Delineation of lipopolysaccharide (LPS)-binding sites on hemoglobin: from in silico predictions to biophysical characterization.

Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and ß subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbß attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.

A potent liver-mediated mechanism for loss of muscle mass during androgen deprivation therapy.

CONTEXT: Androgen deprivation therapy (ADT) in prostate cancer results in muscular atrophy, due to loss of the anabolic actions of testosterone. Recently, we discovered that testosterone acts on the hepatic urea cycle to reduce amino acid nitrogen elimination. We now hypothesize that ADT enhances protein oxidative losses by increasing hepatic urea production, resulting in muscle catabolism. We also investigated whether progressive resistance training (PRT) can offset ADT-induced changes in protein metabolism. OBJECTIVE: To investigate the effect of ADT on whole-body protein metabolism and hepatic urea production with and without a home-based PRT program. DESIGN: A randomized controlled trial. PATIENTS AND INTERVENTION: Twenty-four prostate cancer patients were studied before and after 6 weeks of ADT. Patients were randomized into either usual care (UC) (n = 11) or PRT (n = 13) starting immediately after ADT. MAIN OUTCOME MEASURES: The rate of hepatic urea production was measured by the urea turnover technique using 15N2-urea. Whole-body leucine turnover was measured, and leucine rate of appearance (LRa), an index of protein breakdown and leucine oxidation (Lox), a measure of irreversible protein loss, was calculated. RESULTS: ADT resulted in a significant mean increase in hepatic urea production (from 427.6 ± 18.8 to 486.5 ± 21.3; P < 0.01) regardless of the exercise intervention. Net protein loss, as measured by Lox/Lra, increased by 12.6 ± 4.9% (P < 0.05). PRT preserved lean body mass without affecting hepatic urea production. CONCLUSION: As early as 6 weeks after initiation of ADT, the suppression of testosterone increases protein loss through elevated hepatic urea production. Short-term PRT was unable to offset changes in protein metabolism during a state of profound testosterone deficiency.

Decorin, a growth hormone-regulated protein in humans.

CONTEXT: Growth hormone (GH) stimulates connective tissue and muscle growth, an effect that is potentiated by testosterone. Decorin, a myokine and a connective tissue protein, stimulates connective tissue accretion and muscle hypertrophy. Whether GH and testosterone regulate decorin in humans is not known. OBJECTIVE: To determine whether decorin is stimulated by GH and testosterone. DESIGN: Randomized, placebo-controlled, double-blind study. PARTICIPANTS AND INTERVENTION: 96 recreationally trained athletes (63 men, 33 women) received 8 weeks of treatment followed by a 6-week washout period. Men received placebo, GH (2 mg/day), testosterone (250 mg/week) or combination. Women received either placebo or GH (2 mg/day). MAIN OUTCOME MEASURE: Serum decorin concentration. RESULTS: GH treatment significantly increased mean serum decorin concentration by 12.7 ± 4.2%; P < 0.01. There was a gender difference in the decorin response to GH, with greater increase in men than in women (∆ 16.5 ± 5.3%; P < 0.05 compared to ∆ 9.4 ± 6.5%; P = 0.16). Testosterone did not significantly change serum decorin. Combined GH and testosterone treatment increased mean decorin concentration by 19.5 ± 3.7% (P < 0.05), a change not significantly different from GH alone. CONCLUSION: GH significantly increases circulating decorin, an effect greater in men than in women. Decorin is not affected by testosterone. We conclude that GH positively regulates decorin in humans in a gender-dimorphic manner.

Testosterone prevents protein loss via the hepatic urea cycle in human.

CONTEXT: The urea cycle is a rate-limiting step for amino acid nitrogen elimination. The rate of urea synthesis is a true indicator of whole-body protein catabolism. Testosterone reduces protein and nitrogen loss. The effect of testosterone on hepatic urea synthesis in humans has not been studied. OBJECTIVE: To determine whether testosterone reduces hepatic urea production. DESIGN: An open-label study. PATIENTS AND INTERVENTION: Eight hypogonadal men were studied at baseline, and after two weeks of transdermal testosterone replacement (Testogel, 100 mg/day). MAIN OUTCOMES MEASURES: The rate of hepatic urea synthesis was measured by the urea turnover technique using stable isotope methodology, with 15N2-urea as tracer. Whole-body leucine turnover was measured, from which leucine rate of appearance (LRa), an index of protein breakdown and leucine oxidation (Lox), a measure of irreversible protein loss, were calculated. RESULTS: Testosterone administration significantly reduced the rate of hepatic urea production (from 544.4 ± 71.8 to 431.7 ± 68.3 µmol/min; P < 0.01), which was paralleled by a significant reduction in serum urea concentration. Testosterone treatment significantly reduced net protein loss, as measured by percent Lox/LRa, by 19.3 ± 5.8% (P < 0.05). There was a positive association between Lox and hepatic urea production at baseline (r2 = 0.60, P < 0.05) and after testosterone administration (r2 = 0.59, P < 0.05). CONCLUSION: Testosterone replacement reduces protein loss and hepatic urea synthesis. We conclude that testosterone regulates whole-body protein metabolism by suppressing the urea cycle.

Extracellular haemoglobin upregulates and binds to tissue factor on macrophages: implications for coagulation and oxidative stress.

The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbß binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.