Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide

The cathelicidin antimicrobial peptide bactenecin is a beta-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity. We initially synthesized two bactenecin dimers (antiparallel and parallel) and two monomers (beta-hairpin and linear). Under oxidative folding conditions, reduced linear bactenecin preferentially folded into a dimer forming a ladder-like structure via intermolecular disulfide bonding. As compared to the monomer, the dimer had a greater ability to induce lysis of lipid bilayers and was more rapidly bactericidal. Interestingly, the dimer retained antimicrobial activity at physiological salt concentrations (150 mm NaCl), although the monomer was inactivated. This salt resistance was also seen with bactenecin dimer containing one intermolecular disulfide bond, and the bactenecin dimer appears to undergo multimeric oligomerization at high salt concentrations. Overall, dimeric bactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensation and oligomerization. These characteristics shed light on the features that a peptide would need to serve as an effective therapeutic agent.     

Effect of the central disulfide bond on the unfolding behavior of elongation factor Ts homodimer from Thermus thermophilus

Functionally active elongation factor Ts (EF-Ts) from Thermus thermophilus forms a homodimer. The dimerization interface of EF-Ts is composed of two antiparallel beta-sheets that can be connected by an intermolecular disulfide bond. The stability of EF-Ts from T. thermophilus in the presence and absence of the intermolecular disulfide bond was studied by differential scanning calorimetry and circular dichroism. The ratio of the van't Hoff and calorimetric enthalpies, delta H(vH)/delta H(cal), indicates that EF-Ts undergoes thermal unfolding as a dimer independently of the presence or absence of the disulfide bond. This can be concluded from (1) the presence of residual secondary structure above the thermal transition temperature, (2) the absence of concentration dependence, which would be expected for dissociation of the dimer prior to unfolding of the monomers, and (3) a relatively low heat capacity change (delta Cp) upon unfolding. The retained dimeric structure of the thermally denatured state allowed for the determination of the effect of the intermolecular disulfide bond on the conformational stability of EF-Ts, which is deltadelta G(S-S,SH HS) = 10.5 kJ/mol per monomer at 72.5 degrees C. The possible physiological implications of the dimeric EF-Ts structure and of the intersubunit disulfide bond for the extreme conformational stability of proteins in thermophiles are discussed.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of proteins. It exists as a covalent dimer in solution, with the 15 kDa monomers linked by an interchain disulfide bond through the Cys101 residues. Sedimentation equilibrium and velocity experiments demonstrated that, after removal of the interchain disulfide bond, GDNF remains as a non-covalent dimer and is stable at pH 7.0. To investigate the effect of the intermolecular disulfide on the structure and stability of GDNF, we compared the solution structures of the wild-type protein and a cysteine-101 to alanine (C101A) mutant using Fourier transform infrared (FTIR), FT-Raman and circular dichroism (CD) spectroscopy and sedimentation analysis. The elimination of the intermolecular disulfide bond causes only minor changes (approximately 4%) in the secondary structures of GDNF. The far- and near-UV CD spectra demonstrated that the secondary and tertiary structures were similar for both wild-type and C101A GDNF. Heparin binding and sedimentation velocity experiments also indicated that the folded structure of the wild-type and C101A GDNF are indistinguishable. The thermal stability of GDNF does not appear to be affected by the absence of the interchain disulfide bond and the biological activity of the C101A mutant is identical with that of the wild-type protein. However, small but significant changes in side chain conformations of tyrosine and aliphatic residues were observed by FT-Raman spectroscopy upon removal of the intermolecular disulfide bond, which may reflect structural changes in the area of dimeric contact. By comparing the Raman spectrum of wild-type GDNF with that of the C101A analog, we identified the conformation of the intermolecular disulfide as trans-gauche-trans geometry. These results indicate that GDNF is an active, properly folded molecule in the absence of the interchain disulfide bond.     

Identification and characterization of a novel murine beta-defensin-related gene

Beta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity. We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes. In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins. This defensin-related gene (Defr1) is most highly expressed in testis and heart. The genomic organization is highly similar to Defb3, 4, 5, and 6, and the exon 1 sequence is very highly conserved. A synthetic Defr1 peptide displayed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Burkholderia cepacia. The antimicrobial activity of Defr1 against S. aureus, E.coli, and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. This is the first report of a functional beta defensin that lacks one of the conserved cysteine residues in its predicted mature peptide. This study has major implications for the structure and functions of these important host defense molecules.     

A thiol-based intramolecular redox switch in four-repeat tau controls fibril assembly and disassembly

Oxidative stress has been implicated in the pathogenesis and progression of several tauopathies, including Alzheimer''s disease. The deposition of fibrillar inclusions made of tau protein is one of the pathological hallmarks of these disorders. Although it is becoming increasingly evident that the specific fibril structure may vary from one tauopathy to another and it is recognized that different types of isoforms (three-repeat and four-repeat tau) can be selectively deposited, little is known about the role oxidation may play in aggregation. Four-repeat tau contains two cysteines that can form an intramolecular disulfide bond, resulting in a structurally restrained compact monomer. There is discrepancy as to whether this monomer can aggregate or not. Using isolated four-repeat tau monomers (htau40) with intramolecular disulfide bonds, we demonstrate that these proteins form fibrils. The fibrils are less stable than fibrils formed under reducing conditions but are highly effective in seeding oxidized tau monomers. Conversely, a strong seeding barrier prevents incorporation of reduced tau monomers, tau mimics in which the cysteines have been replaced by alanines or serines, and three-repeat tau (htau23), a single-cysteine isoform. The barrier also holds true when seed and monomer types are reversed, indicating that oxidized and reduced tau are incompatible with each other. Surprisingly, fibrils composed of compact tau disaggregate upon reduction, highlighting the importance of the intramolecular disulfide bond for fibril stability. The findings uncover a novel binary redox switch that controls the aggregation and disaggregation of these fibrils and extend the conformational spectrum of tau aggregates.     

