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AbstractSupplemental data for this article can be accessed here.High mobility group box-1 (HMGb1), an endogenous danger-associated molecular pattern protein (DAMP) whose extracellular release has been associated with sterile injury and various inflammatory diseases and conditions, has been shown to be a valuable clinical drug target. Elucidation of the specific interactions with the HMGb1 receptor, Toll-like receptor 4 (TLR4) and adaptor protein myeloid differentiation factor-2 (MD-2), will lead to more precisely targeted therapeutics. We sought to examine detailed interactions and dynamics of the HMGb1 A-box and B-box fragments, as well as the intact protein using in silicoprotein–protein docking (ZDOCK, ZRANK) and molecular dynamics (Schrödinger Desmond, New York, NY). Mutagenesis and SPR-binding studies allowed us to draw further conclusions regarding the details of the HMGb1–TLR4–MD2 interaction and shed light on the reasons for the opposing biological activities of HMGb1 A-box and B-box fragments. From our findings, we hypothesize that disulfide A-box fragment binds as an anchor toward the TLR4–MD-2 but does not facilitate the TLR4 dimer formation, thereby competing with the HMGb1-binding site and preventing HMGb1-induced signaling and downstream inflammation, whereas the pro-inflammatory B-box fragment retains the MD-2 active conformation and binds to both TLR4 proteins in the complex to aid TLR4 dimer formation, which activates the intracellular signaling for downstream inflammatory pathways and cytokine release.Communicated by Ramaswamy H. Sarma

High mobility group box-1 (HMGb1) protein, a nuclear non-histone protein that is released or secreted from the cell in response to damage or stress, is a sentinel for the immune system that plays a critical role in cell survival/death pathways. This review highlights key features of the endogenous danger-associated molecular pattern (DAMP) protein, HMGb1 in the innate inflammatory response along with various cofactors and receptors that regulate its downstream effects. The evidence demonstrating increased levels of HMGb1 in human inflammatory diseases and conditions is presented, along with a summary of current small molecule or peptide-like antagonists proven to specifically target HMGb1. Additionally, we delineate the measures needed toward validating this protein as a clinically relevant biomarker or bioindicator and as a relevant drug target.

To elucidate the control of hepatic cholesterol metabolism by leptin, rats were administered IV (intravenous) leptin, ICV (intracerebroventricular) leptin or saline. A single low dose of ICV leptin was as effective as a continuous IV infusion of highdose leptin at decreasing the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7α-hydroxylase. These results indicate that the hepatic response to leptin is transduced via the central nervous system.

Background: Typically, a diagnosis of diabetes mellitus is based on elevated circulating blood glucose levels. In an attempt to discover additional markers for the disease and predictors of prognosis, we undertook the characterization of HbA1d3in diabetic and normal patients. Material and Methods: PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the α- and β-globin chains of HbA1d3and characterize their presence in normal and diabetic patients. Results: We report the characterization of HbA1d3as a glutathionylated, minor hemoglobin subfraction that occurs in higher levels in diabetic patients (2.26 ± 0.29 %) than in normal individuals (1.21 ± 0.14%, p< 0.001). The α-chain spectrum displayed a molecular ion of m/z15126 Da, which is consistent with the predicted native mass of the HbA0α-globin chain. By contrast, the mass spectrum of the β-chain showed a mass excess of 307 Da (m/z= 16173 Da) versus that of the native HbA0β-globin chain (m/z= 15866 Da). The native molecular weight of the modified β-globin chain HbA0was regenerated by treatment of HbA1d3with dithiothreitol, consistent with a glutathionylated adduct. Conclusions: We propose that HbA1d3(HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations.