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

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: acetylation; cysteine; cytokine; redox.

Figure 1.. 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.

Figure 2.. 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.

Figure 3.. 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].

Figure 4.. 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].

Figure 5.. 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.