Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, but its pathogenesis remains imprecisely understood and requires further clarification. Recently, the tumor suppressor p53 has received growing attention for its role in metabolic diseases. In this study, we performed in vivo and in vitro experiments to identify the contribution of p53-autophagy regulation to NAFLD. METHODS: Livers from wild-type and p53 knockout mice as well as p53-functional HepG2 cells and p53-dysfunctional Huh7 cells were examined for autophagy status and HMGB1 translocation. In vivo and in vitro NAFLD models were established, and steatosis was detected. In the cell models, autophagy status and steatosis were examined by p53 and/or HMGB1 silencing. RESULTS: First, the silencing of p53 could induce autophagy both in vivo and in vitro. In addition, p53 knockout attenuated high-fat diet-induced NAFLD in mice. Similarly, knockdown of p53 could alleviate palmitate-induced lipid accumulation in cell models. Furthermore, high mobility group box 1 (HMGB1) was proven to contribute to the effect of silencing p53 on alleviating NAFLD in vitro as an autophagy regulator. CONCLUSION: The anti-NAFLD effect of functional p53 silencing is associated with the HMGB1-mediated induction of autophagy.

The effect of high mobility group box-1 protein on cerebral edema, blood-brain barrier, oxidative stress and apoptosis in an experimental traumatic brain injury model.

Traumatic brain injury (TBI) is one of the important reason of morbidity and mortality. While the primary injury due to mechanical impact is unavoidable, the secondary injury which is formed as a result of primary injury and thought to occur due to neuroinflammation in the forefront can be prevented and by this way mortality and morbidity can be reduced. High mobility group box-1 (HMGB1) is a protein that triggers the neuroinflammatory process by being released from the nucleus of necrotic tissues after primary injury. The aim of this study is to investigate the effects of HMGB1 on its receptors TLR4 and RAGE, cerebral edema, blood-brain barrier, oxidative stress and apoptosis causing secondary damage in an experimental traumatic brain injury model. Weighing between 280-320 g, 10 to 12 weeks-old, a total of 30 adult male Sprague-Dawley rats were used for the experiments. The rats were randomly assigned to 3 groups: 1) Control, 2) TBI and 3) TBI + ethyl pyruvate group (n = 10 per group). Right parietal cortical contusion was made by using a weight-dropping TBI method. Brain samples were harvested from pericontusional area at 24 h after TBI. HMGB1, TLR4, RAGE, occludin, claudin-5, ZO-1 levels are investigated by western blot analyses and immunohistochemistry examinations. HMGB-1, TLR4 and RAGE expressions increased after TBI. Major tight junction proteins in the blood-brain barrier: occludin, claudin-5 and ZO-1 expressions decreased after TBI. Brain edema increased after TBI. Also, proapoptotic bax and active caspase 3 expressions increased, antiapoptotic bcl-2 levels decreased after TBI. Total oxidant status and oxidative stress increased, total antioxidant status decreased after TBI. HMGB-1 protein plays a key role in the pathophysiology of traumatic brain injury.

The crystal structure of Capicua HMG-box domain complexed with the ETV5-DNA and its implications for Capicua-mediated cancers.

Capicua (CIC) is a transcriptional repressor and functions downstream of the receptor tyrosine kinase (RTK) signaling pathway. Somatic mutations found in the HMG-box DNA binding domain in CIC have been implicated in several cancers such as oligodendroglioma, oligoastrocytoma, and adenocarcinoma. However, the molecular basis of the DNA binding of CIC and the effect of the somatic mutations found in cancers on DNA binding have not been investigated. Here, we report the crystal structure of the HMG-box domain of CIC complexed with its target DNA, the promoter of Ets Translocation Variant 5 (ETV5). The structure shows that the HMG-box domain has an L-shaped structure and recognizes the minor groove leading to DNA bending. Our structure combined with an electrophoretic mobility shift assay (EMSA) revealed that cancer-associated mutations in the HMG-box domain abrogate the interaction with DNA. These results provide the molecular insight into the DNA binding of CIC and reveal the effects of carcinogenic mutations on DNA binding.

Betaine protects against high-fat-diet-induced liver injury by inhibition of high-mobility group box 1 and Toll-like receptor 4 expression in rats.

BACKGROUND AND OBJECTIVES: Previous studies have shown that betaine prevents alcohol-induced liver injury and improves liver function. The purpose of this study was to investigate the hepatoprotective effects of betaine on nonalcoholic fatty liver disease (NAFLD) and to observe changes of HMGB1/TLR4 signaling. METHODS: Thirty rats were randomly divided into control, model, and betaine groups. The rats in the model and betaine groups were fed a high-fat diet for 12 weeks to induce an animal model of NAFLD. The rats in the betaine group were then intragastrically administered betaine solution at a dose of 400 mg/kg per day for four weeks. Liver histology was examined. Serum levels of ALT, AST, TC, TG, HDL-C, LDL-C, FFA, HMGB1, NF-κB, TLR4, and tHcy were determined and intrahepatic TC, TG, and Hcy levels were assayed. mRNA expression and protein levels of HMGB1, NF-κB, and TLR4 in liver tissue were also determined. RESULTS: Compared with the control group, rats in the model group developed severe liver injury, accompanied by significant increases in serum levels of ALT, AST, TC, TG, LDL-C, FFA, HMGB1, NF-κB, and TLR4, intrahepatic TC, TG, and Hcy content, histological scores for steatosis, inflammation, and necrosis, and mRNA expression and protein levels of HMGB1, NF-κB, and TLR4, and a significant decrease in serum HDL-C (P < 0.05). Compared with the model group, all these indicators were significantly improved by administration of betaine (P < 0.05). CONCLUSIONS: Betaine effectively protects against high-fat-diet-induced NAFLD and improves liver function; the mechanism is probably related to inhibition of HMGB1/TLR4 signaling pathways.

