High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-B and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Tolllike receptor ( HMGB11 (formerly HMG1) was originally described as a non-histone, chromatin-associated nuclear protein (1-4).HMGB1 has a highly conserved sequence among species, with murine HMGB1 differing from the human form by only two amino acids. HMGB1-deficient mice die within a few hours of birth, demonstrating the crucial role of this protein in cellular function. HMGB1 consists of two tandem L-shaped domains, HMGB boxes A and B, each ϳ75 amino acids in length, and a highly acidic carboxyl terminus of 30 amino acids in length.HMGB1 appears to have two distinct functions in cellular systems. First, it has been shown to be an intracellular regulator of transcription, and, second, HMGB1 can occupy an extracellular role in which it promotes tumor metastasis and inflammation (2-9). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (10, 11). Monocytes and macrophages stimulated by lipopolysaccharide (LPS), tumor necrosis factor (TNF)-␣, or interleukin-1 (IL-1) secrete HMGB1 (5, 11). Culture of monocytes with HMGB1 results in the release of TNF-␣, IL-1␣, IL-1␤, IL-1Ra, IL-6, IL-8, macrophage inflammatory protein-1␣, macrophage inflammatory protein-1␤, but not IL-10 or IL-12 (5, 11). Production of proinflammatory cytokines after exposure to HMGB1 occurs with delayed kinetics as compared with LPS-induced stimulation. For example, culture of macrophages with LPS results in increases in TNF-␣ that are apparent within less than 1 h, whereas TNF-␣ synthesis following HMGB1 exposure only begins to occur after 2 h and then persists for as long as 8 h (8, 11). The signaling mechanisms responsible for the delayed expression of proinflammatory cytokines by HMGB1-stimulated cells remain incompletely explained but appear to involve the p38, ERK, JNK, and Akt kinases and to lead to enhanced nuclear translocation of .In recent studies (13), we found that the magnitude and kinetics of cytokine expression and nuclear translocation of NF-B after culture of neutrophils with HMGB1 or LPS were similar, suggesting that overlapping mechanisms of cellular activation might be involved. Comparison of gene expression arrays also demonstrated substantial but not complete homology in response to HMGB1 and LPS. Signaling via the TLR 4 receptor is responsible for LPS-induced activation of the IKK kinase complex, including IKK...

High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-Bdependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS. fluorescence resonance energy transfer; receptor of advanced glycation end products HIGH MOBILITY GROUP BOX 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly by monocytes and macrophages stimulated by LPS, TNF-␣, or IL-1 (2, 44). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (1,44,46), and also appears to be involved in pathophysiological processes associated with cellular necrosis, such as acetaminophen-induced liver injury (34).Although HMGB1 and LPS appear to initiate similar intracellular events, including activation of kinases such as p38, ERK1/2, and Akt and transcriptional factors including NF-B, that lead to production of proinflammatory cytokines, gene arrays demonstrated differences in expression profiles with each of these stimuli (12, 30). Unlike LPS, which primarily increased the activity of IKK-␤, HMGB1 exposure resulted in activation of both IKK-␣ and IKK-␤ (31). In addition, culture of neutrophils lacking Toll-like receptor (TLR)4 with HMGB1, but not with LPS, still resulted in enhanced nuclear translocation of NF-B (31). Such results suggest that the receptors interacting with HMGB1 and leading to cellular activation and gene transcription are likely to be distinct from TLR4, which is responsible for LPS-induced responses (40). Recent data indicate that HMGB1 interacts not only with TLR4 but also with TLR2 and the receptor for advanced glycation end products (RAGE) (31, 46). In particular, a decrease in NF-B-dependent reporter gene expression after transfection with dominantnegative constructs to TLR2, TLR4, or both, demonstr...

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.