Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages.

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

Metabolic and molecular responses to leucine-enriched branched chain amino acid supplementation in the skeletal muscle of alcoholic cirrhosis.

UNLABELLED: Skeletal muscle loss (sarcopenia) is a major clinical complication in alcoholic cirrhosis with no effective therapy. Skeletal muscle autophagic proteolysis and myostatin expression (inhibitor of protein synthesis) are increased in cirrhosis and believed to contribute to anabolic resistance. A prospective study was performed to determine the mechanisms of sarcopenia in alcoholic cirrhosis and potential reversal by leucine. In six well-compensated, stable, alcoholic patients with cirrhosis and eight controls, serial vastus lateralis muscle biopsies were obtained before and 7 hours after a single oral branched chain amino acid mixture enriched with leucine (BCAA/LEU). Primed-constant infusion of l-[ring-(2) H5 ]-phenylalanine was used to quantify whole-body protein breakdown and muscle protein fractional synthesis rate using liquid chromatography/mass spectrometry. Muscle expression of myostatin, mammalian target of rapamycin (mTOR) targets, autophagy markers, protein ubiquitination, and the intracellular amino acid deficiency sensor general control of nutrition 2 were quantified by immunoblots and the leucine exchanger (SLC7A5) and glutamine transporter (SLC38A2), by real-time polymerase chain reaction. Following oral administration, plasma BCAA concentrations showed a similar increase in patients with cirrhosis and controls. Skeletal muscle fractional synthesis rate was 9.63 ± 0.36%/hour in controls and 9.05 ± 0.68%/hour in patients with cirrhosis (P = 0.54). Elevated whole-body protein breakdown in patients with cirrhosis was reduced with BCAA/LEU (P = 0.01). Fasting skeletal muscle molecular markers showed increased myostatin expression, impaired mTOR signaling, and increased autophagy in patients with cirrhosis compared to controls (P < 0.01). The BCAA/LEU supplement did not alter myostatin expression, but mTOR signaling, autophagy measures, and general control of nutrition 2 activation were consistently reversed in cirrhotic muscle (P < 0.01). Expression of SLC7A5 was higher in the basal state in patients with cirrhosis than controls (P < 0.05) but increased with BCAA/LEU only in controls (P < 0.001). CONCLUSIONS: Impaired mTOR1 signaling and increased autophagy in skeletal muscle of patients with alcoholic cirrhosis is acutely reversed by BCAA/LEU.

Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity.

APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins.

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba maté (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of maté tea.

Protective action of Ilex paraguariensis extract against free radical inactivation of paraoxonase-1 in high-density lipoprotein.

Paraoxonase 1 (PON-1), an antioxidant enzyme carried mainly by HDL, has been shown to display cardioprotective effects. In this study, we investigated whether the polyphenol-rich beverage mate, obtained from extract of Ilex paraguariensis (IP), prevented the loss of PON-1 activity from HDL during oxidant stress. The peroxide radical generator 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) induced time- and dose-dependent oxidation of HDL, as measured by lipoperoxide content, which is accompanied by a parallel decrease in the activity of PON-1 (p<0.001). IP extract (2-20 microL/mL) afforded time- and concentration-dependent inhibition of the oxidation, with preservation of apoA-I structure and PON-1 activity. Healthy volunteers drank either 0.5 L of IP extract, 0.5 L of coffee and milk or nothing. PON-1 activity increased an average of 10% above the changes seen when the intake was coffee and milk (p<0.05). In conclusion, we demonstrate that IP extract may, to some extent, prevent the loss of the antiatherogenic function of HDL afforded by PON-1 when the particle is under oxidant stress.