Cysteine Peptides Research Articles

Zinc(ii) and cadmium(ii) complexes of N-terminally free peptides containing two separate cysteinyl binding sites

The novel synthesized cysteine peptides form stable zinc(ii) and cadmium(ii) complexes; the specific sequence makes possible metal induced amide deprotonation.

Effect of Ingestion of Supplements Containing Cystine and Cysteine Peptide Yeast Extract on Skin of Japanese Women: a Double-blind Randomized Controlled Trial

【目的】本試験は, シスチン・システインペプチド酵母エキス混合物含有サプリメントの12週間摂取が女性の肌に及ぼす影響を検証した。【方法】二重盲検ランダム化比較試験。日本人女性にシスチン・システインペプチド酵母エキス混合物含有サプリメントもしくはプラセボ食品を12週間摂取させ, 4週間ごとに肌環境を評価した。【結果】シスチン・システインペプチド酵母エキス混合物群 (CC群) とプラセボ群を比較した結果, 摂取4週後に赤味評価 (面積Lv3) に有意差が認められ, CC群の方が低い測定値を示した (p＝0.021) 。【結論】シスチン・システインペプチド酵母エキス混合物含有サプリメントの摂取は, 摂取4週後に肌の赤みに前向きな影響を与えたといえる。本試験は, 倫理委員会の承認後, 臨床試験登録システムUMIN-CTRに登録し実施した (UMIN000013167) 。

A Comprehensive One-Pot Synthesis of Protected Cysteine and Selenocysteine SPPS Derivatives

A proof-of-principle methodology is presented in which all commercially-available cysteine (Cys) and selenocysteine (Sec) solid phase peptide synthesis (SPPS) derivatives are synthesized in high yield from easily prepared protected dichalcogenide precursors. A Zn-mediated biphasic reduction process applied to a series of four bis-N(α)-protected dichalcogenide compounds allows facile conversion to their corresponding thiol and selenol intermediates followed by insitu S- or Se-alkylation with various electrophiles to directly access twenty one known Cys and Sec SPPS derivatives. Most of these derivatives were able to be precipitated in crude form out of petroleum ether in sufficient purity for direct use as peptide building blocks. Subsequent incorporation of these derivatives into peptide models nicely illustrates their viability and applicability toward SPPS.

Preparation and Functional Exploration of Cysteine Peptides from Fresh Garlic Scales for Improving Bioavailability of Food Legume Iron and Zinc

Abstract Two γ-glutamyl-cysteine peptides (γ-GCPs), ( S C2 R C7)-γ- L -glutamyl- S -allyl- L -cysteine ( 1 ) and ( S C2 R C7)-γ- L -glutamyl- S -propyl- L -cysteine ( 2 ) were isolated from fresh garlic scales using ion-exchange chromatography and preparative HPLC. Their molecular structures were identified by HPLC-MS, CD, 1 H NMR, 13 C NMR, and specific rotation, and confirmed by using corresponding standard compounds. The influence of exogenously adding 1 and 2 on the bioavailability of iron and zinc from food legume was examined with soybean and mung bean, at the level of 0.01 mmol/5 g of legume respectively. The enhancing effect of the two γ-GCPs on bioavailability of iron was generally evidenced in the case of soybean (from 1.88% to 6.73% and 4.42%) and mung bean (from 2.52% to 12.04% and 9.38%). The two γ-GCPs similarly enhanced the bioavailability of zinc from the food legume, with the extent of increase in soybean ranging from 13.37% to 23.95% and 20.58%, and in mung bean from 15.98% to 28.44% and 27.05%. Obviously, both 1 and 2 had a promoting effect on the bioavailability of iron and zinc from food legumes. These findings are of practical value in a food-based strategy to enhance the bioavailability of trace minerals for human health.

Comparison of HNO reactivity with tryptophan and cysteine in small peptides

Recent discoveries of important pharmacological properties have drawn attention to the reactivity of HNO (azanone, nitroxyl) with biologically relevant substrates. Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example, with tryptophan resulting in N-nitrosotryptophan formation. We have investigated the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. Our results point to the more reactive nature of cysteine towards HNO compared with tryptophan.

Semisynthesis of a post-translationally modified protein by using chemical cleavage and activation of an expressed fusion polypeptide.

Herein, we describe a new semisynthetic strategy of a post-translationally modified protein in which the middle region is glycosylated. We designed a single-plasmid coding for a fusion polypeptide, which can provide both an N-terminal α-thioester and a C-terminal cysteine peptide of a target glycoprotein by using chemical-cleavage and activation methods. The use of these resultant peptide derivatives resulted in the successful synthesis of N-glycosylated-interleukin 13.

