Role of disulfide bonds in the structure and activity of human insulin

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Electrostatic influence of local cysteine environments on disulfide exchange kinetics

The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.     

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.     

Enzyme reduction of disulfide bonds by thioredoxin. The reactivity of disulfide bonds in human choriogonadotropin and its subunits.

The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, alpha and beta, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12 muM thioredoxin and 0.1 muM thioredoxin reductase at pH 7 all disulfide bonds in the alpha subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the alpha subunit with thioredoxin followed by S-carboxymethylation with iodol[2-3H]acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the alpha subunit were surface oriented and equally reactive. The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.     

Protein disulfide isomerase-mediated disulfide bonds regulate the gelatinolytic activity and secretion of matrix metalloproteinase-9

Matrix metalloproteinase-9 (MMP-9) is one of the major MMPs that can degrade extracellular matrix. Besides normal physiological functions, MMP-9 is involved in metastasis and tumor angiogenesis. Although several inhibitors of MMP-9 have been identified, in vivo regulators of MMP-9 activation are unknown. In the present study we intended to investigate novel therapeutic target protein(s) that regulate MMP-9 activation and/or secretion. We have identified protein disulfide isomerase as a novel upstream regulator of MMP-9. Mass spectrometric analysis of post-translational modification in MMP-9 confirmed six disulfide bonds in the catalytic domain and one disulfide bond in the hemopexin domain of MMP-9. Establishment of cells that overexpressed wild-type and mutant forms of MMP-9 revealed that 'cysteine-switch' and disulfide bonds within the catalytic domain are necessary for the secretion and intracellular trafficking of MMP-9. However, the disulfide bond of the hemopexin domain and other cysteines have no significant role in secretion. These insights into the secretion of MMP-9 constitute the basis for the development of potential drugs against metastasis.     

Salicylanilide diethyl phosphates: Synthesis,antimicrobial activity and cytotoxicity

A series of 27 salicylanilide diethyl phosphates was prepared as a part of our on-going search for new antimicrobial active drugs. All compounds exhibited in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium strains, with minimum inhibitory concentration (MIC) values of 0.5–62.5 μmol/L. Selected salicylanilide diethyl phosphates also inhibit multidrug-resistant tuberculous strains at the concentration of 1 μmol/L. Salicylanilide diethyl phosphates also exhibited mostly the activity against Gram-positive bacteria (MICs ⩾1.95 μmol/L), whereas their antifungal activity is significantly lower. The IC₅₀ values for Hep G2 cells were within the range of 1.56–33.82 μmol/L, but there is no direct correlation with MICs for mycobacteria.     

Optical activity of disulfide bonds in proteins. 1. Studies on plasmin modified human somatotropin.

The reduction and alkylation of the two disulfide bonds in a preparation of human pituitary growth hormone which had been previously modified by limited proteolysis with the enzyme plasmin have been studied. Quantitative and selective reduction of the carboxyl-terminal disulfide, as well as total reduction of both disulfides, has been achieved in the absence of denaturants. Circular dichroism spectra of the various reduced and reduced-alkylated derivatives have provided sufficient information to allow an estimation of the individual contributions of each disulfide bond to the total optical activity of the protein. These contributions were found to represent a significant portion of the total optical activity between 290 and 250 nm. The carboxyl-termimal bond exhibits negative dichroism with an apparent center near 258 nm ([theta]M,258nm = 2100 deg cm2 dmol-1). By comparison, the contribution of the remaining disulfide is red-shifted to 273 nm, is also negative in sign, and somewhat more intense ([theta]M,273nm = 3200 deg cm2 dmol-1). Circular dichroism measurements have also been used to approximate the rate of reduction of the protein.     

Identification and Biological Characterization of Mouse beta-defensin 14, the orthologue of human beta-defensin 3

beta-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, it became evident that certain members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. We have identified mouse beta-defensin 14 (mBD14, Defb14) as an orthologue of human beta-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cystein linkages, characteristic for the beta-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Interestingly, we also detected mBD14 expression in immature CD11c+ bone marrow-derived dendritic cells. The expression of mBD14 can be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-gamma. Furthermore, expression of mBD14 seems to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. We prepared a recombinant mBD14-Ig fusion protein that retained potent antimicrobial activity against several Escherichia coli strains but not against various Gram-positive Staphylococcus aureus strains. hBD3 and also the newly identified mBD14 were chemotactic for cells expressing the mouse CC-chemokine receptor CCR6. In addition, both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, mBD14, based on structural and functional similarities, appears to be an orthologue of hBD3.     

Influences of disulfide connectivity on structure and antimicrobial activity of tachyplesin I

Tachyplesin I is a potent antimicrobial peptide with broad spectrum of antimicrobial activity. It has 2 disulfide bonds and can form 3 disulfide bond isomers. In this study, the structure and antimicrobial activity of 3 tachyplesin I isomers (tachyplesin I, 3C12C, 3C7C) were investigated using molecular dynamic simulations, circular dichroism structural study, as well as antimicrobial activity and hemolysis assay. Our results suggest that in comparison to the native peptide, the 2 isomers (3C12C, 3C7C) have substantial structural and activity variations. The native peptide is in the ribbon conformation, while 3C12C and 3C7C possess remarkably different secondary structures, which are referred as “globular” and “beads” isomers, respectively. The substantially decreased hemolysis effects for these 2 isomers is accompanied by significantly decreased anti‐gram‐positive bacterial activity.     

Antibacterial activities and conformations of bovine beta-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil beta-defensin BNBD-12, GPLSC(1)GRNGGVC(2)IPIRC(3) PVPMRQIGTC(4) FGRPVKC(5) C(6)RSW with disulfide connectivities C(1)-C(5), C(2)-C(4) and C(3)-C(6) and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt beta-hairpin or beta-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid beta-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in beta-defensins.     

Effect of partial reduction of disulfide bonds on the enzymatic activity of thrombin

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity

BackgroundThimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 μg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains.MethodsThe safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations.ResultsThe viability of all examined cell lines was suppressed entirely in the presence of 4.6 μg/ml (12.5 μM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 μg/ml (5.5, 4.3, 2.7 and 0.8 μM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 μg/ml (7.1, 8.5, 3.5 and 2.4 μM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 μM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 μM) for Aspergillus brasiliensis.ConclusionAccording to our results Thimerosal should be present in culture media at 100 μg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 μg/ml (12.5 μM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.