Full-length HMGB1 Research Articles

Phosphorylation by Protein Kinase C Weakens DNA-Binding Affinity and Folding Stability of the HMGB1 Protein.

The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.

Influence of an Intrinsically Disordered Region on Protein Domains Revealed by NMR-Based Electrostatic Potential Measurements.

Many human proteins possess intrinsically disordered regions containing consecutive aspartate or glutamate residues ("D/E repeats"). Approximately half of them are DNA/RNA-binding proteins. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we investigated the electrostatic properties of D/E repeats and their influence on folded domains within the same protein. Local electrostatic potentials were directly measured for the HMGB1 protein, its isolated D/E repeats, and DNA-binding domains by NMR. The data provide quantitative information about the electrostatic interactions between distinct segments of HMGB1. Due to the interactions between the D/E repeats and the DNA-binding domains, local electrostatic potentials of the DNA-binding domains within the full-length HMGB1 protein were largely negative despite the presence of many positively charged residues. Our NMR data on counterions and electrostatic potentials show that the D/E repeats and DNA have similar electrostatic properties and compete for the DNA-binding domains. The competition promotes dissociation of the protein-DNA complex and influences the molecular behavior of the HMGB1 protein. These effects may be general among the DNA/RNA-binding proteins with D/E repeats.

The motility of breast cancer cells is stimulated by HMGB1/RAGE interaction but the truncated form lacking the C terminus has no effect

HMGB1/RAGE is identified as a ligand-receptor pair that plays an important role in tumorogenesis. HMGB1 and RAGE levels are higher in most human tumors and their overexpression is associated with tumor progression. The causes of breast cancer are still poorly understood. One reason might be the existence of subtypes within various cellular mechanisms as hormone-dependent and hormone -independent malignant processes. We investigated the effect of HMGB1 protein and its truncated form lacking the C terminus on the RAGE expression and cell motility of breast cancer cell lines; MCF7-noninvasive, MDA-MB-231-invasive and normal breast epithelial one MCF10. The results demonstrate that the effects of HMGB1 and HMGB1∆C through RAGE association are observed exclusively for the hormone independent MDA-MB-231 cell line. The mobility of MDA-MB-231 cells was stimulated only by the full length HMGB1. Our results suggest that HMGB1/RAGE signaling should be considered as an essential process for the development of hormone independent breast cancers with great invasive potential. The truncated form plays the role of a blocking molecule that ”locks” the receptor and inactivates it. This makes the tailless molecule a promising therapeutic agent that competes for the biologically active HMGB1 ligand and prevents the downstream signaling through RAGE.

Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain

BackgroundHigh mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. HMGB1 interacts with toll-like receptors (TLRs) but contradictory evidence regarding its identity as a TLR2 ligand persists. The aim of this study was to investigate if highly purified HMGB1 interacts with TLR2 and if so, to determine the functional outcome.MethodsFull length or C-terminal truncated (Δ30) HMGB1 was purified from E.coli. Binding to TLR2-Fc was investigated by direct-ELISA. For the functional studies, proteins alone or in complex with peptidoglycan (PGN) were added to human embryonic kidney (HEK) cells transfected with functional TLR2, TLR 1/2 or TLR 2/6 dimers, macrophages, whole blood or peripheral blood mononuclear cells (PBMCs). Cytokine levels were determined by ELISA.ResultsIn vitro binding experiments revealed that Δ30 HMGB1, lacking the acidic tail domain, but not full length HMGB1 binds dose dependently to TLR2. Control experiments confirmed that the interaction was specific to TLR2 and could be inhibited by enzymatic digestion. Δ30 HMGB1 alone was unable to induce cytokine production via TLR2. However, full length HMGB1 and Δ30 HMGB1 formed complexes with PGN, a known TLR2 ligand, and synergistically potentiated the inflammatory response in PBMCs.ConclusionsWe have demonstrated that TLR2 is a receptor for HMGB1 and this binding is negatively regulated by the C-terminal tail. HMGB1 did not induce functional activation of TLR2 while both full length HMGB1 and Δ30 HMGB1 potentiated the inflammatory activities of the TLR2 ligand PGN. We hypothesize that Δ30 HMGB1 generated in vivo by enzymatic cleavage could act as an enhancer of TLR2-mediated inflammatory activities.

Activation of Toll-Like Receptor 5 Immune Signaling by HMGB1

Human High Mobility Group Box-1 (HMGB-1) is an ubiquitously expressed nuclear protein which primarily functions as an architectural chromatin binding factor that binds to DNA in a structure specific manner. Recently, it has been linked to the pathogenesis of several inflammatory diseases regulating the innate immune system by two specific toll-like receptors (TLRs) such as TLR2 and TLR4. Here we present human HMGB-1 as an inflammatory regulator for TLR5 in immune signaling. The binding modes between these two proteins were demonstrated by a variety of cellular, biophysical and structural studies. The cellular assays revealed that HMGB1/TLR5 interaction invokes an elevation of downstream pro-inflammatory cytokines such as tumor necrosis factor-α, internuclein-8 (IL-8) which are critical in MyD88 dependant inflammatory pathways. Flourescence anisotropy assays revealed the binding constant (Kd) of 2.2uM between these two proteins with a further validation of interaction sites by solution NMR spectroscopy. For NMR structural studies 15N, 13C uniform labeled full length and tailless HMGB1 proteins was expressed and purified from E.coli BL21(DE3)pLysS cells and the 2D HSQC spectra were used for backbone and side chain residue chemical shift mapping from previously published spectra. Change in chemical shifts showed the direct molecular interactions between the acidic tail of HMGB1with the extracellular domain of TLR5 in physiological conditions. Thus, our studies not only provide compelling new insights into the TLR5 signal transduction by HMGB1 but also open the avenue of future therapeutic development.

Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein.

HMGB1 protein and linker histone H1 have overlapping binding sites in the nucleosome. HMGB1 has been implicated in many DNA-dependent processes in chromatin involving binding of specific proteins, including transcription factors, to DNA sites pre-bent by HMGB1. HMGB1 can also act as an extracellular signaling molecule by promoting inflammation, tumor growth a metastasis. Many of the intra- and extracellular functions of HMGB1 depend on redox-sensitive cysteine residues of the protein. Here we report that mild oxidization of HMGB1 (and much less mutation of cysteines involved in disulphide bond formation) can severely compromise the functioning of the protein as a DNA chaperone by inhibiting its ability to unwind or bend DNA. Histone H1 (via the highly basic C-terminal domain) significantly inhibits DNA bending by the full-length HMGB1, and the inhibition is further enhanced upon oxidization of HMGB1. Interestingly, DNA bending by HMGB1 lacking the acidic C-tail (HMGB1ΔC) is much less affected by histone H1, but oxidization rendered DNA bending by HMGB1ΔC and HMGB1 equally prone for inhibition by histone H1. Possible consequences of histone H1-mediated inhibition of DNA bending by HMGB1 of different redox state for the functioning of chromatin are discussed.

Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk (P2001)

Abstract Tumor-induced immune suppression is driven by immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in most patients with cancer. They block T cell activation and drive type 2 immunity. Cross-talk with macrophages enhances MDSC secretion of pro-tumor molecules such as IL-10 and decreases macrophage production of IL-12. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin or Ethyl Pyruvate reduced lung metastases, while treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. BALB/c mice carrying 4T1 tumor cells down-regulated for HMGB1 by shRNA had an extended survival time relative to mice with irrelevant shRNA 4T1 tumor. Treatment of MC38 tumor-bearing mice with the anti-inflammatory A box domain of HMGB1 reduced primary tumor growth. In in vitro experiments, HMGB1 inhibitors decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages, and reduced MDSC suppressive potency for T cells. HMGB1 inhibitors also reduced MDSC differentiation from bone marrow progenitor cells. These findings indicate that full-length HMGB1 drives tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC accumulation.

Abstract 461: Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction.