Localization and functional analysis of HmgB3p, a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila.

The high-mobility-group (HMG)-box domain represents a very versatile protein domain that mediates the DNA-binding of non-sequence-specific and sequence-specific proteins. HMG-box proteins are involved in various nuclear functions, including modulating chromatin structure and genomic stability. In this study, we identified the gene HMGB3 in Tetrahymena thermophila. The predicted HmgB3p contained a single HMG-box, an SK-rich-repeat domain and a neutral phosphorylated C-terminal. HMGB3 was expressed in the growth and starvation stages. Furthermore, HMGB3 showed a higher expression levels during the conjugation stage. HMGB3 knockout strains showed no obvious cytological defects, although initiation of HMGB3 knockout strain mating was delayed and maximum mating was decreased. HA-HmgB3p localized on the micronucleus (MIC) during the vegetative growth and starvation stages. Furthermore, HA-HmgB3p specially decorated the meiotic and mitotic functional MIC during the conjugation stage. Truncated HMGB3 lacking the HMG box domain disappeared from MICs and diffused in the cytoplasm. Overexpressed HmgB3p was abnormally maintained in newly developing macronuclei and affected the viability of progeny. Taken together, these results show that HmgB3p is a germline micronuclear-specific marker protein. It may bind to micronucleus-specific DNA sequences or structures and is likely to have some function specific to micronuclei of T. thermophila.

Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues.

HMGB proteins are abundant non-histone components of eukaryotic chromatin. The biological function of DNA sequence-nonspecific HMGB proteins is obscure. These proteins are composed of one or two conserved HMG box domains, each forming three alpha-helices that fold into a sequence-nonspecific DNA-binding module recognizing the DNA minor groove. Box A and box B homology domains have subtle sequence differences such that box B domains bend DNA strongly while DNA bending by isolated box A domains is weaker. Both box A and box B domains preferentially bind to distorted DNA structures. Here we show using DNA cyclization kinetics assays in vitro and Escherichia coli DNA looping assays in vivo that an isolated HMG box A domain derived from human HMGB2 folds poorly and does not enhance apparent DNA flexibility. Surprisingly, substitution of a small number of cationic residues from the N-terminal leader of a functional yeast box B protein, Nhp6Ap, confers the ability to enhance DNA flexibility. These results demonstrate important roles for cationic leader amino acids in HMGB folding, DNA interaction, and DNA bending.

Full-length SRY protein is essential for DNA binding.

SRY directs testicular development. It has been suggested that the only high-mobility group (HMG) box of the SRY is important for the function of this protein; however, other studies have suggested that the N- and C-terminal regions are also involved in this process. Herein, we analysed and compared in vitro the DNA-binding activity of the full-length SRY and three mutants (HMG box alone, N-terminal less and C-terminal less SRY proteins). DNA-binding capability was analysed by mobility shift assays, optical density and dissociation constant by using pure non-fusion SRY proteins. The structure of the full-length SRY was carried out using a protein molecular model. The HMG box SRY alone and C-terminal less SRY proteins had a statistically diminished DNA binding in comparison with the full-length SRY. In contrast, the affinity for DNA of the N-terminal less SRY was relatively similar to the full-length SRY. Likewise, three-dimensional structure of the full-length SRY suggested that some residues of the C-terminal region of the SRY interact with DNA. We demonstrate the importance that full-length SRY has, particularly the C-terminal region of the protein, in DNA binding in vitro. Likewise, the affinity of the HMG box alone is clearly reduced when compared with the full-length SRY.

The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity.

Trichomes are cytoplasmic extrusions of epidermal cells. The molecular mechanisms that govern the differentiation of trichome-producing cells are conserved across species as distantly related as mice and flies. Several signaling pathways converge onto the regulation of a conserved target gene, shavenbaby (svb, ovo), which, in turn, stimulates trichome formation. The Drosophila ventral epidermis consists of the segmental alternation of two cell types that produce either naked cuticle or trichomes called denticles. The binary choice to produce naked cuticle or denticles is affected by the transcriptional regulation of svb, which is sufficient to cell-autonomously direct denticle formation. The expression of svb is regulated by the opposing gradients of two signaling molecules--the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which activates svb expression, and Wingless (Wg), which represses it. It has remained unclear how these opposing signals are integrated to establish a distinct domain of svb expression. We show that the expression of the high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi, and repressed by Wg, signaling. SoxN is necessary and sufficient to cell-autonomously direct the expression of svb. The closely related protein Dichaete is co-regulated with SoxN and has a partially redundant function in the activation of svb expression. In addition, we show that SoxN and Dichaete function upstream of Wg and antagonize Wg pathway activity. This suggests that the expression of svb in a discreet domain is resolved at the level of SoxN and Dichaete.

