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The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis.

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  • 1. Laboratory of Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.
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    • Yang H 1
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Journal of Leukocyte Biology, 27 Feb 2013, 93(6):865-873
https://doi.org/10.1189/jlb.1212662 PMID: 23446148 PMCID: PMC4051189

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Abstract 

HMGB1 is a ubiquitous nuclear protein present in almost all cell types. In addition to its intracellular functions, HMGB1 can be extracellularly released, where it mediates activation of innate immune responses, including chemotaxis and cytokine release. HMGB1 contains three conserved redox-sensitive cysteines (C23, C45, and C106); modification of these cysteines determines the bioactivity of extracellular HMGB1. Firstly, the cytokine-stimulating activity of HMGB1 requires C23 and C45 to be in a disulfide linkage, at the same time that C106 must remain in its reduced form as a thiol. This distinctive molecular conformation enables HMGB1 to bind and signal via the TLR4/MD-2 complex to induce cytokine release in macrophages. Secondly, for HMGB1 to act as a chemotactic mediator, all three cysteines must be in the reduced form. This all-thiol HMGB1 exerts its chemotactic activity to initiate inflammation by forming a heterocomplex with CXCL12; that complex binds exclusively to CXCR4 to initiate chemotaxis. Thirdly, binding of the HMGB1 to CXCR4 or to TLR4 is completely prevented by all-cysteine oxidation. Also, the initial post-translational redox modifications of HMGB1 are reversible processes, enabling HMGB1 to shift from acting as a chemotactic factor to acting as a cytokine and vice versa. Lastly, post-translational acetylation of key lysine residues within NLSs of HMGB1 affects HMGB1 to promote inflammation; hyperacetylation of HMGB1 shifts its equilibrium from a predominant nuclear location toward a cytosolic and subsequent extracellular presence. Hence, post-translational modifications of HMGB1 determine its role in inflammation and immunity.

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J Leukoc Biol. 2013 Jun; 93(6): 865–873.
PMCID: PMC4051189
PMID: 23446148

The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis

Huan Yang,*,1 Daniel J. Antoine,† Ulf Andersson,‡ and Kevin J. Tracey*
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Abstract

HMGB1 is a ubiquitous nuclear protein present in almost all cell types. In addition to its intracellular functions, HMGB1 can be extracellularly released, where it mediates activation of innate immune responses, including chemotaxis and cytokine release. HMGB1 contains three conserved redox-sensitive cysteines (C23, C45, and C106); modification of these cysteines determines the bioactivity of extracellular HMGB1. Firstly, the cytokine-stimulating activity of HMGB1 requires C23 and C45 to be in a disulfide linkage, at the same time that C106 must remain in its reduced form as a thiol. This distinctive molecular conformation enables HMGB1 to bind and signal via the TLR4/MD-2 complex to induce cytokine release in macrophages. Secondly, for HMGB1 to act as a chemotactic mediator, all three cysteines must be in the reduced form. This all-thiol HMGB1 exerts its chemotactic activity to initiate inflammation by forming a heterocomplex with CXCL12; that complex binds exclusively to CXCR4 to initiate chemotaxis. Thirdly, binding of the HMGB1 to CXCR4 or to TLR4 is completely prevented by all-cysteine oxidation. Also, the initial post-translational redox modifications of HMGB1 are reversible processes, enabling HMGB1 to shift from acting as a chemotactic factor to acting as a cytokine and vice versa. Lastly, post-translational acetylation of key lysine residues within NLSs of HMGB1 affects HMGB1 to promote inflammation; hyperacetylation of HMGB1 shifts its equilibrium from a predominant nuclear location toward a cytosolic and subsequent extracellular presence. Hence, post-translational modifications of HMGB1 determine its role in inflammation and immunity.

Keywords: cytokine, redox, cysteine, acetylation

Introduction

HMGB1 was described originally as a nuclear DNA-binding protein. It is highly conserved evolutionarily and functions as a nuclear cofactor in transcription regulation [1,–4]. Like many other nuclear cofactors, HMGB1 was later discovered to have another role as an intercellular messenger molecule, released from a variety of cells into the extracellular milieu to act on specific cell-surface receptors. In this latter role, HMGB1 is a proinflammatory cytokine that may contribute to many inflammatory diseases, including sepsis [5]. Through engagement with its cell-surface receptors on immune cells, HMGB1 activates intracellular cascades that regulate immune cell functions, including chemotaxis and immune modulation.

