abnormal spermatozoa.

Preformed Seeds Modulate Native Insulin Aggregation Kinetics.

Insulin aggregates under storage conditions via disulfide interchange reaction. It is also known to form aggregates at the site of repeated injections in diabetes patients, leading to injection amyloidosis. This has fueled research in pharmaceutical and biotechnology industry as well as in academia to understand factors that modulate insulin stability and aggregation. The main aim of this study is to understand the factors that modulate aggregation propensity of insulin under conditions close to physiological and measure effect of "seeds" on aggregation kinetics. We explored the aggregation kinetics of insulin at pH 7.2 and 37 °C in the presence of disulfide-reducing agent dithiothreitol (DTT), using spectroscopy (UV-visible, fluorescence, and Fourier transform infrared spectroscopy) and microscopy (scanning electron microscopy, atomic force microscopy) techniques. We prepared insulin "seeds" by incubating disulfide-reduced insulin at pH 7.2 and 37 °C for varying lengths of time (10 min to 12 h). These seeds were added to the native protein and nucleation-dependent aggregation kinetics was measured. Aggregation kinetics was fastest in the presence of 10 min seeds suggesting they were nascent. Interestingly, intermediate seeds (30 min to 4 h incubation) resulted in formation of transient fibrils in 4 h that converted to amorphous aggregates upon longer incubation of 24 h. Overall, the results show that insulin under disulfide reducing conditions at pH and temperature close to physiological favors amorphous aggregate formation and seed "maturity" plays an important role in nucleation dependent aggregation kinetics.

Voltage-Induced Misfolding of Zinc-Replete ALS Mutant Superoxide Dismutase-1.

The monomerization of Cu, Zn superoxide dismutase (SOD1) is an early step along pathways of misfolding linked to amyotrophic lateral sclerosis (ALS). Monomerization requires the reversal of two post-translational modifications that are thermodynamically favorable: (i) dissociation of active-site metal ions and (ii) reduction of intramolecular disulfide bonds. This study found, using amide hydrogen/deuterium (H/D) exchange, capillary electrophoresis, and lysine-acetyl protein charge ladders, that ALS-linked A4V SOD1 rapidly monomerizes and partially unfolds in an external electric field (of physiological strength), without loss of metal ions, exposure to disulfide-reducing agents, or Joule heating. Voltage-induced monomerization was not observed for metal-free A4V SOD1, metal-free WT SOD1, or metal-loaded WT SOD1. Computational modeling suggested a mechanism for this counterintuitive effect: subunit macrodipoles of dimeric SOD1 are antiparallel and amplified 2-fold by metal coordination, which increases torque at the dimer interface as subunits rotate to align with the electric field.

Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin.

Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.

Effects of hesperidin, a flavanone glycoside interaction on the conformation, stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies?

Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP–HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (Ka, ~ 5 × 104 M−1) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.