Peptide macrocyclization through amide-to-amide transpeptidation

Cyclic cysteine peptides are peptide macrocycles endowed with enhanced metabolic stability and potentially, with membrane permeability. They have attracted attention in drug design and interest in their synthesis. The chemical approach for macrocyclization through transpeptidation bears striking similarity to the biological approach using an intein. Both use a similar design of thioester precursors and an amide-to-amide transpeptidation scheme, employing a series of acyl shifts to break and make amide bonds. Here we describe the synthesis of two cyclic cysteine peptides, hedyotide B1 and sunflower trypsin inhibitor-1, highlighting the similarities between the intein-based and chemical amide-to-amide schemes. In our intein-based and chemical schemes, we employed an intein Mxe or a thioethylbutylamido linkage at the C-terminus of their linear precursors, respectively. Our results demonstrated that the chemical approach provides a useful alternative to the intein approach with high efficiency.

Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins

AbstractIn this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent‐labeling studies demonstrated that the installation of a C‐terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site‐specific labeling of cysteine‐containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.

Allenamides as orthogonal handles for selective modification of cysteine in peptides and proteins.

In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site-specific labeling of cysteine-containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.

Arsinous acid as a thiol binding group: potential cysteine peptide tagging functionality that binds a single thiol

A simple aryl arsinous acid (ArAs(CH3)OH) was prepared byorthomercuration ofp-cresol followed by Pd-catalyzed reaction with methylarsenic dibromide, purification as the mercaptoethanol adduct, and deprotection using a silver salt.

Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential

Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients. Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5′-dithiobis-2-nitrobenzoic acid or fluorescamine™ as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides. The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient.

Alternative biomarkers of n-hexane exposure: Characterization of aminoderived pyrroles and thiol-pyrrole conjugates in urine of rats exposed to 2,5-hexanedione

The identification of pyrrole derivatives in urine of rats exposed to 2,5-hexanedione (2,5-HD), was performed to select an adequate peripheral biomarker predictive of 2,5-HD neurotoxicity. Studies on molecular mechanism of 2,5-HD neurotoxicity have revealed that 2,5-hexanedione reacts with free amino groups of lysine in proteins forming primary pyrrole adducts, which may autoxidize and form pyrrole dimers, responsible for protein crosslinking in neurofilaments, or react with sulfhydryl groups of cysteine in peptides and proteins, forming secondary pyrrole adducts, which probably may inhibit the process responsible by 2,5-HD neurotoxicity. In this work, the analysis of excreted 2,5-HD and pyrrole derivatives in urine of rats i.p. treated with 3 doses of 2,5-HD (400mg/kg bw/48h) was performed using ESI-LC–MS/MS. Several pyrrole compounds were identified, namely dimethylpyrrole norleucine (DMPN), cysteine-pyrrole conjugate (DMPN NAC), glutathione-pyrrole conjugate (DMPN GSH) and 2,5-dimethylpyrrole (2,5-DMP). Additionally, free and total 2,5-HD, DMPN and DMPN NAC were quantified. The observed results suggest that DMPN is a sensitive and specific indicator of repeated exposure to 2,5-HD.

Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei . LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrio alginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V . alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibrio penaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.

Glutathione reductase activity with an oxidized methylated glutathione analog

The activity of glutathione reductase with an unnatural analog of oxidized glutathione was explored. The analog, L-γ-glutamyl-2-methyl-L-cysteinyl-glycine disulfide, places an additional methyl group on the alpha position of each of the central cysteine residues, which significantly increases steric bulk near the disulfide bond. Glutathione reductase was completely unable to catalyze the sulfur–sulfur bond reduction of the analog. Additionally, enzyme kinetics experiments indicated that the analog acts as a competitive inhibitor of glutathione reductase. Computational studies confirm that the methylated analog fits within the active site of the enzyme but its disulphide bond geometry is altered, preventing reduction by the enzyme. The substitution of (R)-2-methylcysteine in place of natural (R)-cysteine in peptides constitutes a new strategy for stabilizing disulphide bonds from enzyme-catalyzed degradation.

Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows

Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.