The present understanding of the function of HMGB1 as a nuclear transcription regulator molecule and also as an intercellular messenger has advanced steadily during the last decade. Recent findings in molecular structural functional analyses of HMGB1 revealed that specific post-translational modifications determine the bioactivity of the molecule. Hence, here, we give an abridged review focusing on the mechanism of HMGB1 in sterile and infectious inflammation.

HMGB1

HMGB1 is the first identified member of the HMGB family [4,–10]. The HMGB family contains HMGB1, -2, and -3. (HMGB4 was originally identified as a new member of the family. Later, it was found to be identical to HMGB3 and thus, renamed as HMGB3; [8, 11].) The structure of all-HMGB proteins is highly conserved in the family (>80% sequence identity). The expression of HMGB1 is ubiquitous in almost all cell types examined, whereas the expression of HMGB2 is limited to lymphoid tissues and testis in adult animals; HMGB3 expression is restricted to embryos and hematopoietic stem cells [4,–8]. Among the three HMGB proteins, HMGB1 is the most abundant nonhistone nuclear protein, and it is to some degree also cytoplasmically expressed, as it shuttles back and forth from the nucleus [4, 12]. HMGB1 contains two folded helical DNA-binding motifs, called A and B boxes, and an acidic tail that contains a string of glutamic and aspartic acids. HMGB1 has two NLSs located in the A box (aa 28–44) and in the B box (aa 179–185), respectively (Fig. 1). Four conserved lysine residues are present in NLS1, and five are present in NLS2. They are susceptible to acetylation modification, resulting in nuclear exclusion and subsequent HMGB1 release [12, 13]. In the nucleus, HMGB1 supports the structure of the chromatin by binding DNA in a nonspecific sequence manner and is involved in the transcription regulation of genes [4, 14,–17] (Table 1). Intracellularly, HMGB1 has recently been demonstrated to be involved in autophagy and in PKR/inflammasome activation [13,–16, 18, 27,–30]. It is also a cell-surface, membrane-expressed protein on activated platelets and early neurons involved in neurite outgrowth during development and nerve regeneration [20, 21] (Table 1). Extracellular HMGB1 has become a focus in the field, as it is involved in a variety of immune responses, acting as a prototypic alarm signal [13, 22,–26, 31,–33]. Thus, HMGB1 has compartment-specific functions (Table 1). Structure-functional studies have revealed that the HMGB1 B box expresses the cytokine activity, whereas the A box alone acts as a specific HMGB1 antagonist but still with unresolved mechanisms [33,–35]. HMGB1 contains three cysteine residues at Positions 23, 45, and 106, which are sensitive to redox-dependent modifications (Fig. 1). Recent findings demonstrate that redox and acetyl modifications directly control cytokine and chemotactic activities of HMGB1 (Table 1).

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Structure characterizations of HMGB1.

HMGB1 contains two folded DNA-binding motifs, called A and B boxes. It has an acidic tail that contains a string of glutamic and aspartic acid. HMGB1 is a highly conserved cross-species, with over 98% sequence homology between mouse and human. HMGB1 has three cysteines, with two located at Positions 23 and 45 in A box and one at position 106 in B box. In addition, HMGB1 contains two NLSs, with one located in the A box (aa 28–44) and another located in the B box (aa 179–185). K, Hyperacetylated target lysine.

Table 1.

Compartment-Specific Functions of HMGB1
NucleusDNA binding: transcription regulation [14]
Stabilizing chromatin [15]
Chromosome assembly [16]
Cell replication [14]
DNA repair [17]
IntracellularPKR/inflammasome activation [13]
Autophagy: C106 is required for autophagy induction [18]
Cysteines, disulfide-bonded, are required for autophagy induction [18]
En route for release [1, 5]
Vesicle formation [19]
ExtracellularMembrane-bound: neurite outgrowth [20]
Platelet activation [21]
Extracellular: proangiogenic [22]
Antibacterial [23]
All-cysteine reduced: inflammatory, chemokine-like, chemotaxis [24, 25]
All-cysteine oxidized: noninflammatory [24, 26]
Cysteines, disulfide-bonded: inflammatory, cytokine-like, cytokine-inducing [3, 26]
Lysines hyperacetylated: inflammatory, cytokine-inducing [13]

HMGB1 has separate roles inside and outside of the cells. HMGB1 is highly expressed in the nucleus, where it regulates chromatin structure and gene transcription. HMGB1 is also present in the cytosol, where it is involved in inflammasome activation and autophagy. Extracellular HMGB1 has become a focus in the field, as it is involved in a variety of immune responses, including neurite outgrowth, platelet activation, and cytokine- and chemokine-like activity.

HMGB1 CYTOKINE ACTIVITY

HMGB1 is a cytokine mediator in the pathogenesis of inflammatory diseases

As part of an innate immunity response, HMGB1 can be actively secreted from multiple cell types including macrophages, monocytes, NK cells, DCs, endothelial cells, and platelets (reviewed in ref. [32]). HMGB1 can also be passively released from necrotic or damaged cells; both mechanisms can discharge significant amounts of extracellular HMGB1 [36]. Although apoptotic cells release substantially less HMGB1 compared with necrotic cells, macrophage engulfment of apoptotic cells may induce significant active HMGB1 release [37, 38]. Pyroptosis, programmed necrotic cell death induced by caspase-1, has recently been demonstrated to be an important pathway for active HMGB1 release driven by PKR and inflammasomes [13, 27]. Inflammasome-driven caspase-1 activation mediates pyroptosis and release of IL-1β, IL-18, and HMGB1 [27, 39].

Administration of HMGB1 to normal animals produces systemic inflammatory responses, including fever, weight loss, anorexia, acute lung injury, epithelial barrier dysfunction, arthritis, and death [31, 32]. Antagonistic HMGB1 treatment, based on antibodies, other HMGB1-specific antagonists, or pharmacological agents, has proven successful in a wide range of preclinical inflammatory disease models, resulting in reduced severity of diseases and reduced lethality [1, 5, 9].

HMGB1 is a critical mediator of lethality in sterile and infectious inflammation

Similar inflammatory responses are initiated by insults caused by sterile injury or infection. During infection, innate immunity is activated by foreign molecular products called PAMPs, which include, for example, LPS, dsRNA, and CpG-DNA. During sterile injury or ischemia, the same cells are activated by exposure to endogenous DAMPs, which include molecules such as heat shock proteins, uric acid, annexins, and IL-1α. DAMPs and PAMPs induce the same cascade of inflammation, tissue damage, and multiple organ failure [40]. HMGB1, released by activated immune cells and injured or necrotic cells, plays an important role in host responses to both types of threats; thus, it is a critical mediator in a final common pathway to morbidity and mortality during infection and sterile injury (Fig. 2A) [1, 5, 40].

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Redox-dependent regulation of HMGB1.

(A) Schematic overview of cell necrosis, pyroptosis, and apoptosis-induced HMGB1 release, different cysteine redox states, and relationship to cytokine activity. Necrosis- and pyroptosis-induced HMGB1 (disulfide-bonded form) interacts with MD-2 directly in the TLR4/MD-2 complex to elicit inflammatory responses. Cysteines (at Positions 23, 45, and 106 of HMGB1) are important for this binding interaction and subsequent cytokine activity. Cysteine all-reduced HMGB1 does not have TLR4-dependent cytokine activity, but it binds to CXCL12. This HMGB1-CXCL12 complex acts through CXCR4 and induces neukocytes recruitment and chemotaxis. Cysteine all-oxidized or C106-oxidized HMGB1 (released by apoptotic cells) prevents HMGB1 from having cytokine or chemotactic activity.(B) Redox-dependent effects on HMGB1-induced cytokine release. Inactive HMGB1, created chemically by a formation of mercury thiolate C106 or by mutation of C23, C45, or C106 to other residues (serine or alanine), prevents HMGB1-TLR4-dependent cytokine activity. SH, thiol; S, serine; A, alanine; S-S, disulfide bond; Hg, mercury; SO3H, sulfonic acid.

HMGBs are universal sentinels for nucleicacid-mediated innate-immune responses

HMGB1 and its family members (HMGB2 and -3) are universal sensors for cytosolic nucleic acids [41,–43]. Tian et al. [41]and Ivanov et al. [44] simultaneously arrived at the same conclusion—that HMGB1 is involved in DNA-containing, complex-mediated immune responses via TLR9. Later, Yanai et al. [42] confirmed that HMGB1 binds to all immunogenic nucleic acids examined and mediates immune responses by stimulating the transcription of type 1 IFN, IL-6, and RANTES from immune cells or mouse embryonic fibroblasts. Indeed, HMGB1-deficient cells express much diminished immune responses when stimulated with viral-like DNA or RNA compared with WT controls. Knockdown of all three HMGB proteins inhibits the response to viral nucleic acid stimulation compared with a single HMGB1 knockdown, suggesting that HMGB proteins share the same functionality [42, 43]. Thus, HMGB proteins play an essential role as universal sentinels in nucleic acid-activated innate immune responses but with still unresolved mechanisms.

RECEPTORS MEDIATING HMGB1 ACTIVITY

Family of HMGB1 receptors

Once released into the extracellular milieu, HMGB1 binds to cell-surface receptors to elicit inflammatory responses. Receptors that mediate HMGB1 signaling include the receptor for advanced glycation end products, TLR2, -4, and -9, macrophage antigen-1, syndecan-3, CD24-Siglec-10, CXCR4, and T cell Ig mucin-3 [5, 24, 25, 32, 41, 43, 45, 46]; TLR4 is the primary receptor needed for promotion of macrophage activation, cytokine release, and tissue damage [3]. Apart from a direct receptor interaction, HMGB1 may form heterocomplexes with other molecules, such as IL-1, CXCL12, DNA, RNA, histones, or LPS, which generate synergistic responses compared with those produced by the individual components. These complexes signal via the reciprocal receptors for the HMGB1-partner molecules as the mode of action [1, 25, 32]. HMGB1, acting on its own or in heterocomplex formation, initiates innate immunity responses, including chemotactic activity and release of proinflammatory cytokines, and causes fever, epithelial barrier dysfunction, and acute and chronic inflammation.

Role of MD-2 in HMGB1-TLR4 signaling

TLR4 activity and interaction with its ligands depend on a molecular collaboration with the extracellular adaptor protein MD-2 [47, 48]. With the use of biosensor-based surface plasmon resonance (Biacore, GE Healthcare, Piscataway, NJ), we observed that HMGB1, just like LPS, binds MD-2 with a high affinity (apparent Kd=8 nM), whereas it does not bind to TLR4 alone (Fig. 2A) [49]. Noncytokine-inducing HMGB1, created by formation of mercury thiolate C106, by cysteine substitutions, or by redox modifications, does not allow HMGB1-binding interaction with MD-2, TNF production, or NF-κB nuclear translocation in macrophages. The results demonstrate that HMGB1, like LPS, needs to bind to the MD-2 component of the TLR4/MD-2 complex to elicit cytokine induction, and that the redox state of HMGB1 cysteines controls this binding interaction (Fig. 2B). MD-2 knockdown experiments were performed by transfection of specific siRNA targeting MD-2 in macrophage-like RAW 264.7 cells or human monocytic THP-1 cells. Significantly less TNF release and NF-κB activation with HMGB1 stimulation were observed compared with cells transfected with control siRNA, supporting that MD-2 is required for this mode of HMGB1 signaling in macrophages/monocytes [49]. Gain-of-function experiments confirmed that MD-2 is sufficient to restore HMGB1-induced IL-8 release in human embryonic kidney 293 cells overexpressing TLR4 (our unpublished data). These results reveal that MD-2 is critical for HMGB1-induced TLR4 responses and that all cysteines in HMGB1 are important for the MD-2 interaction to take place (Fig. 2).

REDOX STATE AND CYTOKINE ACTIVITY OF HMGB1

HMGB1 may undergo extensive post-translational modifications, including reversible and terminal cysteine oxidation, acetylation, methylation, ADP ribosylation, glycation, and phosphorylation [1, 4, 5]. Some of these modifications have been demonstrated to influence DNA binding and stability, cellular localization, and the regulation of transcription mediated by HMGB1 [4]. Recent studies emphasize that the redox states of the three conserved cysteine residues with HMGB1 regulate its receptor-binding ability and subsequent biological outcome [18, 24, 26, 28,–30, 50]. As described below, these post-translational redox mechanisms will control the proinflammatory activity of HMGB1 during the pathogenesis of sepsis and other HMGB1-mediated inflammatory diseases.

HMGB1 cytokine activity requires C23 and C45, forming a disulfide link, and C106 in the reduced form (disulfide-bonded HMGB1)

Reduced C106 (thiol) is required for HMGB1 cytokine activity.

