Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.     

Endogenous HMGB1 regulates autophagy

Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.     

Redox state-dependent interaction of HMGB1 and cisplatin-modified DNA

HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.     

The Role of HMGB1 in the Pathogenesis of Inflammatory and Autoimmune Diseases

High-mobility group box 1 (HMGB1) protein is a highly abundant protein that can promote the pathogenesis of inflammatory and autoimmune diseases once it is in an extracellular location. This translocation can occur with immune cell activation as well as cell death, with the conditions for release associated with the expression of different isoforms. These isoforms result from post-translational modifications, with the redox states of three cysteines at positions 23, 45 and 106 critical for activity. Depending on the redox states of these residues, HMGB1 can induce cytokine production via toll-like receptor 4 (TLR4) or promote chemotaxis by binding the chemokine CXCL12 for stimulation via CXCR4. Fully oxidized HMGB1 is inactive. During the course of inflammatory disease, HMGB1 can therefore play a dynamic role depending on its redox state. As a mechanism to generate alarmins, cell death is an important source of HMGB1, although each major cell death form (necrosis, apoptosis, pyroptosis and NETosis) can lead to different isoforms of HMGB1 and variable levels of association of HMGB1 with nucleosomes. The association of HMGB1 with nucleosomes may contribute to the pathogenesis of systemic lupus erythematosus by producing nuclear material whose immunological properties are enhanced by the presence of an alarmin. Since HMGB1 levels in blood or tissue are elevated in many inflammatory and autoimmune diseases, this molecule can serve as a unique biomarker as well as represent a target of novel therapies to block its various activities.     

The disulfide structure of bovine pituitary basic fibroblast growth factor.

Basic fibroblast growth factor has 4 cysteine residues in its amino acid sequence, two of which are perfectly conserved within the fibroblast growth factor family of proteins suggesting a disulfide bond at this position. Furthermore, thiol titration of bovine pituitary basic fibroblast growth factor (bFGF) indicates the presence of two free thiols, which is consistent with an intramolecular disulfide. Direct analysis of natural and recombinant fibroblast growth factor proteins have not confirmed the existence of such a disulfide. Instead, the two nonconserved cysteines of bFGF purified from bovine pituitaries are S-thiolated with glutathione. Inclusion of 75 mM N-ethylmaleimide during the homogenization of the pituitaries effectively blocks the S-thiolation, demonstrating that this modification is an artifact of the purification procedure. Analysis of the N-ethylmaleimide purified bovine pituitary bFGF suggests that the natural protein is in the correct redox state when all 4 cysteines are in the reduced form.     