Stable Conjugates of Peptides with Gold Nanorods for Biomedical Applications with Reduced Effects on Cell Viability

Gold nanorods used in therapy and diagnosis must be nontoxic and stable in biological media and should be specific for the target. The complete combination of these three factors has hindered the use of gold nanorods as carriers in biological and biomedical applications. In this study, we produced a conjugate of gold nanorods with the peptide CLPFFD that recognizes toxic β-amyloid aggregates present in Alzheimer's disease, demonstrates colloidal stability, maintains plasmonic properties, and shows no effects on cell viability in the SH-SY5Y cell line. Furthermore, the irradiation of β-amyloid in the presence of the conjugate with near-infrared region irradiation energy reduces the amyloidogenic process reducing also its cytotoxicity. The nanorods were synthesized following the seed-mediated method in cetyltrimethylammonium bromide (CTAB) and were conjugated with the N-terminal cysteine peptide, CLPFFD. The conjugate was exhaustively characterized using different techniques (Absorption spectroscopy, X-ray photoelectron spectroscopy, electron energy loss spectroscopy, and zeta potential). The effects on cell viability and cell penetration by transmission electron microscopy of the conjugate were evaluated. The chemisorption of the peptide on the surface of gold nanorods increases their stability and reduces their effects on cell viability.

Pressurized whey protein can limit bacterial burden and protein oxidation in Pseudomonas aeruginosa lung infection

BackgroundLung infection caused by Pseudomonas aeruginosa is associated with an exuberant inflammatory response, oxidative stress, and lung damage. Whey protein is a rich source of cysteine, and anti-inflammatory and immune-enhancing peptides. Anti-inflammatory and antioxidant properties of whey are augmented by hyperbaric pressure treatment. In this study, we tested whether dietary supplementation with pressurized whey protein enhances the host ability to clear P. aeruginosa infection compared with native (i.e., unpressurized) whey. MethodsUsing a minimally invasive, non-lethal model of murine (female C57Bl/6) model of P. aeruginosa infection (mucoid strain embedded in agar beads), we studied kinetics of infection, inflammation, and oxidative stress at d 1, 3, and 7 postinfection. A parallel set of mice were fed for 4 wk a semipurified diet containing either native or pressurized whey and subsequently infected with P. aeruginosa. In these mice, the parameters mentioned previously were studied at d 1 and 3 postinfection. ResultsInfection with P. aeruginosa resulted in inflammation and protein oxidation sustained beyond bacterial clearance. Animals that were fed pressurized whey had fewer bacteria at day 3 than mice on native whey. Weight loss or broncho-alveolar lavage cell content were comparable. Airway protein oxidation was attenuated, whereas airway leukocyte bacterial killing ability and oxidative burst in response to opsonized bacteria were increased in the pressurized whey–fed animals. ConclusionsUse of nutritionally derived substances with anti-inflammatory and antioxidant properties, such as pressurized whey, aids in limiting airway bacterial infection, particularly, under conditions of ongoing oxidative stress.

S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

S-Acyl cysteine peptides containing α-, β- or γ-amino acid residues undergo long-range S- to N-acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S → N-acyl transfer relative to intermolecular transacylation is favored increasingly for 9 < 12 < 13 ~ 10-membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations.

The selective peptide reactivity of chemical respiratory allergens under competitive and non-competitive conditions

It is well established that certain chemicals cause respiratory allergy. In common with contact allergens, chemicals that induce sensitization of the respiratory tract must form stable associations with host proteins to elicit an immune response. Measurement of the reactivity of chemical allergens to single nucleophilic peptides is increasingly well-described, and standardized assays have been developed for use in hazard assessment. This study employed standard and modified peptide reactivity assays to investigate the selectivity of chemical respiratory allergens for individual amino acids under competitive and non-competitive conditions. The reactivity of 20 known chemical respiratory sensitizers (including diisocyanates, anhydrides, and reactive dyes) were evaluated for reactivity towards individual peptides containing cysteine, lysine, histidine, arginine, or tyrosine. Respiratory allergens exhibited the common ability to deplete both lysine and cysteine peptides; however, reactivity for histidine, arginine, and tyrosine varied between chemicals, indicating differences in relative binding affinity toward each nucleophile. To evaluate amino acid selectivity for cysteine and lysine under competitive conditions a modified assay was used in which reaction mixtures contained different relative concentrations of the target peptides. Under these reaction conditions, the binding preferences of reference respiratory and contact allergens (dinitrochlorobenzene, dinitrofluorobenzene) were evaluated. Discrete patterns of reactivity were observed showing various levels of competitive selectivity between the two allergen classes.

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol-containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide-α-thioester and an N-terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160-fold) in its fluorescence ratio (I(458)/I(603)). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.