The cytokine-inducing activity of HMGB1 is dependent on the redox state of C106, residing within the B box DNA-binding domain of HMGB1 [35]. C106 expressing a thiol group is mandatory for the HMGB1 binding to TLR4/MD-2. Substituting C106 HMGB1 prevents its binding interaction with TLR4/MD-2 and the subsequent stimulation of cytokine release from macrophages [3]. Furthermore, a synthetic 20-mer peptide containing C106 mediates TNF release in macrophages, whereas replacing C106 with a serine residue abolishes this capacity [3] (Fig. 2). Other modifications of C106, by exposure to mercury forming a mercury thiolate group or terminal oxidation to sulfonate by H2O2 eliminate the cytokine-stimulating activity of HMGB1 (Fig. 2A and B). Together, these results establish that C106 in its reduced state is required for HMGB1 signaling through TLR4 to stimulate cytokine release and inflammation.

A disulfide bond between C23 and C45 is also required for the HMGB1 cytokine activity.

The structure-functional relationship of C23 and C45 for the ability to mediate cytokine induction was, until recently, unknown. LC-MS/MS analysis reveal that the cytokine induction by HMGB1 requires the presence of a disulfide bond between C23 and C45, coinciding with C106 being expressed in its thiol form [26]. Reduction of the C23–C45 disulfide linkage by exposure to the reducing agent DTT or further oxidation using H2O2 to generate sulfonic acid groups completely preventd the cytokine activity of HMGB1 (Fig. 2). Similarly, substituting C23 or C45 with serine or alanine also reduces its cytokine activity markedly (Fig. 2B). A previous report highlights the circumstance that C23 and C45 readily form a disulfide bond that increases the stability of the full-length HMGB1 molecule [51]. Thus, these results clearly demonstrate that specific post-translational redox mechanisms control the cytokine activity of HMGB1 and that the redox states of all three cysteines in HMGB1 are critical for HMGB1 to act like a cytokine.

In vivo studies support the importance of a correct redox state for HMGB1 cytokine activity.

The functional in vivo relevance of redox-modified HMGB1 has been confirmed in experimental animal models. The inflammatory component of APAP-mediated hepatotoxicity is a HMGB1-mediated process that has been used for these studies [36, 52,–54]. Treatment with HMGB1-specific antagonists markedly ameliorates APAP-induced hepatotoxicity. The toxic APAP exposure predominantly generated necrotic hepatocyte cell death in the murine model accompanied by massive systemic release of nuclear HMGB1, which during the early stage of disease, expresses all three cysteines in reduced form mediating chemotaxis. The initial tissue damage is followed by activation of inflammatory cell recruitment and a secondary cytokine storm mediated by the accumulated innate-immune cells that aggravates the liver damage. The cellular activation is caused by disulfide-bonded HMGB1 enabling TLR4/MD-2 signaling. HMGB1 serum levels are also increased markedly during the resolution phase of the injury when the molecule is terminally oxidized, expressing the C106 with a sulfonic acid group [52, 53]. The functional consequences of the course of the redox changes of HMGB1 in vivo are thus in line with those established from in vitro studies.

These studies also demonstrate important functional relationships among metabolism, mode of cell death, and HMGB1 biology. Mice that were fasted for 24 h prior to the APAP overdose could not generate apoptotic hepatocyte cell death during phases of inflammation and tissue repair, because of depletion of basal ATP needed for the energy-dependent programmed cell death driving caspase-3 activation [52]. The lethality and severity of disease were much greater in fasted mice compared with well-fed animals after an APAP overdose. Caspase-3-driven apoptosis in fed mice generated terminal HMGB1 oxidation with no or low inflammatory capacity, a process that did not occur in fasted mice during the necrotic process [52].

All-thiol HMGB1 is a potent chemotactic mediator

The redox state requirements for HMGB1 to induce its potent chemotactic activity to recruit neutrophils and monocytes to inflammatory sites have been recently clarified to be distinctly different than those required for the cytokine-inducing activity [24]. All three cysteines must be fully reduced for HMGB1 to exert chemotaxis. This molecular form of HMGB1 enables a formation of a heterocomplex with the chemokine CXCL12 (stromal cell-derived factor 1), which will signal via the CXCR4 receptor complex in a synergistic mode (Fig. 2A). Terminal oxidation of any of the cysteines by ROS completely abrogates the chemotactic activity. The cysteines per se are not needed for chemotaxis, as they can be substituted with serines with preserved or even enhanced performance regarding leukocyte recruitment [24]. This is thus in great contrast to the requirements for cytokine induction. The vital condition for HMGB1 to enhance chemotaxis is that none of the cysteines is oxidized for reasons that need further investigation.