A Janus Tale of Two Active High Mobility Group Box 1 (HMGB1) Redox States

High mobility group box 1 (HMGB1), the prototypic damage–associated molecular pattern molecule, is released at sites of inflammation and/or tissue damage. There, it promotes cytokine production and chemokine production/cell migration. New work shows that the redox status of HMGB1 distinguishes its cytokine-inducing and chemokine activity. Reduced all-thiol-HMGB1 has sole chemokine activity, whereas disulfide-HMGB1 has only cytokine activity, and oxidized, denatured HMGB1 has neither. Autophagy (programmed cell survival) and apoptosis (programmed cell death) have been implicated in controlling both innate and adaptive immune functions. Reduced HMGB1 protein promotes autophagy, whereas oxidized HMGB1 promotes apoptosis. Thus, the differential activity of HMGB1 in immunity, inflammation and cell death depends on the cellular redox status within tissues.High mobility group box 1 (HMGB1), a nonhistone nuclear factor, acts extracellularly as a damage-associated molecular pattern (DAMP) molecule to modulate inflammation, promoting autophagy and innate immune responses (1–5). HMGB1 has compartment-specific functions: nuclear, intracellular (but extranuclear) and extracellular. Its extracellular functions can now be divided further into cytokine-like or cytokine-inducing, chemokinelike and proangiogenic. Signaling pathways that induce variations on the posttranslational modification, such as phosphorylation and acetylation, have been implicated in the regulation of HMGB1 release. Importantly, HMGB1 contains three cysteines, each of which is susceptible to redox modification (6,7). The redox state of these cysteines is important for the proinflammatory cytokine-stimulating and proautophagic activity of HMGB1 (8–10). Autophagy (literally “self-eating”), a lysosome-mediated catabolic process, contributes to maintenance of intracellular homeostasis and promotes cell survival in response to environmental stress (11–13).Treatment with reduced but not oxidized HMGB1 protein increases autophagy in cancer cells (9). In contrast, oxidized HMGB1 protein activates the caspase-dependent apoptotic cell death pathway (9). Venereau et al.(14) described a new role for redox control of both the cytokine-inducing and chemokine activity of HMGB1 in the setting of sterile inflammation, regulating leukocyte recruitment and their ability to secrete inflammatory cytokines (Figure 1).Open in a separate windowFigure 1Redox control of HMGB1 activity. To act as a DAMP/danger signal and inflammatory mediator, HMGB1 is transported extracellularly by two principal means: active secretion from living inflammatory cells (for example, macrophages) or passive release from necrotic cells. The activities of extracellular HMGB1 are redox dependent. All-thiol-HMGB1 promotes chemokine production and leukocyte recruitment. Disulfide-HMGB1, originating from infiltrating leukocytes, promotes release of proinflammatory cytokines and thus participates in the inflammatory response. Reactive oxygen species produced by leukocytes induces the terminal oxidation of HMGB1, which is inactivated during resolution of inflammation.Structurally, HMGB1 is composed of three domains: two positively charged proximal DNA-binding domains (A box and B box) and a negatively charged carboxyl terminus. Three cysteines are encoded within the molecule: two vicinal cysteines in box A (C23 and C45) and a single one in box B (C106). C23 and C45 can form an intermolecular disulfide bond, whereas C106 is unpaired. Therefore, three different redox forms HMGB1 (all-thiol-HMGB1, disulfide-HMGB1 and oxidized HMGB1) were derived from bacterial expression systems (14). In addition, by using tryptic digests and liquid chromatography tandem mass spectrometric analysis, Venereau et al. observed that recombinant HMGB1 can be reversibly oxidized and reduced in the presence of electron donors (for example, dithiothreitol) or acceptors (oxygen) (14).Next, Venereau et al. assessed whether individual redox forms of HMGB1 have a differential role in cytokine-stimulating and chemoattractant activities (14). They found that disulfide-HMGB1 induced activation of the nuclear factor (NF)-κB pathway and production of proinflammatory cytokines (for example, tumor necrosis factors-α, interleukin [IL]-6 and IL-8) in fibroblasts and macrophages. Interestingly, all-thiol-HMGB1 failed to induce a proinflammatory response. In contrast, all-thiol-HMGB1, but