The effect of long term calorie restriction on in vivo hepatic proteostatis: a novel combination of dynamic and quantitative proteomics.

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic (2)H(2)O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (≈ 80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Measurement of human plasma proteome dynamics with (2)H(2)O and liquid chromatography tandem mass spectrometry.

Dysfunction of protein turnover is a feature of many human diseases, and proteins are substrates in important biological processes. Currently, no method exists for the measurement of global protein turnover (i.e., proteome dynamics) that can be applied in humans. Here we describe the use of metabolic labeling with deuterium ((2)H) from (2)H(2)O and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of mass isotopomer patterns to measure protein turnover. We show that the positions available for (2)H label incorporation in vivo can be calculated using peptide sequence. The isotopic incorporation values calculated by combinatorial analysis of mass isotopomer patterns in peptides correlate very closely with values established for individual amino acids. Inpatient and outpatient heavy water labeling protocols resulted in (2)H label incorporation sufficient for reproducible quantitation in humans. Replacement rates were similar for peptides deriving from the same protein. Using a kinetic model to account for the time course of each individual's (2)H(2)O enrichment curves, dynamics of approximately 100 proteins with half-lives ranging from 0.4 to 40 days were measured using 8 µl of plasma. The measured rates were consistent with literature values. This method can be used to measure in vivo proteome homeostasis in humans in disease and during therapeutic interventions.

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach.

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.

Alcohol-induced interactive phosphorylation of Src and toll-like receptor regulates the secretion of inflammatory mediators by human astrocytes.

Secretion of pro-inflammatory molecules by astrocytes after alcohol treatment was shown to be associated with neuroinflammation. We hypothesized that activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX-2) by ethanol in astrocytes enhanced the secretion of inflammatory agents via the interactive tyrosine phosphorylation of toll-like receptor 4 (TLR4) and Src kinase. To test this hypothesis, we treated primary human astrocytes with 20 mM ethanol for 48 h at 37°C. Ethanol exposure elevated cytochrome P450-2E1 activity, reactive oxygen species levels, and secretion of prostaglandin E2 (PGE2) in these cells. Secretion of PGE2 was associated with induction of cPLA2 activity and protein content as well as COX-2 protein level in a Src phosphorylation-dependent manner that occurred by enhanced transcription. Immunoprecipitation and Western blot analyses indicated that the interactive tyrosine phosphorylation of TLR4-Src complex at the cell membrane triggered the activation of cPLA2 and COX-2 in the cytoplasm through a Src signaling intermediate. Inhibition of ethanol metabolism, blockage of Src activity, or inactivation of TLR4 prevented the activation of cPLA2 and COX-2 as well as diminished PGE2 production, suggesting that interactive phosphorylation of TLR4-Src regulated the pro-inflammatory response in astrocytes. Experiments with small interfering RNA knockdown of TLR4 in human astrocytes confirmed that silencing expression also abolished the interactive phosphorylation of both TLR4 and Src in the presence of ethanol.