The redox state of HMGB1 and its cytokine activity is a reversible process

DTT-exposed HMGB1 expressing all-thiol cysteines does not stimulate TNF production in cultured macrophages. However, mild oxidation with low concentrations of H2O2 of this DTT-exposed HMGB1 restored the TNF-inducing activity by inducing a disulfide bridge between C23 and C45 in the A box domain (disulfide-bonded form), demonstrating that the cytokine activity of HMGB1 is reversible (Fig. 3). This feature of HMGB1 has clinical implication, as there is a shift of redox states of HMGB1 during tissue injury exemplified in the APAP-induced liver-injury studies. After APAP administration, the initially released HMGB1 isoform is the chemotactic, all-thiol form, whereas later, the predominant form of serum HMGB1 during the onset of severe hepatic inflammation is the inflammatory (disulfide-bonded) form. As hepatic inflammation resolves, the predominant form of circulating HMGB1 contains the C106 terminally oxidized [26]. Thus, the inflammatory forms of serum HMGB1 are correlated with hepatic inflammation during APAP-induced hepatic toxicity.

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Redox modification of HMGB1 is reversible.

DTT-exposed HMGB1 did not stimulate TNF. The same HMGB1 treated with H2O2 (mild; 50 μM for 2 h) could renature the TNF-inducing activity. The nature of HMGB1 was examined by using tryptic digestion, LC-MS/MS [26].

Redox state dynamics of HMGB1 during cell death and injury

The redox state of intracellular and extracellular HMGB1 in cell death and injury is dynamic. Studies have shown that intracellular HMGB1 in a resting cell is predominantly an all-cysteine-reduced form, whereas secreted HMGB1 contains both all-thiol and disulfide-bonded forms [24]. Also, we found that disulfide-bonded HMGB1 is present predominantly in serum of mice with APAP-induced liver toxicity, and this correlates with the severity of disease [26, 52]. Elevated intracellular levels of ROS by H2O2 or SOD1 (Cu, Zn SOD) siRNA promote HMGB1 release in several types of cells, including macrophages, suggesting that ROS plays a critical role in the induction of HMGB1 release or active secretion [28, 55,–58]. Natural or synthetic antioxidants inhibit cell death and inflammatory responses that have been shown to inhibit HMGB1 release in several disease models [29]. Collectively, these studies indicate that the redox status of HMGB1 is modulated in a dynamic microenvironment [59].

ACETYLATION AND HMGB1 CYTOKINE ACTIVITY

Hyperacetylation

Acetylation of key lysine residues within the two NLS sites of HMGB1 is a decisive regulatory mechanism for intracellular shuttling of HMGB1, leading to active release of HMGB1 from activated monocytes and macrophages [12, 13] and possibly from other cell types too. Hyperacetylation of the NLS-located lysines shifts the equilibrium of HMGB1 from its predominant nuclear position toward a cytoplasmic accumulation by preventing the nuclear re-entry of HMGB1 in its continuous shuttle between the nucleus and the cytosol (Fig. 4).

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Acetylation and redox status of HMGB1 released by macrophages in response to inflammasome activation (MSU, ATP, ALU) or by necrosis (freeze/thaw).

Pyrotosis, induced by MSU, ATP, or ALU, causes PKR/inflammasome activation and HMGB1 translocating from nucleus to cytosol/extracellular release. MS analysis revealed that HMGB1 thus released is acetylated on NLS1 and -2 regions, whereas necrosis (induced by repeated freeze/thaw)-induced HMGB1 release is not acetylated. MS characterization of the redox status of three cysteines of HMGB1 showed that pyroptosis- and necrosis-induced HMGB1 contains the cytokine-stimulating (disulfide-bonded) and reduced (by necrosis) forms of HMGB1 [13].