not disulfide-HMGB1, had chemoattractant activity in fibroblasts. These findings prompted them to determine whether HMGB1 inhibitors, such as box A and monoclonal antibody PDH1.1, block the chemoattractant and/or cytokine-inducing activities of HMGB1. Unexpectedly, these inhibitors prevented cell migration but not cytokine production, although they are widely used as HMGB1-targeting agents in experimental inflammatory diseases.Reactive oxygen species oxidize the HMGB1 released from dying cells, thereby neutralizing its stimulatory activity and promoting tolerance in immune cells (15,16). In addition, oxidation of C106 or lack of a disulfide bridge between C23 and C45 then causes HMGB1 to lose its proinflammatory effects in macrophages (8). Venereau et al. found that terminal oxidation by hydrogen peroxide results in the loss of both the cytokine-stimulating and chemoattractant activities of HMBG1. Moreover, the authors found that the three HMGB1 cysteine residues were required for the cytokine-stimulating activity but not for the chemoattractant activity of HMGB1. Cysteine mutant HMGB1 promotes fibroblast migration, but not cytokine expression in macrophages (14). Collectively, these findings establish a crucial role for redox in the regulation of HMGB1 activity in inflammation and migration.What is the redox state of HMGB1 in the pathogenesis of individual diseases? The redox state of HMGB1 from the human acute monocytic leukemia cell line THP-1 was measured in the presence or absence of lipopolysaccharide (LPS) and necrotic medium in vitro. Intracellular HMGB1 was all-thiol-HMGB1, whereas secreted HMGB1 contained both all-thiol- and disulfide-HMGB1 (14). Furthermore, disulfide-HMGB1 was present later and time-dependently increased in cardiotoxin-injured muscles in vivo, confirming that the redox state of HMGB1 is altered during tissue damage and inflammation. HMGB1 protein with all three cysteines mutated to serine are resistant to oxidation and induce leukocyte recruitment without inducing cytokine production (14). The activities of HMGB1 are thus redox-dependent and can be modified within the injured tissues after HMGB1 release. Therefore, release of dynamic redox-regulated HMGB1 contributes to the orderly orchestrated recruitment of leukocytes, activation of cytokine release and subsequent resolution of inflammation.Several issues remain unresolved regarding the redox control of HMGB1 activity. First, HMGB1 is specifically recognized by several cell surface receptors (2), including Toll-like receptor (TLR)-4 and the receptor for advanced glycation end products (RAGE), but most recently was joined by T-cell immunoglobulin and mucin domain 3 (TIM-3) (17). Initial studies suggest that reduced C106 is necessary for the binding of HMGB1 to one of its receptors, TLR4, to stimulate cytokine release (8). HMGB1-induced recruitment of inflammatory cells depends on forming a complex with CXCL12 and signaling via CXCR4 (18). Moreover, RAGE is required for reduced HMGB1-mediated autophagy, but not oxidized HMGB1-induced apoptosis (9). All-thiol-HMGB1, but not disulfide-HMGB1, binds CXCL12 (14). The influence of HMGB1 receptors (for example, RAGE, TLR4, TLR2, CD24, TIM-3 and triggering receptor expressed on myeloid cells 1 [TREM1]) on biological activities of individual redox forms of HMGB1 remains to be carefully investigated. Second, HMGB1 forms highly inflammatory complexes with DNA, lipoteichoic acid, LPS, IL-1β, chemokine (C-X-C motif) ligand 12 (CXCL12)/ stromal cell–derived factor-1 (SDF-1) and nucleosomes (19). There is great interest in determining whether the individual redox forms of HMGB1 have varying affinity profiles active in inflammation and immunity. Third, HMGB1 has multiple intracellular and extracellular functions in health and disease, including cancer (1,2,6,20). Additional studies will be needed to determine whether redox is required for other functions of HMGB1, such as regeneration and cellular differentiation as well as the complex interactions between autophagy and immunity (5). One additional unanswered question is where and how the formation of the disulfide takes place and whether there is an enzyme specific for regulating this. This is important, knowing that the nuclear form is mostly all thiol. Finally, the development and performance of a simple, sensitive method for the detection of individual HMGB1 redox state isoforms in clinical specimens remains to be accomplished.     

