Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA.

Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.

Mutual promotion of co-condensation of KRAS G-quadruplex and a well-folded protein HMGB1.

Liquid-liquid phase separation (LLPS) of G-quadruplex (GQ) is involved in many crucial cellular processes, while the quadruplex-folding and their functions are typically modulated by specific DNA-binding proteins. However, the regulatory mechanism of binding proteins, particularly the well-folded proteins, on the LLPS of GQs is largely unknown. Here, we investigated the effect of HMGB1 on the condensation of a G-quadruplex of KRAS promoter (GQKRAS). The results show that these two rigid macro-biomolecules undergo co-condensation through a mutual promotion manner, while neither of them can form LLPS alone. Fluidity measurements confirm that the liquid-like droplets are highly dynamic. HMGB1 facilitates and stabilizes the quadruplex folding of GQKRAS, and this process enhances their co-condensation. The KRAS promoter DNA retains quadruplex folding in the droplets; interference with the GQ-folding disrupts the co-condensation of GQKRAS/HMGB1. Mechanistic studies reveal that electrostatic interaction is a key driving force of the interaction and co-condensation of GQKRAS/HMGB1; meanwhile, the recognition of two macro-biomolecules plays a crucial role in this process. This result indicates that the phase separation of GQs can be modulated by DNA binding proteins, and this process could also be an efficient way to recruit specific DNA binding proteins.

The multifunctional protein HMGB1: 50 years of discovery.

Fifty years since the initial discovery of HMGB1 in 1973 as a structural protein of chromatin, HMGB1 is now known to regulate diverse biological processes depending on its subcellular or extracellular localization. These functions include promoting DNA damage repair in the nucleus, sensing nucleic acids and inducing innate immune responses and autophagy in the cytosol and binding protein partners in the extracellular environment and stimulating immunoreceptors. In addition, HMGB1 is a broad sensor of cellular stress that balances cell death and survival responses essential for cellular homeostasis and tissue maintenance. HMGB1 is also an important mediator secreted by immune cells that is involved in a range of pathological conditions, including infectious diseases, ischaemia-reperfusion injury, autoimmunity, cardiovascular and neurodegenerative diseases, metabolic disorders and cancer. In this Review, we discuss the signalling mechanisms, cellular functions and clinical relevance of HMGB1 and describe strategies to modify its release and biological activities in the setting of various diseases.

Impact of HMGB1 binding on the structural alterations of platinum drug-treated single dsDNA molecule.

High mobility group B1 (HMGB1) is an architectural protein that recognizes the DNA damage sites formed by the platinum anticancer drugs. However, the impact of HMGB1 binding on the structural alterations of the platinum drug-treated single dsDNA molecules has remained largely unknown. Herein, the structural alterations induced by the platinum drugs, the mononuclear cisplatin and it's analog the trinuclear BBR3464, have been probed in presence of HMGB1, by atomic force microscopy (AFM) and AFM-based force spectroscopy. It is observed that the drug-induced DNA loop formation enhanced upon HMGB1 binding, most likely as a result of HMGB1-induced increase in DNA conformational flexibility that allowed the drug-binding sites to come close and form double adducts, thereby resulting in enhanced loop formation via inter-helix cross-linking. Since HMGB1 enhances DNA flexibility, the near-reversible structural transitions as observed in the force-extension curves (for 1 h drug treatment), generally occurred at lower forces in presence of HMGB1. The DNA structural integrity was largely lost after 24 h drug treatment as no reversible transition could be observed. The Young's modulus of the dsDNA molecules, as estimated from the force-extension analysis, increased upon drug treatment, due to formation of the drug-induced covalent cross-links and consequent reduction in DNA flexibility. The Young's modulus increased further in presence of HMGB1 due to HMGB1-induced enhancement in DNA flexibility that could ease formation of the drug-induced covalent cross-links. To our knowledge, this is the first report that shows an increase in the stiffness of the platinum drug-treated DNA molecules in presence of HMGB1.

Interaction of bioactive kaempferol with HMGB1: Investigation by multi-spectroscopic and molecular simulation methods.

Chronic and persistent inflammation associated with excessive high mobility group protein 1 (HMGB1) is a risk factor for various diseases. Dietary intake of kaempferol has been proven to be effective in reducing HMGB1 levels and the degree of inflammation, but the structural mechanism remains unclear. In this context, we first investigated the interaction between bioactive kaempferol and HMGB1 using multi-spectroscopic and molecular simulation techniques. The surface plasmon resonance (SPR) data indicated that kaempferol binds directly to HMGB1 with a Kd value of 2.89 × 10-5 M. Binding of kaempferol with HMGB1 led to the intrinsic fluorescence quenching and modest secondary structure change of HMGB1 supported by fluorescence spectrometry and circular dichroism (CD). Using dynamic light scattering (DLS), it was found that kaempferol induced the aggregation of HMGB1 protein complex to form larger particles. On HMGB1-activated RAW264.7 cells, kaempferol co-incubation exhibited a remarkable inhibitory effect on nitric oxide (NO) release with an IC50 value of 5.02 µM, which was lower than that of quercetin. In silico, kaempferol binds to HMGB1 mainly through hydrogen bonds and hydrophobic forces. Collectively, our study showed kaempferol as a potential HMGB1 inhibitor, mainly acting by direct binding to HMGB1 and inducing its conformational changes, which provides clues for the treatment of chronic inflammation by kaempferol.