Serum levels of acetylated HMGB1 in acetaminiphen-induced hepatotoxicity in patients have been demonstrated to be a sensitive and specific biomarker to predict the clinical outcome [53]. HMGB1 released in vivo following liver ischemia/reperfusion in mice has been demonstrated to be hyperacetylated, and hepatocytes exposed to oxidative stress in vitro release hyperacetylated HMGB1 [39]. These effects are indicated to be caused by decreased histone deacetylase activity failing to remove acetyl groups adequately from lysine residues.

When determining the impact of post-translational modifications on HMGB1 structure-function relationships, it is important to note that acetylation of nearby lysine residues might change the electrostatic potential and affect the pKa of cysteine thiol groups; however, no lysine amino group is present within 8 Å from the thiol groups in the three-dimensional structure of the HMGB1, and therefore, an impact on the regulation of the proinflammatory function of HMGB1 is unlikely [51].

Pyroptosis and hyperacetylation

The recent published findings by Lu et al. [13] demonstrate that hyperacetylated HMGB1 is a novel biomarker for pyroptosis (Fig. 4). In primary mouse macrophages stimulated with prototypical danger signals, such as ATP, MSU, or ALU, HMGB1 is released extracellularly via activation of PKR and the inflammasome system [27, 39]. Analyses by MS/MS revealed that HMGB1 thus released was highly acetylated at NLS1 and -2 regions. In comparison, HMGB1 released from macrophage cultures by necrosis induced by freeze-thawing was not hyperacetylated in the NLSs (Fig. 4). Redox status of three cysteines of HMGB1 revealed that HMGB1 released by pyroptosis may contain active (disulfide-bonded) and terminally oxidized forms, whereas HMGB1 releases during necrosis may contain MD-2/TLR4-binding or all-thiol forms (Fig. 4). Thus, multiple observations indicate that HMGB1 released during inflammasome activation is acetylated [12, 13, 60]. Inflammasomes are multiprotein complexes that promote the secretion of the proinflammatory cytokines IL-1β and IL-18, as well as pyroptosis [27, 39]. Our data further demonstrate that HMGB1 is a novel tool to identify and quantify pyroptosis, a central physiological process that until now, lacks a suitable biomarker. A more recent study showed that priming did not greatly affect the magnitude of HMGB1 release from macrophages during pyroptosis, whereas the priming by various cell-surface TLR agonists resulted in a switch of released HMGB1 from a chemotactic (cysteine all-thiol) form to a TLR4-agonist form (disulfide bonded) from macrophages [61].

REDOX MODIFICATION OF HMGB1 AND AUTOPHAGY

Autophagy is a lysosome-mediated, self-eating process that is important for cell survival during stress [18, 28,–30]. HMGB1 is a critical regulator of autophagy. Stimuli that enhance ROS can also increase HMGB1 translocation from the nucleus to the cytosol and thereby, enhance autophagic flux [28]. Cysteine modifications of HMGB1 alter its activity of inducing autophagy [18, 28,–30]. Mutation of C106 within HMGB1, but not the neighboring C23 and C45, promotes HMGB1 cytosolic translocation and autophagy. Treatment with cysteine all-reduced, but not oxidized, HMGB1 increases autophagy in cancer cells [30]. Moreover, the disulfide bridge between C23 and C45 of HMGB1 is required for its binding to Beclin 1 to sustain the autophagy process [62]. Thus, HMGB1, regulated by post-translational redox modifications, plays an important role in autophagy that enhances cell survival in response to cell stress.