Basic and acidic regions flanking the HMG-box domain of maize HMGB1 and HMGB5 modulate the stimulatory effect on the DNA binding of transcription factor Dof2.

The chromatin-associated high-mobility group (HMG) proteins of the plant HMGB family are characterized by a central HMG-box domain that is flanked by a basic N-terminal and an acidic C-terminal domain. By functional interaction with certain transcription factors, HMGB proteins contribute to transcriptional regulation. Previous work has shown that the maize HMGB5 protein is markedly more efficient than other HMGB proteins in stimulating the binding of transcription factor Dof2 to DNA target sites. Here we examine the structural requirements that determine the particular efficiency of HMGB5. The HMG-box domains of HMGB1 and HMGB5 (which mediate the interaction with Dof2) promoted Dof2-DNA binding to a similar extent, indicating that the terminal domains modulate the interaction with Dof2. Analysis of full-length, truncated, and chimeric HMGB1/5 proteins revealed that the acidic C-terminal domains positively influence the stimulation of Dof2-DNA binding, while the basic N-terminal domains have a rather negative effect. In particular, the C-terminal domain of HMGB5 has a striking positive effect and may account for the efficient stimulation mediated by full-length HMGB5. Interestingly, recombinant HMGB protein variants that have a relatively low affinity for linear DNA (such as proteins lacking the basic N-terminal domain) efficiently assist Dof2-DNA binding.

SRY and human sex determination: the basic tail of the HMG box functions as a kinetic clamp to augment DNA bending.

Human testis-determining factor SRY contains a high-mobility-group (HMG) box, an alpha-helical DNA-binding domain that binds within an expanded minor groove to induce DNA bending. This motif is flanked on the C-terminal end by a basic tail, which functions both as a nuclear localization signal and accessory DNA-binding element. Whereas the HMG box is broadly conserved among otherwise unrelated transcription factors, tails differ in sequence and mode of DNA binding. Contrasting examples are provided by SRY and lymphoid enhancer factor 1 (LEF-1): whereas the SRY tail remains in the minor groove distal to the HMG box, the LEF-1 tail binds back across the center of the bent DNA site. The LEF-1 tail relieves electrostatic repulsion that would otherwise be incurred within the compressed major groove to enable sharp DNA bending with high affinity. Here, we demonstrate that the analogous SRY tail functions as a "kinetic clamp" to regulate the lifetime of the bent DNA complex. As in LEF-1, partial truncation of the distal SRY tail reduces specific DNA affinity and DNA bending, but these perturbations are modest: binding is reduced by only 1.8-fold, and bending by only 7-10 degrees . "Tailed" and truncated SRY complexes exhibit similar structures (as probed by NMR) and distributions of long-range conformational substates (as probed by time-resolved fluorescence resonance energy transfer). Surprisingly, however, the SRY tail retards dissociation of the protein-DNA complex by 20-fold. The marked and compensating changes in rates of association and dissociation observed on tail truncation, disproportionate to perturbations in affinity or structure, suggest that this accessory element functions as a kinetic clamp to regulate the lifetime of the SRY-DNA complex. We speculate that the kinetic stability of a bent DNA complex is critical to the assembly and maintenance of a sex-specific transcriptional pre-initiation complex.

Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal.

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

Evidence for involvement of HMGB1 protein in human DNA mismatch repair.

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.

The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9].

Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown.

Distorted DNA structures induced by HMGB2 possess a high affinity for HMGB2.

HMGB2 (HMG2) protein binds with DNA duplex in a sequence-nonspecific manner, then bends and unwinds the DNA. In DNA cyclization analyses for the bending activity of HMGB2, two unidentified bands, denoted alpha and beta, were observed in addition to monomer circular DNA (1C) on the gel. Re-electrophoresis and proteinase K digestion revealed that alpha and beta are complexes of circularized probe DNA (seeming 1C) with HMGB2 (K(d) approximately 10(-10) M). The DNA components of alpha and beta (alpha- and beta-DNA) showed higher affinities to HMGB2 than did the linear probe DNA (K(d) approximately 10(-7) M). The DNAs have distorted structures containing partial single-stranded regions. Nicked circular molecules presumably due to severe DNA distortion by HMGB2 were observed in alpha- and beta-DNA, in addition to closed circular double-stranded molecules. The alpha and beta bands were not formed in the presence of sole DNA binding regions which are necessary for DNA bending, indicating that the acidic C-tail in the HMGB2 molecule is necessary for inducing the peculiar distorted structures of higher affinity to HMGB2. HMGB2 binds with linker DNA and/or the entry and exit of nucleosomes fixed at both ends likewise mini-circles similar to alpha-DNA and beta-DNA. Thus, the distorted structures present in alpha-DNA and beta-DNA should be important in considering the functional mechanisms in which HMGB2 participates.