Expression, Purification and Functional Analysis of Hexahistidine-tagged Human Glutathione S-Transferase P1-1 and its Cysteinyl Mutants

The bacterial expression and purification of human glutathione S-transferase P1-1(hGST P1-1), as a hexahistidine-tagged polypeptide was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two (C47A/C101A), three (C14A/C47A/C101A) or all four (C14A/C47A/ C101A/C169A) cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, hexahistidine-tagged hGST P1-1 (His₆-hGST P1-1) treated with 1 mM H₂O₂ showed at least three extra bands, in addition to the native His₆-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed. These authors contributed equally to this work.     

Proteomic analysis of the oxidation of cysteine residues in human age-related nuclear cataract lenses

Loss of protein thiols is a key feature associated with the onset of age-related nuclear cataract (ARNC), however, little is known about the specific sites of oxidation of the crystallins. We investigated cysteine residues in ARNC lenses and compared them with age-matched normal lenses. Proteomic analysis of tryptic digests revealed ten cysteine residues in older normal lenses that showed no significant oxidation compared to foetal counterparts (Cys 170 in betaA1/3-crystallin, Cys 32 in betaA4-crystallin, Cys 79 in betaB1-crystallin, Cys 22, Cys 78/79, C153 in gammaC-crystallin and Cys 22, Cys 24 and Cys 26 in gammaS-crystallin). Although these thiols were not oxidised in normal lenses past the 6th decade, they were present largely as disulphides in the ARNC lenses. By contrast, two cysteine residues, Cys 41 in gammaC-crystallin and Cys 18 in gammaD-crystallin, were not oxidised, even in advanced ARNC lenses. These cysteines are buried deep within the protein and any unfolding associated with cataract must be insufficient to expose them to the oxidative environment present in the centre of advanced ARNC lenses. The vast majority of the loss of protein thiol observed in such lenses is due to disulphide bond formation.     

Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.     

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase.

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.     

REDOX Reaction at ASK1-Cys250 Is Essential for Activation of JNK and Induction of Apoptosis

ASK1 cysteine oxidation allows JNK activation upon oxidative stress. Trx1 negatively regulates this pathway by reducing the oxidized cysteines of ASK1. However, precisely how oxidized ASK1 is involved in JNK activation and how Trx1 regulates ASK1 oxidoreduction remains elusive. Here, we describe two different thiol reductase activities of Trx1 on ASK1. First, in H₂O₂-treated cells, Trx1 reduces the various disulfide bonds generated between cysteines of ASK1 by a rapid and transient action. Second, in untreated cells, Trx1 shows a more stable thiol reductase activity on cysteine 250 (Cys250) of ASK1. After H₂O₂ treatment, Trx1 dissociates from Cys250, which is not sufficient to activate the ASK1-JNK pathway. Indeed, in untreated cells, a Cys250 to alanine mutant of ASK1 (C250A), which cannot bind Trx1, does not constitutively activate JNK. On the other hand, in H₂O₂-treated cells, this mutant (C250A) fails to activate JNK and does not induce apoptosis, although it remains fully phosphorylated on Threonine 838 (Thr838) in its activation loop. Overall, our data show that Cys250 is essential for H₂O₂-dependent signaling downstream from ASK1 but at a step subsequent to the phosphorylation of ASK1 Thr838. They also clarify the thiol reductase function of Trx1 on ASK1 activity.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1)

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.     

人谷胱甘肽S-转移酶pi（GSTp）中半胱氨酸对其在细胞氧化应激下功能的影响

利用PCR定点突变技术构建人GSTp三种半胱氨酸突变体C^47/101、C^14/47/101和C^14/47/101/169。将GSTp野生型和突变体表达质粒转染293细胞,以CDNB为底物测定胞内GST的转移酶活性,结果显示各类突变体均明显抑制了细胞内源性GST的催化活性,具有显著的负显性(dominant negative)突变体的功能;将GSTp野生型和突变体与c-Jun、NF-κB和p53的报告基因载体共转染,通过萤光素酶活性测定发现突变体C^47/101和C^14/47/101/169能明显激活c-Jun和p21的转录活性;Western印迹分析显示突变体均能上调细胞内p21蛋白的水平,细胞存活率的测定表明GSTp突变体能增强293细胞对H202刺激的敏感性;实验结果表明半胱氨酸残基对于维持GSTp在对抗细胞氧化应激过程中的保护作用至关重要。     

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site.     

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.     

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.     

Mass spectrometric identification of N- and O-glycosylation sites of full-length rat selenoprotein P and determination of selenide-sulfide and disulfide linkages in the shortest isoform

Rat selenoprotein P is an extracellular glycoprotein of 366 amino acid residues that is rich in cysteine and selenocysteine. Plasma contains four isoforms that differ principally by length at the C-terminal end. Mass spectrometry was used to identify sites of glycosylation on the full-length protein. Of the potential N-glycosylation sites, three located at residues 64, 155, and 169 were occupied, while the two at residues 351 and 356 were not occupied. Threonine 346 was variably O-glycosylated. Thus, full-length selenoprotein P is both N- and O-glycosylated. The shortest isoform of selenoprotein P, which terminates at residue 244, was analyzed for selenide-sulfide and disulfide linkages. In this isoform, a single selenocysteine and seven cysteines are present. Mass spectrometric analysis indicated that a selenide-sulfide bond exists between Sec40 and Cys43. Two disulfides were also detected as Cys149-Cys167 and Cys153-Cys156. The finding of a selenide-sulfide bond in the shortest isoform is compatible with a redox function of this pair that might be analogous to the selenol-thiol pair near the C terminus of animal thioredoxin reductase. The disulfide formed by Cys153-Cys156 also has some characteristics of a redox active pair.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO

Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.     

Thiol-disulfide exchanges modulate aldo–keto reductase family 1 member B10 activity and sensitivity to inhibitors

The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and the glutathione (GSH) and free amino acid cysteine (Cys) are critical cellular thiol anti-oxidants, protecting proteins from irreversible oxidative damage. In this study, we found that aldo–keto reductase family 1 member B10 (AKR1B10) contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299. Exposing AKR1B10 to ROS mixtures resulted in significant decrease of its free sulfhydryl groups, up to 40–50% in the presence of physiological thiol cysteine at 0.5 or 1.0 mM; and accordingly, AKR1B10 enzymatic activity was reversibly decreased, in parallel with the oxidation of the sulfhydryl groups. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Together our results showed for the first time that AKR1B10's enzymatic activity and inhibitor sensitivity are modulated by thiol/disulfide exchanges.     

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.     

A single free cysteine residue and disulfide bond contribute to the thermostability of Aspergillus saitoi 1,2-alpha-mannosidase

Aspergillus saitoi 1,2-alpha-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T(m) values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 degrees C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.     

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.