METHODS FOR DETECTING POST-TRANSLATIONAL MODIFICATIONS OF HMGB1 AND THEIR LIMITATIONS

The introduction of protein MS-based analysis, coupled with molecular techniques and immunological readouts, has helped elucidate the structure-function-based relationship associated with redox-dependent modifications of cysteine residues or acetylation modifications on lysines of HMGB1. With respect to redox, the precise determination of chemical species in published reports has been made possible through the enzymatic cleavage of HMGB1 following differential alkylation of thiol groups (followed by reduced and then alkylated disulfide bonds). These peptide mixtures are then fractionated by nano-LC and analyzed by MS/MS. This approach not only permits the determination of particular post-translational modifications but can also pinpoint the precise amino acid modified. However, despite the accuracy and sensitivity of defining post-translational modifications on peptides by MS, several limitations exist. Firstly, published methods are low-throughput and not amenable to high-throughput screening. Secondly, these methods are expensive and dependent on skilled, trained analysts and a specialized proteomic laboratory that might not be accessible to all researchers. Unfortunately, to date, there are no specific antibodies to identify different functional isoforms of HMGB1, and MS/MS-based analysis presently remains the only option for accurate identification. Until the development of ELISA-based assays, which offer a more accessible and higher throughput option, there will be a trade-off between speed of analysis and precision of identification of the analyte. Thirdly, the differential ionization of peptides also represents a challenge for the absolute and relative qualification of different peptides (with defined redox or acetyl modifications) across sample sets and regarding HMGB1. This represents an area of unmet need and one not addressed by current published reports. However, advances in isobaric tag for relative and absolute quantitation technology and the use of heavily labeled peptide standards will further permit such analysis in future studies. Now that precise chemical characterization of redox-dependent changes in HMGB1 or acetyl modifications can be related to biological function, a focus of research should focus on the methods for absolute quantification. Currently, few reports have been published that describe accurate quantification of a post-translationally modified HMGB1 isoform by MS [53]. This is fundamental to efforts in translational research so that basic findings may yield clinical impact, for example, the potential of HMGB1 functional isoforms to represent disease-specific biomarkers.

SUMMARY

In summary, HMGB1 is a molecule with many faces showing different activities with redox modifications, with or without hyperacetylation, during inflammation and apoptosis. The cytokine-stimulating HMGB1 is a disulfide-bonded form (a disulfide bond between C23 and C45, and C106 remains in its reduced thiol form). This form of HMGB1 binds to MD-2 in the TLR4/MD-2 complex and induces TNF release and NF-κB activation in cultured macrophages [26]. HMGB1 C106 in thiol form is necessary for this TLR4/MD-2 interaction and subsequent cytokine stimulation. Modifications of C106, by formation of mercury thiolate C106 or by mutation of C106 to another amino acid residue, prevent interaction with the TLR4/MD-2 complex and the induction of cytokine release. Furthermore, HMGB1 requires a C23–C45 disulfide bond for its cytokine activity. Mutations of C45 or C23 also abolish the cytokine activity of HMGB1 [26]. Thus, HMGB1 simultaneously needs to have C106 in its reduced thiol form and C23–C45 in a disulfide linkage to bind to TLR4/MD-2 for subsequent cytokine stimulation. In addition, the all-thiol form of HMGB1 acts only as a chemotactic mediator. All-oxidized HMGB1 has no cytokine-stimulating or chemotactic activity. Pyroptosis-induced HMGB1 is cytokine-stimulating, contains a disulfide-bonded form, and is also hyperacetylated HMGB1.

The data discussed in this review demonstrate that the post-translational modifications are crucial for HMGB1 functionality as a mediator during infection and sterile injury (Fig. 5). These recent advances and the advent of MS-based analysis reveal a novel mechanism to regulate the activities of HMGB1 and suggest that HMGB1 might be a key therapeutic target for redox modification during inflammation and its resolution. This information is central to gain insights into the function of HMGB1 for controlling immune responses.

An external file that holds a picture, illustration, etc. Object name is zgb0061358450005.jpg
Schematic presentation of HMGB1 with different redox or acetylation status and corresponding immune responses.

Tissue damage induces the release of HMGB1 with all-cysteines reduced, whereas this form of HMGB1 does not stimulate cytokine release; it recruits leukocytes to the site of injury. During infection or later stage of injury, HMGB1 released is acetylated or disulfide-bonded, and it stimulates cytokine release.

ACKNOWLEDGMENTS

This work was supported by grants from NIGMS (to K.J.T.), from NIGMS (GM098446; to H.Y.), and from the Swedish Medical Research Council (to U.A.). D.J.A. is supported by a Wellcome Trust Research Fellowship and the Medical Research Council (UK), grant number G0700654.

Footnotes

ALU
adjuvant aluminum
APAP
acetaminophen
C23/45/106
cysteine 23/45/106
DAMP
damage-associated molecular pattern
H2O2
hydrogen peroxide
HMGB1
high-mobility group box 1
LC
liquid chromatography
MD-2
myeloid differentiation protein 2
MS/MS
tandem mass spectrometry
MSU
monosodium urate
NIGMS
National Institute for General Medical Science
NLS
nuclear localization sequence
PKR
dsRNA-dependent protein kinase
siRNA
small interfering RNA

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