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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 functions 1 – 3 . Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus 4 , 5 . This suggests that mutations in disordered proteins may alter condensate properties and function 6 – 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. Frameshift mutations that create arginine-rich basic tails in transcription factors and other proteins can lead to altered phase separation in the nucleolus, which in turn leads to syndromes such as brachyphalangy, polydactyly and tibial aplasia.

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.Fifty years since the discovery of HMGB1 protein, its physiological and pathological roles have been extensively studied. This Review covers the structure, localization and functions of HMGB1 in immune responses, including historical foundations and recent advances.

HMGB protein sentinels The chromosomal HMGB (high-mobility group box) proteins HMGB1, HMGB2 and HMGB3 are shown here to be essential for all nucleic-acid receptor-mediated activation of innate immune responses. HMGBs bound to all immunogenic nucleic acids tested — whether considered ligands for Toll-like receptors or for cytosolic receptors — suggesting that they may have a physiological role as universal sentinels for intracellular nucleic acids. Activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities. This activation is known to be mediated by transmembrane Toll-like receptors and cytosolic receptors; however, it remains unclear whether a mechanism exists that integrates these two nucleic-acid-sensing systems. High-mobility group box (HMGB) proteins 1, 2 and 3 are now shown to function as universal sentinels for nucleic-acid-mediated innate immune responses. The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors 1 , 2 . However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1 -/- and Hmgb2 -/- mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-κB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

During infection, vertebrates develop \"sickness syndrome,\" characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.

High mobility group box 1 (HMGB1) is a nonhistone chromatin-associated protein that has been widely reported to play a pivotal role in the pathogenesis of hematopoietic malignancies. As a representative damage-associated molecular pattern (DAMP), HMGB1 normally exists inside cells but can be secreted into the extracellular environment through passive or active release. Extracellular HMGB1 binds with several different receptors and interactors to mediate the proliferation, differentiation, mobilization, and senescence of hematopoietic stem cells (HSCs). HMGB1 is also involved in the formation of the inflammatory bone marrow (BM) microenvironment by activating proinflammatory signaling pathways. Moreover, HMGB1-dependent autophagy induces chemotherapy resistance in leukemia and multiple myeloma. In this review, we systematically summarize the emerging roles of HMGB1 in carcinogenesis, progression, prognosis, and potential clinical applications in different hematopoietic malignancies. In summary, targeting the regulation of HMGB1 activity in HSCs and the BM microenvironment is highly beneficial in the diagnosis and treatment of various hematopoietic malignancies.

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2–EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis. Inflammation involves a Warburg effect that switches cellular metabolism to glycolysis. Here the authors show this switch drives IL-1β, IL-18 and HMGB1 release from macrophages by activating the NLRP3 and AIM2 inflammasomes via protein kinase R phosphorylation, a pathway that can be inhibited to prevent sepsis in mice.

Recurrent spontaneous abortion (RSA) is a common complication of pregnancy that affects the physical and mental health of pregnant women, and approximately 50% of the mechanisms are unclear. Our previous studies have found that high mobility group box 1 (HMGB1) molecules are highly expressed at the maternal-fetal interface of unexplained recurrent spontaneous abortion (URSA) patients. The purpose of this study was to further detect the expression of HMGB1 and pyroptosis in decidual tissue of URSA patients, and explore the potential mechanism of the protective role of HMGB1 in URSA patients and mouse model. The decidua tissues of 75 URSA patients and 75 women who actively terminated pregnancy were collected, and URSA mouse models were established and treated with HMGB1 inhibitor-aspirin. The expression of HMGB1, and their receptors (RAGE, TLR2, TLR4), pyroptosis-associated proteins (NLRP-3, caspase-1, GSDMD) and NF-κB was examined at the maternal-fetal interface of human and mouse. Our study found that HMGB1, NLRP-3, Caspase-1, GSDMD, RAGE, TLR2 and TLR4 were highly expressed and NF-κB signaling pathway were activated in the decidua tissue of URSA group. Moreover, immune cell disorder and co-localization of HMGB1 and macrophages were found at the maternal-fetal interface of URSA mice. However, HMGB1, TLR2, TLR4, NF-κB, and pyroptosis-associated proteins can be down-regulated by administering low-dose aspirin. These data may indicate that highly expressed HMGB1 was actively secreted by macrophages and then activated pyroptosis through the TLR2/TLR4-NF-κB pathway to cause aseptic inflammation, leading to the occurrence and development of URSA. Moreover, low-dose aspirin can reduce HMGB1 protein levels of serum and decidual in URSA.

Muscle cell death in polymyositis is induced by CD8 + cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8 + cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8 + cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes. Polymyositis (PM) is a chronic inflammatory myopathy characterized by progressive muscle weakness. Here the authors showed that muscle fibers in PM undergo necroptosis and aggravate inflammation via releasing pro-inflammatory molecules such as HMGB1.

High Mobility Group Box 1 (HMGB1) and Procathepsin L (pCTS-L) are crucial inflammatory mediators, yet their immunomodulating properties in human immune cells have not been systematically compared. This study employed RNA-sequencing to comparatively analyze their transcriptional effects on primary human peripheral blood mononuclear cells (PBMCs). Our findings demonstrate that while both mediators elicited significant transcriptional changes indicative of robust inflammatory responses, HMGB1 consistently induced a more extensive and diversified inflammatory program. Specifically, at a lower concentration of 0.5 µg/ml, HMGB1 triggered nearly four times more differentially expressed genes (DEGs) than pCTS-L (2.0 µg/ml). Despite this quantitative difference, an overlap of 412 DEGs (272 upregulated, 140 downregulated) revealed shared core inflammatory pathways, including the extensive upregulation of pro-inflammatory cytokines (e.g., IL1A, IL1B, and IL6), chemokines (e.g., CCL2 and CXCL1), and S100 proteins (e.g., S100A8, S100A9, and S100A12). Both mediators also converged on activating the non-canonical NF-κB pathway, evidenced by NFKB2 and RELB upregulation, suggesting a common underlying regulatory mechanism. Notably, HMGB1 uniquely upregulated CASP4 and CASP5—key components of the non-canonical inflammasome pathway—and a broader spectrum of cytokines and chemokines (e.g., IL23A, CXCL5). These findings delineate the distinct yet overlapping roles of HMGB1 and pCTS-L in orchestrating immune responses, offering a foundation for targeted therapeutic development for inflammatory diseases.

High circulating levels of lactate and high mobility group box-1 (HMGB1) are associated with the severity and mortality of sepsis. However, it is unclear whether lactate could promote HMGB1 release during sepsis. The present study demonstrated a novel role of lactate in HMGB1 lactylation and acetylation in macrophages during polymicrobial sepsis. We found that macrophages can uptake extracellular lactate via monocarboxylate transporters (MCTs) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. We also observed that lactate stimulates HMGB1 acetylation by Hippo/YAP-mediated suppression of deacetylase SIRT1 and β-arrestin2-mediated recruitment of acetylases p300/CBP to the nucleus via G protein-coupled receptor 81 (GPR81). The lactylated/acetylated HMGB1 is released from macrophages via exosome secretion which increases endothelium permeability. In vivo reduction of lactate production and/or inhibition of GPR81-mediated signaling decreases circulating exosomal HMGB1 levels and improves survival outcome in polymicrobial sepsis. Our results provide the basis for targeting lactate/lactate-associated signaling to combat sepsis.