HMGB1 Human
Description
Molecular Structure and Domains
HMGB1 is a 215-amino-acid protein with three distinct regions:
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HMG A and B boxes: Two DNA-binding domains (residues 9–79 and 95–163) that adopt an L-shaped structure to bend DNA and stabilize nucleosomes .
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C-terminal acidic tail: A negatively charged region (residues 186–215) rich in aspartic and glutamic acids, critical for protein interactions and anti-inflammatory activity .
Post-translational modifications (acetylation, phosphorylation, oxidation) dictate HMGB1’s subcellular localization and function . For example, hyperacetylation promotes cytoplasmic translocation and secretion .
Nuclear Roles
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Chromatin remodeling: Bends DNA to enhance transcription factor access .
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DNA repair: Facilitates V(D)J recombination via interaction with RAG endonuclease .
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Autophagy regulation: Binds Beclin-1 to sustain autophagy under stress .
Extracellular Roles
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Damage-associated molecular pattern (DAMP): Released during necrosis or secreted by immune cells, activating TLR2/4/9 and RAGE receptors to drive inflammation .
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Chemoattractant: Fully reduced HMGB1 recruits neutrophils and monocytes .
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Cytokine synergy: Binds IL-1β, LPS, and CXCL12 to amplify immune responses .
Pathological Implications
HMGB1 is implicated in diverse diseases due to its dual roles in homeostasis and inflammation:
Therapeutic Targeting
Strategies to modulate HMGB1 activity include:
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Neutralizing antibodies: Block extracellular HMGB1 in TBI and intracerebral hemorrhage .
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Small-molecule inhibitors: Glycyrrhizic acid suppresses HMGB1-mediated microglial activation .
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Redox-state modulation: Targeting C23/C45 disulfide bonds to inhibit cytokine activity .
Key Research Findings
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Redox-dependent activity:
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Structural insights:
Clinical Relevance
While preclinical studies highlight HMGB1’s potential as a biomarker and therapeutic target, clinical trials remain limited. Challenges include optimizing delivery of HMGB1 inhibitors and managing its dual roles in homeostasis and pathology .
Product Specs
MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERW
KTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPS
AFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLK
EKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEEEDEED
EDEEEDDDDELEHHHHHH
Q&A
What is the molecular structure of human HMGB1?
Human HMGB1 is expressed as a 30 kDa, 215 amino acid single chain polypeptide containing three distinct domains: two N-terminal globular, positively charged DNA-binding domains (HMG boxes A and B), each approximately 70 amino acids in length, and a negatively charged 30 amino acid C-terminal region composed exclusively of aspartic acid and glutamic acid residues. The protein contains nuclear localization sequences (NLS) at residues 27-43 and 178-184. HMGB1 undergoes extensive post-translational modifications, with acetylation occurring on up to 17 lysine residues, which significantly impacts its cellular localization and function .
How does HMGB1 function in normal cellular physiology?
HMGB1 was originally identified as a nuclear protein that can bend DNA, stabilizing nucleosome formation and regulating gene expression when recruited by specific DNA binding proteins. Intracellularly, HMGB1 functions as a DNA chaperone, facilitating various nuclear processes including transcription, replication, and DNA repair. Additionally, HMGB1 can interact with and stimulate the enzymatic activity of topoisomerase IIα (topo IIα), influencing DNA topology during these processes . It is expressed at high levels in almost all cells, underscoring its fundamental importance in cellular homeostasis.
What is the significance of extracellular HMGB1?
When released extracellularly, HMGB1 functions as a damage-associated molecular pattern (DAMP) molecule, serving as a critical mediator of inflammation. Extracellular HMGB1 is a key biomarker of immunogenic cell death, a specialized form of cell death that results in an immune response . Following trauma or tissue damage, HMGB1 is rapidly released into the circulation within hours, initiating and perpetuating inflammatory cascades that can lead to systemic inflammation and organ dysfunction . This extracellular function makes HMGB1 an important biomarker and potential therapeutic target in various inflammatory conditions.
What are the validated methods for quantifying HMGB1 in human samples?
Several established methods exist for HMGB1 detection, each with distinct advantages and limitations:
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Western blotting: Provides qualitative visualization of HMGB1 but offers limited quantification capabilities.
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ELISA (Enzyme-Linked Immunosorbent Assay): The most widely used format for HMGB1 detection in human plasma/serum. Sandwich ELISA utilizing matched antibody pairs offers good sensitivity and specificity .