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

Nuclear Fructose-1,6-Bisphosphate Inhibits Tumor Growth and Sensitizes Chemotherapy by Targeting HMGB1.

Metabolites are important for cell fate determination. Fructose-1,6-bisphosphate (F1,6P) is the rate-limiting product in glycolysis and the rate-limiting substrate in gluconeogenesis. Here, it is discovered that the nuclear-accumulated F1,6P impairs cancer cell viability by directly binding to high mobility group box 1 (HMGB1), the most abundant non-histone chromosome structural protein with paradoxical roles in tumor development. F1,6P disrupts the association between the HMGB1 A-box and C-tail by targeting K43/K44 residues, inhibits HMGB1 oligomerization, and stabilizes P53 protein by increasing P53-HMGB1 interaction. Moreover, F1,6P lowers the affinity of HMGB1 for DNA and DNA adducts, which sensitizes cancer cells to chemotherapeutic drug(s)-induced DNA replication stress and DNA damage. Concordantly, F1,6P resensitizes cancer cells with chemotherapy resistance, impairs tumor growth and enhances chemosensitivity in mice, and impedes the growth of human tumor organoids. These findings reveal a novel role for nuclear-accumulated F1,6P and underscore the potential utility of F1,6P as a drug for cancer therapy.

The clinical value of high mobility group box-1 and CRP/Alb ratio in the diagnosis and evaluation of sepsis in children.

OBJECTIVE: To explore the clinical value of high mobility group box-1 (HMGB-1), C-reactive protein (CRP), procalcitonin (PCT), and CRP to albumin (Alb) ratio in the diagnosis and evaluation of the severity of sepsis in children. PATIENTS AND METHODS: A total of 90 children, 50 with sepsis and 40 with general infection, whose symptoms did not meet the criteria for diagnosis of sepsis, were admitted to the Pediatrics Department of Jingzhou Central Hospital in Hubei Province between November 2021 and December 2022, were enrolled and selected as experimental and control group, respectively. The serum of two groups was collected within 24 hours after admission, the levels of HMGB-1 were detected by enzyme-linked immunosorbent assay (ELISA), and CRP, PCT, Alb, and hospitalization days were recorded. The differences in indicators between the two groups were compared, and correlation analysis was performed between hospitalization days and various indicators. The receiver operating characteristic (ROC) curve was drawn to evaluate the independent or combined value of CRP, PCT, HMGB-1, and CRP/Alb ratio in the early diagnosis of sepsis in children. RESULTS: These four indicators of children with sepsis were significantly higher than those in the general infection group (all p=0.000). The levels of CRP, PCT and CRP/Alb ratio were significantly positively correlated with the hospitalization days (r=0.329, 0.333, 0.329; p=0.02, 0.01, 0.002). The area under curve (AUC) of CRP, PCT, HMGB-1, and CRP/Alb ratio for the diagnosis of sepsis in children was 0.798, 0.817, 0.838, 0.809, respectively, and that of the combination of four indicators was 0.952. CONCLUSIONS: CRP, PCT, HMGB-1, and CRP/Alb ratio resulted as effective indicators for early diagnosis and evaluation of childhood sepsis, having a higher value in combined diagnosis.

Correlation of HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis with Amniotic Fluid Fecal Dye.