Structural and redox properties of the leaderless DsbE (CcmG) protein: both active-site cysteines of the reduced form are involved in its function in the Escherichia coli periplasm

The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, N-terminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL-). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E'0) for the active thiol/ disulfide was determined to be -0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active-site cysteines and the structure of the reduced form.     

Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa 118) is bonded to cysteine 4 (aa 127). Thus, the biochemical analysis of gD-2 agrees with the genetic analysis of gD-1. A similar disulfide bond arrangement is postulated to exist in other gD homologs.     

Redox status of high‐mobility group box 1 performs a dual role in angiogenesis of colorectal carcinoma

During inflammation, high‐mobility group box 1 in reduced all‐thiol form (at‐HMGB1) takes charge of chemoattractant activity, whereas only disulfide‐HMGB1 (ds‐HMGB1) has cytokine activity. Also as pro‐angiogenic inducer, the role of HMGB1 in different redox states has never been defined in tumour angiogenesis. To verify which redox states of HMGB1 induces angiogenesis in colorectal carcinoma. To measure the expression of VEGF‐A and angiogenic properties of the endothelial cells (ECs), at‐HMGB1 or ds‐HMGB1 was added to cell medium, further with their special inhibitors (DPH1.1 mAb and 2G7 mAb) and antibodies of corresponding receptors (RAGE Ab and TLR4 Ab). Also, a co‐culture system and conditioned medium from tumour cells were applied to mimic tumour microenvironment. HMGB1 triggered VEGF‐A secretion mainly through its disulfide form interacting with TLR4, while co‐operation of at‐HMGB1 and RAGE mediated migratory capacity of ECs. Functional inhibition of HMGB1 and its receptors abrogated HMGB1‐induced angiogenic properties of ECs co‐cultured with tumour cells. HMGB1 orchestrates the key events of tumour angiogenesis, migration of ECs and their induction to secrete VEGF‐A, by adopting distinct redox states.     

High Systemic Levels of the Cytokine-Inducing HMGB1 Isoform Secreted in Severe Macrophage Activation Syndrome

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.     

Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments

Green fluorescent protein (GFP) indicators were previously developed that rapidly and quantitatively respond to changes in the thiol/disulfide equilibrium within subcellular compartments. In these indicators, surface-exposed cysteines residues were introduced so as to form a labile redox-active disulfide that in turn controls the emission properties of the internal chromophore. The biosensors have been shown to be effective reporters of the thiol/disulfide status within reducing compartments such as the mitochondria and cytosol for several cell types. However, due to the high thermodynamic stability of the introduced disulfide bond, the indicators are not useful for quantitative analysis within more oxidizing compartments such as the endoplasmic reticulum. Here we report the development of a new family of GFP-based redox indicators (roGFP1-iX) in which the thermodynamic stability of the disulfide is substantially lowered by insertion of a single amino acid into the main chain, adjacent to cysteine 147. The insertions result in indicators with midpoint potentials of -229 to -246 mV and are thus better suited for study of relatively oxidizing subcellular compartments. Atomic resolution crystallographic analyses suggest that two important factors act to destabilize the disulfide linkage in roGFP1-iX. In the oxidized state, an unusual non-proline cis-peptide bond adjacent to one of the cysteines introduces geometric strain into the system, while in the reduced state, a dramatic loop opening lowers the effective concentration of the reacting species.     

Characterization of the redox activity and disulfide bond formation in apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1's redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1's redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1-TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.     

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.     

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

S-Glutathionylation regulates HDL-associated paraoxonase 1 (PON1) activity

HDL-associated paraoxonase 1 (PON1) undergoes inactivation under oxidative stress and is preserved by dietary antioxidants. PON1 cysteines can affect PON1 enzymatic activities. S-Glutathionylation, a redox regulatory mechanism characterized by the formation of a mixed disulfide between a protein thiol and oxidized glutathione (GSSG), was shown to preserve some enzymes from irreversible inactivation under pathological conditions. We questioned whether PON1 activity is regulated by S-glutathionylation. Incubation of PON1 or HDL with GSSG indeed resulted in a dose-dependent inactivation of PON1 activities, including its physiological activity to increase HDL-mediated macrophage cholesterol efflux. This PON1 inactivation was associated with the formation of a mixed disulfide bond between GSSG and PON1's cysteine residue(s), as detected by immunoblotting with anti-glutathione IgG. PON1 activity was recovered following the addition of a reducing agent, DL-Dithiothreitol (DTT), to the PON1-SSG complex. We thus conclude that HDL-associated serum PON1 can undergo S-glutathionylation under oxidative stress with a consequent reversible inactivation.     

Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases.