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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #1)

Sulfur- and seleno-containing amino acids by Maria Wróbel; Martha H. Stipanuk; Noriuki Nagahara (pp. 1-2).

Abstracts presented at the 12th International Congress on Amino Acids, Peptides and Proteins (pp. 1-86).

Remembering Professor Toshihiko Ubuka (1934–2008) by Maria Wróbel; Takayoshi Ubuka; Satoko Ubuka (pp. 3-5).

Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents by Arthur J. L. Cooper; Boris F. Krasnikov; Zoya V. Niatsetskaya; John T. Pinto; Patrick S. Callery; Maria T. Villar; Antonio Artigues; Sam A. Bruschi (pp. 7-27).

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1–15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M _r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

Keywords: Cysteine S-conjugates; Cysteine S-conjugate β-lyases; S-(1,2-Dichlorovinyl)-l-cysteine; Glutamine transaminase K; Mitochondrial aspartate aminotransferase; S-(1,1,2,2-tetrafluoroethyl)-l-cysteine

Chemopreventive mechanisms of α-keto acid metabolites of naturally occurring organoselenium compounds by John T. Pinto; Jeong-In Lee; Raghu Sinha; Melanie E. MacEwan; Arthur J. L. Cooper (pp. 29-41).

Previous studies on the chemopreventive mechanisms of dietary selenium have focused on its incorporation into antioxidative selenoproteins, such as glutathione peroxidase and thioredoxin reductase. Several studies, however, have revealed that dietary selenium in the form of l-selenomethionine and the 21st amino acid, selenocysteine, also have intrinsic anti-cancer properties. Biochemical mechanisms previously investigated to contribute to their anticancer effects involve β- and γ-lyase reactions. Some pyridoxal 5′-phosphate (PLP)-containing enzymes can catalyze a β-lyase reaction with Se-methyl-l-selenocysteine (MSC) generating pyruvate and ammonia. Other PLP-enzymes can catalyze a γ-lyase reaction with l-selenomethionine (SM) generating α-ketobutyrate and ammonia. In both cases, a purported third product is methylselenol (CH₃SeH). Although not directly quantifiable, as a result of its extreme hydrophobicity and high vapor pressure, CH₃SeH has been indirectly observed to act through the alteration of protein-sulfhydryl moieties on redox-responsive signal and transcription factors, thereby maintaining a non-proliferative intracellular environment. We have considered the possibility that α-keto acid analogues of MSC (i.e., methylselenopyruvate; MSP) and SM (i.e., α-keto-γ-methylselenobutyrate; KMSB), generated via a transamination and/or l-amino acid oxidase reaction may also be chemoprotective. Indeed, these compounds were shown to increase the level of histone-H3 acetylation in human prostate and colon cancer cells. MSP and KMSB structurally resemble butyrate, an inhibitor of several histone deacetylases. Thus, the seleno α-keto acid metabolites of MSC and SM, along with CH₃SeH derived from β- and γ-lyase reactions, may be potential direct-acting metabolites of organoselenium that lead to de-repression of silenced tumor suppressor proteins and/or regulation of genes and signaling molecules.

Keywords: Prostate cancer; Se-Methyl-l-selenocysteine; l-Selenomethionine; Histone deacetylase; Methylselenopyruvate; α-Keto-γ-methylselenobutyrate; Glutamine transaminase K; Glutamine transaminase L; l-Amino acid oxidase

Latest news about the sulfurtransferase protein family of higher plants by Jutta Papenbrock; Sebastian Guretzki; Melina Henne (pp. 43-57).

Sulfurtransferases/rhodaneses (Str) comprise a group of enzymes widely distributed in all phyla which catalyze in vitro the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. The best characterized Str is bovine rhodanese (EC 2.8.1.1) which catalyses in vitro the transfer of a sulfane sulfur atom from thiosulfate to cyanide, leading to the formation of sulfite and thiocyanate. Plants as well as other organisms contain many proteins carrying a typical rhodanese pattern or domain forming multi-protein families (MPF). Despite the presence of Str activities in many living organisms, the physiological role of the members of this MPF has not been established unambiguously. While in mammals these proteins are involved in the elimination of toxic cyanogenic compounds, their ubiquity suggests additional physiological functions. In plants, Str are localized in the cytoplasm, mitochondria, plastids, and nucleus. Str probably also transfer reduced sulfur onto substrates as large as peptides or proteins. Several studies in different organisms demonstrate a protein–protein interaction with members of the thioredoxin MPF indicating a role of Str in maintenance of the cellular redox homeostasis. The increased expression of several members of the Str MPF in various stress conditions could be a response to oxidative stress. In summary, data indicate that Str are involved in various essential metabolic reactions.

Keywords: Arabidopsis thaliana ; 3-Mercaptopyruvate; Oxidative stress; Thioredoxin; Thiosulfate

Intermolecular disulfide bond to modulate protein function as a redox-sensing switch by N. Nagahara (pp. 59-72).

Recently, redox-regulated biological reactions have been elucidated. In the regulation of these reactions, redox-sensing molecular switches function as unique biological machineries that modulate the functional proteins present in enzymes, transcriptional factors, sensor proteins, and transcriptional factor modulators. The redox-sensing cysteine residues and the disulfide bond formed between these cysteine residues serve as redox-sensing molecular switches; these switches sense cellular oxidizing factors such as oxygen, reactive oxygen species, and cellular reducing factors such as thioredoxin (Trx), glutathione (GSH), and their family molecules. Depending on the redox status, the switch directly modulates the protein function via the “locking and unlocking” of the critically functional residue or indirectly modulates the protein function via “protein conformational changes,” which affects the functioning of a distantly located critical residue in an allostery-like fashion or a topology change. Redox-sensing switches can be classified into two types—intramolecular (intrasubunit) and intermolecular (intersubunit) ones. Further, depending on the sensing specificity to reducing factors, the switch subtype is classified into Trx, GSH, or their family molecules-specific type. This review focused on the intermolecular redox-sensing switches found in various proteins.

Keywords: Disulfide bond; Glutathione; Redox-sensitive cysteine; Redox-sensing switch; Thioredoxin

Differing views of the role of selenium in thioredoxin reductase by Robert J. Hondal; Erik L. Ruggles (pp. 73-89).

This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pK _a and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO₂ ⁻, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO₂ ⁻ to Cys-SH).

Keywords: Selenocysteine; Seleninic acid; Sulfinic acid; Nucleophile; Leaving group; Electrophile

Thiol dioxygenases: unique families of cupin proteins by Martha H. Stipanuk; Chad R. Simmons; P. Andrew Karplus; John E. Dominy Jr. (pp. 91-102).

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.

Keywords: Cysteamine dioxygenase; Cysteine dioxygenase; Cupin proteins; Domain of unknown function 1637 (DUF1637); Iron-dependent enzymes; Thiols

Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy by S. Melino; R. Sabelli; M. Paci (pp. 103-112).

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.

Keywords: Organosulfur compounds; Alk(en)yl thiosulfate; Garlic; Glutathione; S-transferase; Detoxification; Tumor

Hydrogen sulfide: its production, release and functions by Hideo Kimura (pp. 113-121).

Hydrogen sulfide (H₂S), which is a well-known toxic gas, has been recognized as a signal molecule as well as a cytoprotectant. It is produced by three enzymes, cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase along with cysteine aminotransferase. In addition to an immediate release of H₂S from producing enzymes, it can be stored as bound sulfane sulfur, which may release H₂S in response to physiological stimuli. As a signal molecule, it modulates neuronal transmission, relaxes smooth muscle, regulates release of insulin and is involved in inflammation. Because of its reputation as a toxic gas, the function as a cytoprotectant has been overlooked: the nervous system and cardiovascular system are protected from oxidative stress. In this review, enzymatic production, release mechanism and functions of H₂S are focused on.

Keywords: H₂S; Cystathionine β-synthase; Cystathionine γ-lyase; 3-Mercaptopyruvate sulfurtransferase; Bound sulfane sulfur; Neuromodulator; Neuroprotectant; Cardioprotectant; Smooth muscle relaxant; EDRF

Protein cysteine S-guanylation and electrophilic signal transduction by endogenous nitro-nucleotides by Khandaker Ahtesham Ahmed; Tomohiro Sawa; Takaaki Akaike (pp. 123-130).

Nitric oxide (NO), a gaseous free radical that is synthesized in organisms by nitric oxide synthases, participates in a critical fashion in the regulation of diverse physiological functions such as vascular and neuronal signal transduction, host defense, and cell death regulation. Two major pathways of NO signaling involve production of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and posttranslational modification (PTM) of redox-sensitive cysteine thiols of proteins. We recently clarified the physiological formation of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) as the first demonstration, since the discovery of cGMP more than 40 years ago, of a new second messenger derived from cGMP in mammals. 8-Nitro-cGMP is electrophilic and reacts efficiently with sulfhydryls of proteins to produce a novel PTM via cGMP adduction, a process that we named protein S-guanylation. 8-Nitro-cGMP may regulate electrophilic signaling on the basis of its electrophilicity through induction of S-guanylation of redox sensor proteins. Examples include S-guanylation of the redox sensor protein Kelch-like ECH-associated protein 1 (Keap1), which leads to activation of NF-E2-related factor 2 (Nrf2)-dependent expression of antioxidant and cytoprotective genes. This S-guanylation-mediated activation of an antioxidant adaptive response may play an important role in cytoprotection during bacterial infections and oxidative stress. Identification of new redox-sensitive proteins as targets for S-guanylation may help development of novel therapeutics for oxidative stress- and inflammation-related disorders and vascular diseases as well as understanding of cellular protection against oxidative stress.

Keywords: Nitric oxide; Reactive oxygen species; Oxidative stress; Posttranslational modification; Redox signal; ROS signal; Electrophilic signal; Adaptive response

Potential therapeutic advantage of ribose-cysteine in the inhibition of astrocytoma cell proliferation by H. Jurkowska; T. Uchacz; J. Roberts; M. Wróbel (pp. 131-139).

It has been observed that astrocyte and astrocytoma cells differ in their response to d-ribose-l-cysteine (RibCys) in the culture medium. RibCys, a prodrug of l-cysteine, elevates the level of cysteine and glutathione in both astrocytoma and astrocyte cultures. It also affects the activity of two sulfurtransferases, 3-mercaptopyruvate sulfurtransferase and rhodanese, involved in the metabolism of sulfane sulfur-containing compounds and in consequence exerts an effect on the level of sulfane sulfur. Under conditions, in which the raised level of sulfane sulfur was accompanied by an elevated activity of 3-mercaptopyruvate sulfurtransferase, the proliferation of the human astrocytome U373 line was decreased. The experiments were simultaneously performed with murine astrocytes to compare the behavior of normal cells under similar conditions. In murine astrocytes, RibCys was capable of increasing cellular proliferation, and was accompanied by a diminished level of sulfane sulfur and unchanged activity of the two sulfurtransferases. Thus, RibCys might offer a therapeutic advantage in the inhibition of astrocytoma cell proliferation. Besides, in the absence of oxidative stress, measured as the ratio of GSH/GSSG, the obtained results confirm that the fall in the level of sulfane sulfur is associated with increasing proliferation of cells, whereas a rise in the level causes a decrease in the proliferation of U373 cells.

Keywords: Astrocytes; Cysteine; Glutathione; 3-Mercaptopyruvate sulfurtransferase; Ribose-cysteine; Rhodanese; Sulfane sulfur; U373 cells

Mobilization of sulfane sulfur from cysteine desulfurases to the Azotobacter vinelandii sulfurtransferase RhdA by Francesca Cartini; William Remelli; Patricia C. Dos Santos; Jutta Papenbrock; Silvia Pagani; Fabio Forlani (pp. 141-150).

Mobilization of the l-cysteine sulfur for the persulfuration of the rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the functional results about rhodanese-like proteins and RhdA itself, it is suggested that RhdA-dependent modulation of l-cysteine levels must deal with a redox-related process.

Keywords: RhdA; Sulfurtransferase; Cysteine desulfurase; l-Cysteine; Thiosulfate; Azotobacter vinelandii

The expression and activity of cystathionine-γ-lyase and 3-mercaptopyruvate sulfurtransferase in human neoplastic cell lines by Halina Jurkowska; Wojciech Placha; Noriuki Nagahara; Maria Wróbel (pp. 151-158).

The expression and activity of cystathionine γ-lyase (CST) and 3-mercaptopyruvate sulfurtransferase (MPST) were investigated in the human neoplastic cells lines: astrocytoma U373, neuroblastoma SH-SY5Y, melanoma A375, and melanoma WM35. Gene expression analysis demonstrated that the investigated neoplastic cells showed the expression of MPST and what is particularly interesting, the expression of CST. The presence of CST in these cells was confirmed using RT-PCR and western blot analysis. However, in U373 cells, a very low activity of CST was detected. In all the investigated cell lines, the activity of MPST was higher than that of CST, which suggests that in these cells, the main pathway of sulfane sulfur formation is the MPST-catalyzed reaction. RP-HPLC analysis showed a large disparity between the level of cystathionine and GSH in the investigated neoplastic cells. In SH-SY5Y cells, the low level of GSH and low GSH/GSSG ratio corresponded with the highest CST activity. Further investigations could aim at verifying whether the stimulation of CST, at the level of protein or gene expression, could change the proliferation of neoplastic cells.

Keywords: Cystathionine-γ-lyase; 3-Mercaptopyruvate sulfurtransferase; Sulfane sulfur; Glutathione; Astrocytoma U373; Neuroblastoma SH-SY5Y; Melanoma A375; Melanoma WM35

Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium by Angelos K. Sikalidis; Jeong-In Lee; Martha H. Stipanuk (pp. 159-171).

The integrated stress response (ISR), a defense mechanism cells employ when under stress (e.g., amino acid deprivation), causes suppression of global protein synthesis along with the paradoxical increased expression of a host of proteins that are useful in combating various stresses. Genes that were similarly differentially expressed under conditions of either leucine- or cysteine-depletion were identified. Many of the genes known to contain an amino acid response element and to be induced in response to eIF2α phosphorylation and ATF4 heterodimer binding (ATF3, C/EBPβ, SLC7A1, SLC7A11, and TRIB3), as well as others shown to be induced downstream of eIF2α phosphorylation (C/EBPγ, CARS, SARS, CLCN3, CBX4, and PPP1R15A) were among the upregulated genes. Evidence for the induction of the ISR in these cells also included the increased phosphorylation of eIF2α and increased protein abundance of ATF4, ATF3, and ASNS in cysteine- and leucine-depleted cells. Based on genes highly differentially expressed in both leucine- and cysteine-deficient cells, a list of 67 downregulated and 53 upregulated genes is suggested as likely targets of essential amino acid deprivation in mammalian cells.

Keywords: Amino acid deprivation; Integrated stress response; eIF2α; ATF4; HepG2/C3A

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation by N. Sawada; N. Nagahara; F. Arisaka; K. Mitsuoka; M. Minami (pp. 173-180).

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys²²³ is transferred in the active center to be coordinated by Cys¹²², Cys¹²⁴, and Cys¹³² (Sawada et al. in J Biol Inorg Chem 10:199–207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f ₀. It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys¹²², Cys¹²⁴, and Cys¹³² and coordination with zinc ion is closely linked to change in the oligomeric state.

Keywords: Analytical ultracentrifugation; Conformational change; Enzyme activity regulation; Negative staining electron microscopy; Porphobilinogen synthase

Quantum mechanical origin of the conformational preferences of 4-thiaproline and its S-oxides by Amit Choudhary; Khian Hong Pua; Ronald T. Raines (pp. 181-186).

The saturated ring and secondary amine of proline spawn equilibria between pyrrolidine ring puckers as well as peptide bond isomers. These conformational equilibria can be modulated by alterations to the chemical architecture of proline. For example, C^γ in the pyrrolidine ring can be replaced with sulfur, which can be oxidized either stereoselectively to yield diastereomeric S-oxides or completely to yield a sulfone. Here, the thiazolidine ring and peptide bond conformations of 4-thiaproline and its S-oxides were analyzed in an Ac–Xaa–OMe system using NMR spectroscopy, X-ray crystallography, and hybrid density functional theory. The results indicate that the ring pucker of the S-oxides is governed by the gauche effect, and the prolyl peptide bond conformation is determined by the strength of the n → π* interaction between the amide oxygen and the ester carbonyl group. These findings, which are consistent with those of isologous 4-hydroxyprolines and 4-fluoroprolines, substantiate the importance of electron delocalization in amino acid conformation.

Keywords: Collagen; n → π* interaction; Stereoelectronic effect; Thiazolidine

An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine by Rafał Głowacki; Edward Bald; Hieronim Jakubowski (pp. 187-194).

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with ε-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein’s function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3–5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples.

Keywords: Homocysteine-thiolactone; Protein N-linked homocysteine; HPLC; o-Phthaldialdehyde; Human plasma; Urine

Versatile synthesis of α-substituted taurines from nitroolefins by Chuanxiang Xu; Jiaxi Xu (pp. 195-203).

A series of 1-substituted and 1,1-disubstituted taurines were synthesized from nitroolefins via the Michael addition with sodium ethylxanthate, oxidation with performic acid, and reduction with hydrogen in the presence of palladium on carbon powder. The current route is a versatile and salt-free method for synthesis of both aliphatic and aromatic 1-substituted and 1,1-disubstituted taurines.

Keywords: Amino acid; Aminoalkanesulfonic acid; Nitroolefin; Synthesis; Taurine; Xanthate

S-Propargyl-cysteine (SPRC) attenuated lipopolysaccharide-induced inflammatory response in H9c2 cells involved in a hydrogen sulfide-dependent mechanism by Li-Long Pan; Xin-Hua Liu; Qi-Hai Gong; Yi-Zhun Zhu (pp. 205-215).

The present study attempts to investigate the effects of S-propargyl-cysteine (SPRC), a sulfur-containing amino acid, on lipopolysaccharide (LPS)-induced inflammatory response in H9c2 cardiac myocytes. We found that SPRC prevented nuclear factor-κB (NF-κB) activation assessed by NF-κB p65 phosphorylation and IκBα degradation, suppressed LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and intracellular reactive oxygen species (ROS) production. Furthermore, incubation of H9c2 cells with SPRC induced phosphorylation of Akt in a time- and concentration-dependent manner. In addition, SPRC attenuated LPS-induced mRNA and protein expression of tumor necrosis factor-α (TNF-α), and mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS). The effects of SPRC were abolished by cystathionine γ-lyase [CSE-an enzyme that synthesizes hydrogen sulfide (H₂S)] inhibitor, dl-propargylglycine (PAG), SPRC-induced Akt phosphorylation and TNF-α release was also abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, SPRC also increased LPS-induced down-regulation expression of CSE and H₂S level in H9c2 cells. PAG abolished SPRC-induced up-regulation of H₂S level. Therefore, we concluded that SPRC produced an anti-inflammatory effect in LPS-stimulated H9c2 cells partly through the CSE/H₂S pathway by impairing IκBα/NF-κB signaling and by activating PI3K/Akt signaling pathway.

Keywords: S-Propargyl-cysteine; Nuclear factor-κB; Inflammatory response; Hydrogen sulfide; Reactive oxygen species

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.41, #1)