Role of the propeptide in controlling conformation and assembly state of hepatitis B virus e-antigen

Hepatitis B virus “e-antigen” (HBeAg) is thought to be a soluble dimeric protein that is associated with chronic infection. It shares 149 residues with the viral capsid protein “core-antigen” (HBcAg), but has an additional 10-residue, hydrophobic, cysteine-containing amino-terminal propeptide whose presence correlates with physical, serological, and immunological differences between the two proteins. In HBcAg dimers, the subunits pair by forming a four-helix bundle stabilized by an intermolecular disulfide bond. The structure of HBeAg is probably similar but, instead, has two intramolecular disulfide bonds involving the propeptide. To compare the proteins directly and thereby clarify the role of the propeptide, we identified mutations and solution conditions that render both proteins as either soluble dimers or assembled capsids. Thermally induced unfolding monitored by circular dichroism, and electrophoresis of oxidized and reduced dimers, showed that the propeptide has a destabilizing effect and that the intramolecular disulfide bond forms preferentially and blocks the formation of the intermolecular disulfide bond that otherwise stabilizes the dimer. The HBeAg capsids are less regular than the HBcAg capsids; nevertheless, cryo-electron microscopy reconstructions confirm that they are constructed of dimers resembling those of HBcAg capsids. In them, a portion of the propeptide is visible near the dimer interface, suggesting that it intercalates there, consistent with the known formation of a disulfide bond between C(− 7) in the propeptide and C61 in the dimer interface. However, this intercalation distorts the dimer into an assembly-reluctant conformation.     

Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1)

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.     

Analytical ultracentrifugation studies on chloroplastic fructose bisphosphatase

Analytical ultracentrifugation studies performed on spinach chloroplast fructose bisphosphatase show that the tetrameric oxidized (inactive) or reduced (active) enzyme dissociates into inactive dimers and monomers at alkaline pH. The dissociation process is, at least, partially reversible if the enzyme is dimeric. Moreover, the oxidized inactive tetrameric enzyme is less prone to dissociation into dimers and monomers than the reduced active tetramer. The irreversibility of the dissociation process may be explained by a sulfhydryl-disulfide interchange. Together with the findings from previously published sulfhydryl group titration experiments (J. Pradel et al., Eur. J. Biochem., 113 (1981) 507), the above results suggest that the activation of the oxidized tetramer involves the reduction of two inter-protomeric disulfide bonds.     

The location of an engineered inter-subunit disulfide bond in factor for inversion stimulation (FIS) affects the denaturation pathway and cooperativity

Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: De novo sequencing and analysis of tandem mass spectrometry data

Eight intact antimicrobial peptides were identified from the skin of Odorrana jingdongensis by de novo sequencing following low energy ESI CID Q-TOF MS/MS in positive-mode with the help of Edman degradation and structural similarity analysis. We devised exact mass measurements to discriminate the K/Q amino acid residue in the peptides between 2.0kDa to 3.8kDa. Moreover, the cleavage at the CS bond at the side chain of Met was observed in all the spectra of the peptides containing Met residue. And we found unusual cleavages within the intramolecular disulfide loop with high frequency. Our data revealed that the cleavage pathways are significantly different from those reported previously which are similar to the cycle peptide cleavage mode followed by the secondary cleavage at the CS bond on oxidized Cys. Thus, our results highly suggest that ion series generated from the cleavages within the intramolecular disulfide loop should be considered in both the top-down sequencing and the disulfide bridge location with the presence of a relatively high intensity of MH(+)-28 ion marker. Furthermore, our activity data implied that different AMPs may use different strategies to kill microbes.     

Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies. Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105. Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies. These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography. Human IL-2 produced in inclusion bodies in E. coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds. Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively. Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.     

An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys(61) and Cys(177) produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.     

Effect of disulfide crosslinking on thermal transitions and chaperone-like activity of human small heat shock protein HspB1

Temperature-induced conformational changes of reduced and oxidized HspB1 crosslinked by disulfide bond between single Cys137 of neighboring monomers were analyzed by means of different techniques. Heating of reduced HspB1 was accompanied by irreversible changes of Trp fluorescence, whereas oxidized HspB1 underwent completely reversible changes of fluorescence. Increase of the temperature in the range of 20–70 °C was accompanied by self-association of both reduced and oxidized protein. Further increase of the temperature led to formation of heterogeneous mixture of large self-associated complexes of reduced HspB1 and to formation of smaller and less heterogeneous complexes of oxidized HspB1. Heat-induced changes of oligomeric state of reduced HspB1 were only partially reversible, whereas the corresponding changes of oligomeric state of oxidized HspB1 were almost completely reversible. Oxidation resulted in decrease of chaperone-like activity of HspB1. It is concluded that oxidative stress, inducing formation of disulfide bond, can affect stability and conformational mobility of human HspB1.     

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.