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EMSA (Electrophoretic Mobility Shift Assay): Based on HMGB1's high affinity for hemicatenated DNA, this method is sensitive but laborious, time-consuming, and shows cross-reactivity with other DNA-binding proteins .
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Electrochemical immunosensors: Newer approach using impedance measurements with antibody-coated gold electrodes, offering rapid detection (approximately 19 minutes total assay time) with a limit of detection near 2 ng/mL .
What sample preparation is critical for accurate HMGB1 measurement in human plasma?
Perchloric acid (PCA) extraction is essential for accurate measurement of HMGB1 in human plasma. Recent experiments have demonstrated that PCA-extraction results in nearly 100% solubility of HMGB1 protein while removing most contaminants. This preparation step is critical, as preliminary ELISA experiments showed that PCA-extracted human patient plasma contained 2-3 times more detectable HMGB1 than non-extracted plasma . The necessity of this pretreatment highlights the challenges in accurately measuring HMGB1 in complex biological matrices, where interacting proteins or inhibitory factors may mask detection.
How does the Lumit® HMGB1 Immunoassay work, and what are its performance characteristics?
The Lumit® HMGB1 Immunoassay utilizes a novel NanoBiT® Luciferase-based approach:
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Principle: Primary antibodies specific to HMGB1 are labeled with LgBiT and SmBiT subunits of NanoBiT® Luciferase. When these antibodies bind to HMGB1, the subunits are brought together to form an active luciferase enzyme.
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Signal generation: Addition of an optimized substrate generates a bright luminescence signal proportional to HMGB1 levels.
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Performance characteristics:
| Species | Limit of Detection | Dynamic Range | Assay Time | Sample Type |
|---|---|---|---|---|
| Human HMGB1 | 1 ng/ml | 4-1,000 ng/ml | 90 minutes | Cell culture samples |
| Mouse HMGB1 | 3 ng/ml | 3-2,187 ng/ml | 90 minutes | Cell culture samples |
The assay can be used for both human and mouse HMGB1 due to their high homology, with minimal performance differences between species .
What is the time course of HMGB1 elevation following trauma in humans?
Unlike in sepsis where HMGB1 is considered a late mediator, trauma induces rapid HMGB1 elevation in humans. Plasma HMGB1 levels increase significantly within 1 hour of traumatic injury, with marked elevations occurring from 2 to 6 hours post-injury (median 526.18 ng/mL). These elevated levels remain significantly above control values for at least 136 hours after injury . This rapid release pattern suggests that HMGB1 is an early mediator in the inflammatory response to trauma, contrasting with its role in sepsis where levels peak days after onset.
How does HMGB1 contribute to inflammation-mediated diseases?
HMGB1 contributes to inflammation-mediated diseases through multiple mechanisms:
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Activation of pattern recognition receptors: Extracellular HMGB1 activates TLR2, TLR4, and RAGE receptors, triggering pro-inflammatory signaling cascades.
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Amplification of cytokine production: HMGB1 stimulates the release of pro-inflammatory cytokines including TNF-α, IL-1, and IL-6.
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Promotion of leukocyte recruitment: HMGB1 enhances endothelial activation and leukocyte adhesion/migration.
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Induction of tissue damage: Persistent HMGB1 signaling contributes to tissue injury and organ dysfunction.
In sepsis, HMGB1 levels correlate with the degree of organ dysfunction and can predict mortality, with levels remaining elevated for up to 7 days after admission, well after other cytokine levels have normalized . This prolonged elevation supports HMGB1's role as a sustained mediator of systemic inflammation in septic patients.
How does HMGB1 regulate gene expression through interactions with topoisomerase IIα?
HMGB1 can up-regulate cellular expression of topoisomerase IIα (topo IIα) through a complex regulatory mechanism:
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Promoter activation: HMGB1 up-regulates the activity of the human topo IIα promoter, but this effect is dependent on the absence of functional retinoblastoma protein (pRb). This was demonstrated using luciferase gene reporter assays .
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DNA bending requirement: The ability of HMGB1 to bend DNA is essential for this transactivation, as mutant HMGB1 incapable of DNA bending failed to up-regulate topo IIα promoter activity .
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Transcription factor modulation: HMGB1 appears to transactivate the topo IIα promoter by modulating the binding of transcription factor NF-Y to the promoter .
