Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism.

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.

Deciphering the Duality of Clock and Growth Metabolism in a Cell Autonomous System Using NMR Profiling of the Secretome.

Oscillations in circadian metabolism are crucial to the well being of organism. Our understanding of metabolic rhythms has been greatly enhanced by recent advances in high-throughput systems biology experimental techniques and data analysis. In an in vitro setting, metabolite rhythms can be measured by time-dependent sampling over an experimental period spanning one or more days at sufficent resolution to elucidate rhythms. We hypothesized that cellular metabolic effects over such a time course would be influenced by both oscillatory and circadian-independent cell metabolic effects. Here we use nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling of mammalian cell culture media of synchronized U2 OS cells containing an intact transcriptional clock. The experiment was conducted over 48 h, typical for circadian biology studies, and samples collected at 2 h resolution to unravel such non-oscillatory effects. Our data suggest specific metabolic activities exist that change continuously over time in this settting and we demonstrate that the non-oscillatory effects are generally monotonic and possible to model with multivariate regression. Deconvolution of such non-circadian persistent changes are of paramount importance to consider while studying circadian metabolic oscillations.

Binding sites for interaction of peroxiredoxin 6 with surfactant protein A.

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H(+)-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell.

p67(phox) terminates the phospholipase A(2)-derived signal for activation of NADPH oxidase (NOX2).

The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67(phox) and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67(phox) and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67(phox) with nonphosphorylated Prdx6 was relatively weak. Association of p67(phox) and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67(phox) bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67(phox) did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67(phox); the calculated dissociation constant (Kd) of the p67(phox): phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67(phox) with siRNA. These data indicate that p67(phox) binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.-Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67(phox) terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2).