BACKGROUND: This paper aims to investigate the correlation between high mobility group protein-1 (HMG-b1), antioxidant enzyme-1 (paraoxon-1, PON-1), monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, MCP-1), P. gingivalis, and MSAF. MATERIALS AND METHODS: The total sample size comprised of 73 cases in both groups. These patients were further subdivided into 2 groups: the MSAF group and the control group. 38 women were in the MSAF group and 35 women with term amniotic fluid serum were in the control group. The MSAF group was selected as a full-term singleton amniotic fluid fecal infection group. Clinical data were collected, and specimens were collected. Fecal staining of amniotic fluid and full-term amniotic fluid removes the placenta and umbilical cord blood. The expression of HMGB1 in the placenta was observed by immune-histochemical staining of MSAF and control groups. The content of PON-1 in cord blood was determined by ELISA. RESULTS: Correlation between maternal and neonatal clinical data and MSAF was done; MSAF group mean gestational age was 41.38 ± 1.40 weeks; control group mean gestational age was 39.20 ± 1.24 weeks. This study found no correlation between the birth weight, maternal age, sex, first/transmaternal, hyperthyroidism, hypothyroidism, and anemia between the MSAF and control group with nonsignificant P value (P > 0.05). However, the fatal age, gestational diabetes, gestational hypertension, umbilical cord abnormalities, placental abnormalities, and neonatal asphyxia factors were statistically different with a significant P value of <0.05 between both groups. HMGB1 and Periodontal P. gingivalis are mostly expressed in placental trophoblast, vascular endothelial cells, and amniotic epithelial and interstitial cells. After HE staining of 72 placentas by HE in MSAF and control, 6 had acute chorioamnionitis (5.1 control), 32 had chronic (23.9), 35 had abnormal placentas, and three in MSAF had chorionic columnar metaplasia. In immune-histochemistry experiments, the HMGB1 expression intensity of placental tissue was higher in the MSAF group (P < 0.05); however, the level of PON-1 was lower in the MSAF group as compared to the controls (P < 0.05). CONCLUSIONS: Gestational age and placental abnormalities are clinical high-risk factors for MSAF. HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis may be involved in the development of MSAF, suggesting an oxidative/antioxidant imbalance with inflammation, and may be one of the mechanisms for MSAF development.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

Proteins Binding to the Carbohydrate HNK-1: Common Origins?

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.

2-O, 3-O desulfated heparin (ODSH) increases bacterial clearance and attenuates lung injury in cystic fibrosis by restoring HMGB1-compromised macrophage function.

BACKGROUND: High mobility group box 1 protein (HMGB1) is an alarmin following its release by immune cells upon cellular activation or stress. High levels of extracellular HMGB1 play a critical role in impairing the clearance of invading pulmonary pathogens and dying neutrophils in the injured lungs of cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS). A heparin derivative, 2-O, 3-O desulfated heparin (ODSH), has been shown to inhibit HMGB1 release from a macrophage cell line and is efficacious in increasing bacterial clearance in a mouse model of pneumonia. Thus, we hypothesized that ODSH can attenuate the bacterial burden and inflammatory lung injury in CF and we conducted experiments to determine the underlying mechanisms. METHODS: We determined the effects of ODSH on lung injury produced by Pseudomonas aeruginosa (PA) infection in CF mice with the transmembrane conductance regulator gene knockout (CFTR-/-). Mice were given ODSH or normal saline intraperitoneally, followed by the determination of the bacterial load and lung injury in the airways and lung tissues. ODSH binding to HMGB1 was determined using surface plasmon resonance and in silico docking analysis of the interaction of the pentasaccharide form of ODSH with HMGB1. RESULTS: CF mice given 25 mg/kg i.p. of ODSH had significantly lower PA-induced lung injury compared to mice given vehicle alone. The CF mice infected with PA had decreased levels of nitric oxide (NO), increased levels of airway HMGB1 and HMGB1-impaired macrophage phagocytic function. ODSH partially attenuated the PA-induced alteration in the levels of NO and airway HMGB1 in CF mice. In addition, ODSH reversed HMGB1-impaired macrophage phagocytic function. These effects of ODSH subsequently decreased the bacterial burden in the CF lungs. In a surface plasmon resonance assay, ODSH interacted with HMGB1 with high affinity (KD = 3.89 × 10-8 M) and induced conformational changes that may decrease HMGB1's binding to its membrane receptors, thus attenuating HMGB1-induced macrophage dysfunction. CONCLUSIONS: The results suggest that ODSH can significantly decrease bacterial infection-induced lung injury in CF mice by decreasing both HMGB1-mediated impairment of macrophage function and the interaction of HMGB1 with membrane receptors. Thus, ODSH could represent a novel approach for treating CF and ARDS patients that have HMGB1-mediated lung injury.

Specific detection of high mobility group box 1 degradation product with a novel ELISA.

BACKGROUND: During sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and ultimately into systemic circulation, where it mediates systemic inflammation and remote organ failure. The proinflammatory effects of HMGB1 can be suppressed by recombinant thrombomodulin (rTM), in part through a mechanism involving thrombin-rTM-mediated degradation of HMGB1. Given that HMGB1 is proinflammatory but the HMGB1 degradation product (desHMGB1) is not, an analytical method that discriminates between these two molecules may provide a more in-depth understanding of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency. METHODS: A peptide that has a shared amino-terminal structure with desHMGB1 was synthesized. C3H/lpr mice were immunized with the desHMGB1 peptide conjugate, and antibody-secreting hybridoma cells were developed using conventional methods. The reactivity and specificity of the antibodies were then analyzed using antigen-coated enzyme-linked immunosorbent assay (ELISA) as well as antibody-coated ELISA. Next, plasma desHMGB1 levels were examined in a cecal ligation and puncture (CLP)-induced septic mouse model treated with rTM. RESULTS: Through a series of screening steps, we obtained a monoclonal antibody that recognized desHMGB1 but did not recognize intact HMGB1. ELISA using this antibody specifically detected desHMGB1, which was significantly increased in CLP-induced septic mice treated with rTM compared with those treated with saline. CONCLUSIONS: In this study, we obtained a desHMGB1-specific monoclonal antibody. ELISA using the novel monoclonal antibody may be an option for the in-depth analysis of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency.