Abstract Cancer immunotherapy aims to harness the power of the immune system to fight advanced stages of cancer. A main hindrance to cancer immunotherapy is tumor-induced immune suppression. Within the tumor microenvironment are a plethora of immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in nearly all types of cancer blocking T cell activation and shifting immunity towards a type 2 tumor-promoting phenotype through cross-talk with macrophages that enhances the secretion of pro-tumor molecules such as IL-10, and simultaneously drops IL-12 production by macrophages. MDSC accumulation is induced by pro-inflammatory cytokines including IL-1β, IL-6 and VEGF as well as alarmins such as S100A8/9. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Inhibition of HMGB1 in vitro by chemical inhibitors Glycyrrhizin and Ethyl Pyruvate decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages. Ethyl Pyruvate also reduced MDSC suppressive capacity in transgenic T cell suppression assays, suggesting that HMGB1 regulates multiple aspects of MDSC function. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin and Ethyl Pyruvate reduced lung metastases and did not impact primary tumor progression. Treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. HMGB1 consists of two domains, the pro-inflammatory B box domain and an anti-inflammatory A box domain. Treatment of C57BL/6 mice with MC38 tumor with A box significantly reduced primary tumor growth and decreased MDSC levels in the blood. These findings indicate that full-length HMGB1 drives inflammation and tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC. These results suggest that HMGB1 drives MDSC accumulation and tumor progression and that A box can be used to target MDSC-mediated tumor-induced immune suppression. (Research supported by NIH RO1CA115880 and RO1CA84232) Citation Format: Katherine Parker, Suzanne Rosenberg, Pratima Sinha, Huan Yang, Kevin Tracey, Jianhua Li. Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2013-461

The DNA Binding and Bending Activities of Truncated Tail-less HMGB1 protein are Differentially Affected by Lys-2 and Lys-81 Residues and Their Acetylation

The role of lysines 2 and 81 as target sites for acetylation in full-length HMGB1 and truncated tail-less protein, respectively, has been studied by mutation analysis for the abilities of these proteins to bind and bend DNA. The DNA bending ability of truncated tail-less HMGB1 containing Lys-2 mutated to alanine does not differ from that of the wild-type protein, while the same mutation of Lys-81 reduced the bending capacity of the mutant protein. These data demonstrate that Lys-81 is critical for the DNA bending ability of truncated HMGB1. Such a conclusion is further confirmed by the experiments carried out with CBP-acetylated proteins: acetylation of Lys-2 in mutant protein K81/A81 alleviated DNA bending and induced DNA end-joining. On the contrary, the acetylation of Lys-81 in the mutant K2/A2 enhanced the bending potential of HMGB1∆C. Regarding the ability of HMGB1 to specifically bind bent DNA, the individual mutations of either K2 or K81 as well as the double mutation of both residues to alanine were found to completely abolish binding of truncated tail-less HMGB1 to cisplatin-modified DNA. We conclude that unlike the case with the bending ability of truncated HMGB1, where Lys-81 has a primary function, Lys-2 and Lys-81 are both critical for the protein's binding to cisplatin-modified DNA. The mutation K2/A2 in full-length HMGB1 and acidic tail removal induce the same conformational changes. Any further substitutions at the acetylable lysines in the truncated form of HMGB1 do not have an additional effect.

Tail-Mediated Collapse of HMGB1 Is Dynamic and Occurs via Differential Binding of the Acidic Tail to the A and B Domains

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1–DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box–tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. 15N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a “sandwich”-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein–protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Distinct Domains in HMGB1 Are Involved in Specific Intramolecular and Nucleosomal Interactions

HMGB1 is composed of two DNA-binding domains and a long acidic tail at the C-terminus. The acidic tail interacts with the DNA-binding domains of HMGB1 and with core histone H3 in the nucleosome. These interactions are important for modulation of the DNA and chromatin binding activities of HMGB1, as well as biological functions of HMGB1. However, the interactions are not fully characterized, because the tertiary structure of full-length HMGB1 is still unknown. Here we use chemical cross-linking, mass spectrometry, and epitope masking analysis to perform a detailed characterization of the inter- and intramolecular protein interactions of the acidic tail of HMGB1. We show that specific regions of the acidic tail participate in intramolecular interactions with Lys2 of HMGB1 and in intermolecular interactions with Lys36 and Lys37 of histone H3. The acidic tail is oriented by its location adjacent to the C-terminus of helix III of DNA-binding HMG box A in the HMGB1 molecule. These results suggest that the acidic tail modulates the biological functions of HMGB1 through these specific interactions.

