Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice.

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

Genetic code expansion in the mouse brain.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

Hepatocytes respond differently to major dietary trans fatty acid isomers, elaidic acid and trans-vaccenic acid.

BACKGROUND: It has been discussed if the adverse health effect associated with the ingestion of trans fatty acids correlates with the food source, as the composition of the isomers varies in different foods. We have investigated the hepatocellular responses to the predominant trans fatty acid isomers in industrially produced partially hydrogenated vegetable oils (elaidic acid) and products of ruminant origin (trans-vaccenic acid). RESULTS: The responses of HepG2-SF cells exposed to 100 µM fatty acids during 7 days were examined. Elaidic acid decreased the cellular proliferation rate while trans-vaccenic acid had no effect. Analysis of cellular triacylglycerol fractions showed, that both trans fatty acids were metabolized by HepG2-SF cells, although elaidic acid, to a higher degree than trans-vaccenic, accumulated in the triacylglycerol fraction. Proteome analysis revealed that the overlap of differentially regulated proteins only contained four proteins, suggesting that the two trans fatty acid isomers affect the cells in different ways. The data are available via ProteomeXchange with identifier PXD000760. CONCLUSIONS: Our investigations revealed that the hepatocellular response to the two most abundant dietary positional C18:1 trans fatty acid isomers differ substantially. In addition, the results suggest that trans-vaccenic acid does not affect cholesterol metabolism adversely compared to elaidic acid.

Labeling and identifying cell-specific proteomes in the mouse brain.

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain.

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing.

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse's active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.

Effects of elaidic acid on lipid metabolism in HepG2 cells, investigated by an integrated approach of lipidomics, transcriptomics and proteomics.

Trans fatty acid consumption in the human diet can cause adverse health effects, such as cardiovascular disease, which is associated with higher total cholesterol, a higher low density lipoprotein-cholesterol level and a decreased high density lipoprotein-cholesterol level. The aim of the study was to elucidate the hepatic response to the most abundant trans fatty acid in the human diet, elaidic acid, to help explain clinical findings on the relationship between trans fatty acids and cardiovascular disease. The human HepG2 cell line was used as a model to investigate the hepatic response to elaidic acid in a combined proteomic, transcriptomic and lipidomic approach. We found many of the proteins responsible for cholesterol synthesis up-regulated together with several proteins involved in the esterification and hepatic import/export of cholesterol. Furthermore, a profound remodeling of the cellular membrane occurred at the phospholipid level. Our findings contribute to the explanation on how trans fatty acids from the diet can cause modifications in plasma cholesterol levels by inducing abundance changes in several hepatic proteins and the hepatic membrane composition.

TSG-6 transfers proteins between glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism.

Studies of the interaction between Bikunin proteins, tumor necrosis factor-stimulated gene-6 protein (TSG-6), and glycosaminoglycans have revealed a unique catalytic activity where TSG-6/heavy chain 2 transfer heavy chain subunits between glycosaminoglycan chains. The activity is mediated by TSG-6/heavy chain 2 and involves a transient SDS stable interaction between TSG-6 and the heavy chain to be transferred. The focus of this study was to characterize the molecular structure of this cross-link to gain further insight into the catalytic mechanism. The result showed that the C-terminal Asp residue of the heavy chains forms an ester bond to Ser(28) beta-carbon of TSG-6 suggesting that this residue plays a role during catalysis.

The transfer of heavy chains from bikunin proteins to hyaluronan requires both TSG-6 and HC2.

Tumor necrosis factor-stimulated gene-6 protein (TSG-6) is involved in the transfer of heavy chains (HCs) from inter-alpha-inhibitor (IalphaI), pre-alpha-inhibitor, and as shown here HC2.bikunin to hyaluronan through the formation of covalent HC.TSG-6 intermediates. In contrast to IalphaI and HC2.bikunin, pre-alpha-inhibitor does not form a covalent complex in vitro using purified proteins but needs the presence of another factor (Rugg, M. S., Willis, A. C., Mukhopadhyay, D., Hascall, V. C., Fries, E., Fülöp, C., Milner, C. M., and Day, A. J. (2005) J. Biol. Chem. 280, 25674-25686). In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including IalphaI, HC2.bikunin, and free HC2 promoted the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.