Disulfide and Fully Reduced HMGB1 Induce Different Macrophage Polarization and Migration Patterns.

Macrophage plasticity enables cells to obtain different functions over a broad proinflammatory and repairing spectrum. In different conditions, macrophages can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity, to polarize towards a pro- (M1) or anti-inflammatory (M2) phenotype. In this study, we investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) induced by different HMGB1 redox isoforms in depth. Our results demonstrate that disulfide HMGB1 (dsHMGB1) induces a unique macrophage phenotype that secretes pro-inflammatory cytokines, rather than inducing metabolic changes leading to nitric oxide production. Fully reduced HMGB1 (frHMGB1) did not induce macrophage polarization. The migrating function of BMDMs was measured by scratch assay after the stimulation with dsHMGB1 and frHMGB1. Both dsHMGB1 and frHMGB1 induced cell migration. We found that dsHMGB1 mediates cytokine secretion and cellular motility, mainly through toll-like receptor 4 (TLR4). Importantly, our data shows that dsHMGB1 and frHMGB1 induce distinct BMDM polarization phenotypes, and that dsHMGB1 induces a unique phenotype differing from the classical proinflammatory macrophage phenotype.

Dynamic Autoinhibition of the HMGB1 Protein via Electrostatic Fuzzy Interactions of Intrinsically Disordered Regions.

Highly negatively charged segments containing only aspartate or glutamate residues ("D/E repeats") are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40-900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

Paeonol attenuates inflammation by confining HMGB1 to the nucleus.

Inflammation is a biological process that exists in a large number of diseases. If the magnitude or duration of inflammation becomes uncontrolled, inflammation may cause pathological damage to the host. HMGB1 and NF-κB have been shown to play pivotal roles in inflammation-related diseases. New drugs aimed at inhibiting HMGB1 expression have become a key research focus. In the present study, we showed that paeonol (Pae), the main active component of Paeonia suffruticosa, decreases the expression of inflammatory cytokines and inhibits the translocation of HMGB1 induced by lipopolysaccharide (LPS). By constructing HMGB1-overexpressing (HMGB1+ ) and HMGB1-mutant (HMGB1m ) RAW264.7 cells, we found that the nuclear HMGB1 could induce an LPS-tolerant state in RAW264.7 cells and that paeonol had no influence on the expression of inflammatory cytokines in HMGB1m RAW264.7 cells. In addition, the anti-inflammatory property of paeonol was lost in HMGB1 conditional knockout mice, indicating that HMGB1 is a target of paeonol and a mediator through which paeonol exerts its anti-inflammatory function. Additionally, we also found that HMGB1 and P50 competitively bound with P65, thus inactivating the NF-κB pathway. Our research confirmed the anti-inflammation property of paeonol and suggests that inhibiting the translocation of HMGB1 could be a new strategy for treating inflammation.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

High-mobility group box 1 is a driver of inflammation throughout pregnancy.

A proinflammatory response driven by high-mobility group box 1 (HMGB1) is important for the success of both the early stages of pregnancy and parturition initiation. However, the tight regulation of HMGB1 within these two stages is critical, as increased HMGB1 can manifest into pregnancy-related pathologies. Although during the early stages of pregnancy HMGB1 is critical for the development and implantation of the embryo, and uterine decidualization, high levels within the uterine cavity have been linked to pregnancy failure. In addition, chronic inflammation, resultant from increased HMGB1 within the maternal circulation and gestational tissues, also increases the risk for preterm labor, preterm birth, or infant mortality. Due to the link between HMGB1 and several pregnancy pathologies, the possibility of leveraging HMGB1 as a biomarker has been assessed. However, data are limited that demonstrate how known HMGB1 inhibitors could reduce inflammation within pregnancy. Thus, further research is warranted to improve our understanding of the potential of HMGB1 as a therapeutic target to reduce inflammation within pregnancy. This review aims to describe what is understood about the role of HMGB1 that drives inflammation throughout pregnancy and highlight its potential as a biomarker and therapeutic target within this context.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions.

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5'→3' (T5 Exo) and 3'→5' direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.