Immunological Significance of HMGB1 Post-Translational Modification and Redox Biology (1)
The human HMGB1 protein has 215 amino acid (aa) residues (MW: 25–30 kDa) that form two homologous DNA-binding domains (A-box, 1–79 aa; B-box, 89–162 aa) and a negatively charged C-terminal acidic tail (186–215 aa). HMGB1 is located in the nucleus as a result of bipartite nuclear localization signals (NLS; NLS1, 28–44 aa; NLS2, 179–185 aa) mediated by the nuclear importin karyopherin (KAP)-α1; however, the affinity between the two molecules is decreased by HMGB1 phosphorylation. The 201–205 aa residues in the C-terminal acidic tail play a crucial role in the antibacterial activity of HMGB1. HMGB1 residues 89–108 bind to Toll-like receptor (TLR) 4 and increase pro-inflammatory signaling, whereas residues 150–183 interact with the receptor for advanced glycation end products (RAGE) to regulate cell migration and stimulate inflammation. Residues 1–15 and 80–96 have been found to inhibit lipopolysaccharide (LPS)-induced cytokine production in a subclinical endotoxemia mouse model. Cys23 and Cys45 can form an intramolecular disulfide bond depending on the reactive oxygen species (ROS) concentration and environmental conditions under which HMGB1 binds to its ligands. The half-life of all-thiol-HMGB1 ranges from ~17 min in human serum and saliva to 3 h in cell culture medium. All-thiol-HMGB1 can exert chemoattractive effects by binding to CXCR4 and can prompt autophagy by binding to RAGE. Disulfide-HMGB1 can exert pro-inflammatory effects by binding to TLR4. Fully oxidized-HMGB1 is inert. Cys106 plays a crucial role in the translocation of HMGB1 from the nucleus to the cytosol. C23-C45 oxidation induces a shift between helix I and helix II in the A-box domain that reduces DNA binding affinity by altering the orientation of Phe37, resulting in cytoplasmic translocation. HMGB1 can also affect transcription in the nucleus, requiring rapid transition between the all-thiol-and disulfide forms of HMGB1.
HMGB1 can be passively released during various forms of cell death, including pyroptosis, apoptosis, autophagy, necroptosis, and necrosis. Apoptotic cells induce HMGB1 release. Necrosis is a form of premature cell death caused by the loss of membrane integrity, intracellular organelle swelling, and ATP depletion, and it has been shown that HMGB1 is passively released by necrotic or damaged cells.
HMGB1 has two acetylation clusters at Lys27–29 and Lys181–183, and it has been shown that nuclear localization is unaffected by mutating either Lys cluster. The poly(ADP-ribose) polymerase-1 (PARP1) induces the cytoplasmic translocation and extracellular secretion of HMGB1 by catalyzing its acetylation. Acetylated HMGB1 (six lysine residues for glutamines) can increase TNF-α production in RAW264.7 cells and reduces DC maturation. In HMGB1, Ser35, 39, 42, and 46 in NLS1, 181 in NLS2, and 53 close to NLS1 have been shown to be phosphorylated in macrophages after TNF-α and okadaic acid treatment, while Ser39, 53, and 182 of HMGB1 are phosphorylated by PKC-ζ in colon cancer cells. Lys42 in HMGB1 can be mono-methylated which significantly reduces its DNA binding activity, causing its translocation from the nucleus to the cytoplasm. Lys112 has also been found to be mono-methylated in HMGB1 and contribute toward its cytoplasmic localization. HMGB1 derived from calf thymus undergoes O-linked GlcNac glycosylation with sugars such as Fuc, Man, GalNH2, GlcNH2, and Gal monosaccharides. HMGB1 can also undergo N-linked glycosylation at two consensus (Asn37 and Asn137) residues and one non-consensus (Asn135) residue. N-glycosylation of HMGB1 is induced by PMA, TSA and LPS.
HMGB1 can take three different oxidation forms: an all-thiol form, disulfide form (Cys23-Cys45 intramolecular disulfide bond with Cys106 thiol form), and a fully oxidized form. During the active secretion of HMGB1, the Cys23-Cys45 intramolecular disulfide bond induces HMGB1 cytoplasmic translocation. The majority of HMGB1 released from necrotic cells being in an all-thiol state but that released from apoptotic cells being in a fully oxidized form. HMGB1 phosphorylation by PMA and/or acetylation by TSA promotes its nuclear transport and extracellular secretion.
By treating mouse BMDMs with 100 ng/mL of LPS, HMGB1 oxidation increased with time, with oxidation first detectable after just 30 min, and disulfide-HMGB1 was maintained for up to 4 h and then gradually decreased after 8 h. Despite rapid HMGB1 oxidation in the nucleus, its secretion began only after 4 h and increased up to 16 h.
HMGB1 containing three thiol-form cysteines exerts chemoattractive effects by forming a heterocomplex with CXCL12, which then binds to CXCR4 and induces cell migration. All-thiol-HMGB1 can also bind to RAGE and promote autophagy by inhibiting mTOR and promoting Beclin 1-Ptdlns3KC3 complex formation. Disulfide-HMGB1 stimulates cytokine production and inflammation by forming a complex with CD14 and MD-2 via TLR4. The disulfide bond between C23-C45 and the thiol form of C106 residues are not only required for binding TLR4 but also inducing the translocation of NF-κB and release of TNF-α.
Strategies for suppressing HMGB1 secretion can be divided into three categories: (1) small molecules inhibiting HMGB1 release; (2) neutralizing HMGB1 itself; and (3) blocking HMGB1 receptors.
