• Korean Chemical Engineering Research
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• v.55 no.1
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• pp.27-33
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• 2017

The determination of disulfide bonds is important for comprehensive understanding of the chemical structure of protein. So far, many strategies for the disulfide bond analysis have been suggested in terms of speed and sensitivity. However, most of these strategies have not considered free thiol residues in the target protein in the process of determining the disulfide bond. We suggested the strategy which was composed of four steps for the identification of disulfide bonds; the first step was the prediction of possible disulfide bonds, the second step was the determination of free cysteine residues, the third step was the analysis of disulfide bond using a high-resolution mass spectrometry, and the final step was the determination of disulfide bonds based on the comprehensive verification. In this study, we performed the characterization of disulfide bonds for the recombinant protein (HRPE1), where 1 and 5 inter- and intra-chain disulfide bonds were identified, respectively.

• Food Science and Preservation
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• v.5 no.1
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• pp.65-71
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• 1998

The changes in gel characteristics of soy protein as a result of various reagents that alter specific interactions which affect the formation and textural properties of gels, were studied. The reagents were added to 15% soy protein solutions prior to heat treatment. The gels were not formed with urea, indicating that hydrogen bonds significantly contributed to the formation and hardness of soy protein gel. Hydrophobic interactions and disulfide bonds compensated for hydrogen bonds and the contributions of electrostatic interactions to gel hardness are relatively insignificant. The farce primarily responsible for gel cohesiveness appeared to be disulfide bonds, because a significant decrease in cohesiveness was found only with the presence of N-ethylmaleimide. Adhesiveness decreased only with the addition of urea, and thus the contribution of hydrogen bonding to adhesiveness of gel could be concluded to be resent. However, adhesiveness was suggested to be interpreted not only wile molecular forces involved in gel formation but also with hydration properties of protein.

• BMB Reports
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• v.47 no.11
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• pp.625-630
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• 2014

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin's ${\alpha}$-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin's ${\alpha}$-helical region is highly homologous to those of other insect defensins.

• Textile Coloration and Finishing
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• v.2 no.2
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• pp.14-19
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• 1990

The setting behavior of disulfide-enriched wool and disulfide-crosslinked silk obtained by treatment with a disulfide-containing crosslinking agent, bis $(\beta-isocyanatoethyl)$ dissulfide was compared with that of untreated wool and silk under the absence and the presence of a reducing agent. Rearrangment of secondary bonds facilitated by cleavage of crosslinks as well as rearrangement of crosslinks itself seems to play an important role in the set stability.

• Textile Coloration and Finishing
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• v.1 no.1
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• pp.1-6
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• 1989

The reaction of silk with a disulfide-containing crosslinking agent, i.e. bis($\beta$-isocyanatoethyl)disulfide(BIED), was studied in an attempt to obtain disulfide-crosslinked silk. The setting properties of disulfide-crosslinked silk fibers were studied. The permanent set values of single fibers were evaluated after the set fibers were relaxed in boiling water. When single fibers were set in boiling water or in boiling alkaline solution, the permanent set values of BIED-treated silk fibers were less than those of untreated silk fibers. When the fibers were treated with 2% thioglycolic acid solution at $60^\circ{C}$ followed by oxidation, settability of BIED-treated silk was better than that of untreated silk. The rearrangement of secondary bonds faciliated by cleavage of crosslinks as well as the rearrangement of crosslinks itself seems to be an important role in the set stability.

• Mass Spectrometry Letters
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• v.1 no.1
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• pp.5-8
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• 2010

In the present study, collisionally-activated dissociation (CAD) experiments were performed under low energy collision conditions in six peptides containing a disulfide bond. Fragments produced as a result of the cleavage of a disulfide bond were obtained after CAD in four peptides (bactenecin, TGF-$\alpha$, cortistantin, and linearly linked peptide, Scheme 1) with basic amino acid residues. In contrast, the CAD analysis of two peptides with no basic residue (oxytocin and tocinoic acid) rarely produced fragments indicative of cleavage of a disulfide bond. These results are consistent with the mobile proton model suggested by the McLuckey and O'air groups (ref. 22 and 23); nonmobile protons sequestered at basic amino acid residues appear to promote the cleavage of disulfide bonds.

• Molecules and Cells
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• v.44 no.10
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• pp.758-769
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• 2021

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I_CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I_CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I_CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I_CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

• Journal of Life Science
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• v.11 no.5
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• pp.415-422
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• 2001

Many secreted proteins have disulfide bonds that are important for their structure and function. Protein disulfide isomerase (PDI, EC 5.3.1.4.), an enzyme that catalyzes the formation and rearrangement of thiol/disulfide exchange reactions, is a resident of the endoplasmic reticulum (ER). The subcellular localization and its function as catalyst of disulfide bond formation in the biosynthesis of secretory and cell membrane proteins suggest that PDI plays a key role in the secretory pathway. We have isolated a cDNA encoding protein disulfide isomerase from Bombyx mori(bPDI). It has been characterized under ER stress conditions (dominantly induced by calcium ionophore A23187, tunicamycin and DTT), which is known to cause an accumulation of unfolded proteins in the ER. Furthermore, It has also been examined for tissue distribution(pronounced at the fat body), hormonal regulation (juvenile hormone, insulin and juvenile +transferrin; however, it is not effected by transferrin alone), and the effect of exogenous bacteria (peak at 16 h after infection) on the bPDI mRNA expression. The results suggest that bPDI is a member of the ER stress protein group, and it may play an important role in exogenous bacterial infection in fat body, and that homones regulate its expression.

• Molecules and Cells
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• v.24 no.2
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• pp.261-267
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• 2007

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.

• BMB Reports
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• v.50 no.8
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• pp.401-410
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• 2017

The protein disulfide isomerase (PDI) family is a group of multifunctional endoplasmic reticulum (ER) enzymes that mediate the formation of disulfide bonds, catalyze the cysteine-based redox reactions and assist the quality control of client proteins. Recent structural and functional studies have demonstrated that PDI members not only play an essential role in the proteostasis in the ER but also exert diverse effects in numerous human disorders including cancer and neurodegenerative diseases. Increasing evidence suggests that PDI is actively involved in the proliferation, survival, and metastasis of several types of cancer cells. Although the molecular mechanism by which PDI contributes to tumorigenesis and metastasis remains to be understood, PDI is now emerging as a new therapeutic target for cancer treatment. In fact, several attempts have been made to develop PDI inhibitors as anti-cancer drugs. In this review, we discuss the properties and diverse functions of human PDI proteins and focus on recent findings regarding their roles in the state of diseases including cancer and neurodegeneration.