Construction and characterization of the HMGB1 mutant as a competitive antagonist to HMGB1 induced cytokines release

Recent studies indicate that the High Mobility Group Box-1 protein (HMGB1) acts as a potent proinflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. The proinflammatory cytokine activity of HMGB1 has become a therapeutic target. In this study, we cloned the cDNA encoding human HMGB1 and constructed HMGB1 mutants using a one-step opposite direction PCR. The binding of the HMGB1 mutants to THP-1 cell and the cytokine activities of these HMGB1 mutants were observed. Results showed that the HMGB1 Mut (102–105), one of the HMGB1 mutants, in which amino acids 102–105 (FFLF) were replaced with two Glys, significantly decreased the full-length HMGB1 protein induced TNF-α release in human monocyte cultures. The results indicate that we have developed a novel recombinant HMGB1 mutant that competitively antagonizes the proinflammatory activity of HMGB1. This may be of significant importance in providing a new anti-inflammatory strategy for the treatment of severe sepsis and other inflammatory disorders.

Activation of Poly(ADP)-ribose Polymerase (PARP-1) Induces Release of the Pro-inflammatory Mediator HMGB1 from the Nucleus

Necrotic cells release inflammatory mediators that activate cytokine production from innate immune cells. One mediator of this activation is high mobility group box 1 protein (HMGB1). HMGB1 is normally a chromatin-associated protein and is sequestered at condensed chromatin during apoptosis. How it is released from chromatin during necrotic cell death is not known. Here we show that after DNA-alkylating damage, the activation of poly(ADP)-ribose polymerase (PARP) regulates the translocation of HMGB1 from the nucleus to the cytosol. This displaced HMGB1 is subject to release if the cell then loses plasma membrane integrity as a result of necrosis. Both full-length HMGB1 and a truncated form of HMGB1 lacking the highly conserved glutamate-rich C-terminal tail can induce macrophage activation and tumor necrosis factor-alpha production. However, displacement of HMGB1 from the nucleus following PARP activation requires the presence of the glutamate-rich C-terminal tail. Although the C-terminal tail is not the sole substrate for PARP modification of HMGB1, it appears to be required to destabilize HMGB1 association with chromatin following PARP-dependent chromatin modifications. These data suggest that PARP-dependent nuclear-to-cytosolic translocation of HMGB1 serves to establish the ability of cells to release this potent inflammatory mediator upon subsequent necrotic death.

Determinants of Specific Binding of HMGB1 Protein to Hemicatenated DNA Loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

In vitro acetylation of HMGB-1 and -2 proteins by CBP: the role of the acidic tail.

Histone acetyltransferases CBP, PCAF, and Tip60 have been tested for their ability to in vitro acetylate HMGB-1 and -2 proteins and their truncated forms lacking the C-terminal tail. It was found that these proteins were substrates for CBP only. Analyses of modified proteins by electrophoresis, amino acid sequencing, and mass spectrometry showed that full-length HMGB-1 and -2 were monoacetylated at Lys2. Removal of the C terminus resulted in (i) an increased incorporation of radiolabeled acetate within the proteins to a level close to that observed with histones H3/H4 and (ii) creation of a novel target site at Lys81. Acetylated and nonmodified HMGB-1 and -2 protein lacking the acidic tail were compared relative to their binding affinity to distorted DNA and the ability to bend linear DNA. Both proteins showed similar affinities to cisplatin-damaged DNA; the acetylated protein, however, was 3-fold more effective in inducing ligase-mediated circularization of a 111-bp DNA fragment. The alterations in the acetylation pattern of HMGB-1 and -2 upon removal of the C-terminal tail are regarded as a means by which the acidic domain modulates some properties of these proteins.