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HMGB1-pRb interaction: Despite HMGB1 binding to both wild-type and mutant pRb, a higher fraction of HMGB1 (approximately 2-3 fold) associates with wild-type pRb than with mutant pRb. This interaction may explain the inhibitory effect of pRb on HMGB1-mediated transactivation .
This regulatory mechanism may contribute to the previously observed correlation between increased levels of HMGB1 and topo IIα in tumors.
What experimental approaches can be used to silence HMGB1 expression?
HMGB1 expression can be effectively silenced using short hairpin RNA (shRNA) technology:
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Vector system: Plasmid pcDNA-Zeo(-)-U6 under the control of a human U6 promoter can be used to express shRNAs that specifically cleave HMGB1 mRNA in human cells .
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Target sequences:
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Selection process: For stable transfection, cells can be grown in media containing 300 μg/ml Zeocin and selected for 2-3 weeks .
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Control: A scrambled sequence not related to any human sequence (GAGAGGACAAGAGATGTATT) should be used as a negative control .
This approach has been demonstrated to result in diminished expression of topo IIα, supporting the role of HMGB1/2 in modulating cellular activity of the topo IIα gene.
How can HMGB1 be utilized as a biomarker in inflammatory and trauma conditions?
HMGB1 serves as a valuable biomarker in various conditions:
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Trauma assessment: Rapid elevation within 1 hour of trauma makes HMGB1 an early indicator of tissue damage. Levels peak from 2-6 hours post-injury (median 526.18 ng/mL) and remain elevated for over 5 days .
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Sepsis monitoring: In sepsis, HMGB1 levels correlate with organ dysfunction severity and predict mortality, with divergence between survivors and non-survivors becoming apparent by day 3 after admission .
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Immunogenic cell death evaluation: HMGB1 is a key biomarker for immunogenic cell death, making it valuable for assessing certain cancer therapies .
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Practical considerations: For optimal sensitivity, PCA extraction of plasma is recommended prior to measurement, as this step has been shown to increase detectable HMGB1 by 2-3 fold .
What complementary assays should be considered alongside HMGB1 measurement?
For comprehensive assessment of immunogenic cell death and inflammatory responses, several complementary assays should be considered:
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RealTime-Glo™ Extracellular ATP Assay: Kinetically measures the release of ATP from dying cells, providing real-time assessment of immunogenic cell death induction. When used alongside HMGB1 measurement, it provides a more complete picture of the immunogenic cell death process .
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IL-1β measurement: Quantification of IL-1β release can assess inflammasome activation, which often accompanies HMGB1 release in inflammatory conditions .
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Cell viability assays: Correlating HMGB1 release with cell death parameters helps distinguish passive release from necrotic cells versus active secretion.
When combined, these assays provide a comprehensive assessment of damage-associated molecular pattern (DAMP) release and subsequent inflammatory activation.
Product Science Overview
HMGB1 contains two DNA-binding domains known as HMG boxes (A-box and B-box) and an acidic C-terminal tail . These domains allow HMGB1 to bind to DNA and facilitate various nuclear functions, including:
- Transcriptional Regulation: HMGB1 interacts with transcription factors and other regulatory proteins to modulate gene expression .
- DNA Repair: It plays a role in the repair of damaged DNA by stabilizing nucleosomes and facilitating the access of repair proteins .
- Chromatin Remodeling: HMGB1 helps in the assembly and disassembly of nucleosomes, thereby influencing chromatin structure and function .
HMGB1 has been implicated in several pathological conditions, including cancer, sepsis, and autoimmune diseases . Its role in these conditions is attributed to its ability to modulate inflammation and immune responses. For instance:
- Cancer: HMGB1 can act as both an oncogene and a tumor suppressor, depending on the context. It regulates nucleosomal structure, DNA damage repair, and genomic stability within the cell, while also playing a role in immune cell functions .
- Sepsis: Elevated levels of extracellular HMGB1 are associated with the progression of sepsis, making it a potential therapeutic target .
- Autoimmune Diseases: HMGB1’s involvement in inflammatory processes makes it a key player in the pathogenesis of autoimmune diseases .
Human recombinant HMGB1 is produced using recombinant DNA technology, which involves cloning the HMGB1 gene into an expression vector and introducing it into a host cell (such as E. coli) for protein production. This recombinant protein is used in various research applications to study its biological functions and potential therapeutic uses.