High mobility group 1 B-box mediates activation of human endothelium.

Severe sepsis and septic shock is a consequence of a generalized inflammatory systemic response because of an invasive infection that may result in acute organ dysfunction. Mortality is high despite access to modern intensive care units. The nuclear DNA binding protein high mobility group 1 (HMGB1) protein has recently been suggested to act as a late mediator of septic shock via its function as a macrophage-derived pro-inflammatory cytokine (J Exp Med 2000; 192: 565, Science1999; 285: 248). We investigated the pro-inflammatory activities of the A-box and the B-box of HMGB1 on human umbilical venular endothelial cells (HUVEC). The HUVEC obtained from healthy donors were used for experiments. Recombinant human full-length HMGB1, A-box and B-box were cloned by polymerase chain reaction (PCR) amplification from a human brain quick-clone cDNA. The activation of HUVEC was studied regarding (i) upregulation of adhesion molecules, (ii) the release of cytokines and chemokines, (iii) the adhesion of neutrophils to HUVEC, (iv) the activation of signalling transduction pathways and (v) the involvement of the receptor for advanced glycation end-products (RAGE). The full-length protein and the B-box of HMGB1 dose-dependently activate HUVEC to upregulate adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and to release IL-8 and G-CSF. The activation of HUVEC could be inhibited to 50% by antibodies directed towards the RAGE. HMGB1-mediated HUVEC stimulation resulted in phosphorylation of the ELK-1 signal transduction protein and a nuclear translocation of p65 plus c-Rel, suggesting that HMGB1 signalling is regulated in endothelial cells through NF-kappaB. The HMGB1 acts as a potent pro-inflammatory cytokine on HUVEC and the activity is mainly mediated through the B-box of the protein. HMGB1 may be a key factor mediating part of the pro-inflammatory response occurring in septic shock and severe inflammation.

Cloning and characterization of rice HMGB1 gene

We isolated a 918 bp long full-length rice HMGB1 cDNA, which has an open reading frame of 471 bp encoding 157 amino acids, with a central domain of high sequence similarity to the HMG-box domain of other plant HMGB1 proteins. RNA gel blot analysis indicated that rice HMGB1 gene is constitutively expressed in various tissues and organs. Southern hybridization and sequence analyses suggested that a single copy of the HMGB1 gene composed of seven exons and six introns exists in rice. We have also cloned a 1755 bp long 5′ flanking region of the rice HMGB1 gene, which can be regarded as its promoter. 5′ deletion analysis of this promoter indicated that positive cis-elements residing between −1400 and −1115 are important to enhance quantitative expression, whereas negative cis-elements between −1755 and −1400 and between −1115 and −351 inhibit expression.

The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription.

p53 function is modulated by several covalent and non-covalent modifiers. The architectural DNA- binding protein, High Mobility Group protein B-1 is a unique activator of p53. HMGB-1 protein is structured into two HMG-box domains, namely A-box and B-box, connected to a long highly acidic C-terminal domain. Here we report that both the C-terminal domain and A-box of HMGB-1 are critical for stimulation of p53-mediated DNA binding to its cognate site. Though deletion of these domains showed minimal effect in activation of p53-mediated transcription from the DNA template as compared to full-length HMGB-1, truncation of both the domains indeed showed significant reduction of transcriptional activation from the chromatin template as observed in DNA binding. Using transient transfection assays we showed that the C-terminal acidic domain and A-box of HMGB-1 are critical for the enhancement of the p53-mediated transactivation in vivo. Furthermore, the C-terminal domain and A-box deleted HMGB-1 could not activate p53-dependent apoptosis above the basal level. In conclusion, these results elucidate the role of acidic C-terminal domain and A-box of HMGB-1 in p53-mediated transcriptional activation and its further downstream effect.