Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological lightdark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.circadian rhythm | proteomics | liver metabolism | posttranslational regulation | protein secretion L ight and heat, the principle energy sources for life, are only periodically available with a period of 1 d. Consequently, organisms acquired a timing system to adapt their physiology and anticipate these diurnal variations. In mammals, this circadian clock influences most aspects of physiology and behavior (1). In humans, perturbations of this clock lead to pathologies, including metabolic and vascular diseases. Although the oscillatory clockwork is cell-autonomous, timing on the scale of organisms uses a hierarchal organization: a master clock within the suprachiasmatic nuclei (SCN) of the hypothalamus receives light input through the retina and communicates timing signals to slave oscillators in other peripheral tissues (2).In mammals, the molecular oscillator uses interconnected transcriptional and translational feedback loops, in which multiple layers of control, including temporal posttranscriptional and posttranslational regulation, play important roles (3). An active area of chronobiology aims at understanding how the temporal signals from the core oscillator are relayed to clock output function. In this context, genome-wide rhythms in mRNA accumulation were characterized in several models. In general, around 10% of the genes, encoding many enzymes involved in different aspects of cellular metabolism, show rhythmic mRNA accumulation, establishing the role of the circadian clock in temporally gating rhythmic physiology (4).However, comparatively little is known on the temporal accumulation of proteins, despite increasing evidence suggesting that posttranscriptional mechanisms also contr...

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation.

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SummaryDiurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membraneassociated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity. Molecular & Cellular Proteomics 6:1459 -1472, 2007.The halotolerant alga Dunaliella salina uses a unique osmoregulatory mechanism and is able to proliferate in environments with extreme salt content (1). This is achieved through remarkable changes in metabolism and ion transport brought about by up-regulation or down-regulation of multiple enzymes and membrane proteins. Elucidation of the molecular basis for this remarkable adaptation might provide tools to improve the resistance of crop plants to the progressive salinization of soils, which is already a major limitation for agricultural productivity worldwide. By characterizing the changes in the soluble subproteome of D. salina, we demonstrated previously that the alga responds to hypersaline conditions by up-regulating key enzymes in photosynthetic CO 2 fixation and in energy metabolism that divert carbon metabolism to massive synthesis of glycerol, the osmotic element in Dunaliella (2).Salinity stress destabilizes biological membranes and affects the solubility of many essential substrates and ions (3). Adaptation to salinity stress might also alter the composition and organization of the membrane proteome associated with the stabilization and enhancement of ion transporters. Early studies revealed the accumulation of two carbonic anhydrases, dCAI 1 and dCAII (4, 5), and a transferrin-like protein (6) that presumably compensated the impaired availability of bicarbonate and iron under high salt. Salt-induced changes in organellar membranes, such as the expression of ER fatty acid elongase, were also reported (7). High salinity also affects sodium transport (8), lipid organization (9, 10), and activation of PM pr...

Social insects frequently engage in oral fluid exchange – trophallaxis – between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers’ food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes.DOI: http://dx.doi.org/10.7554/eLife.20375.001

LC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, combined with data processing, stringent, and sequence-similarity database searching tools, was employed in a layered manner to identify proteins in organisms with unsequenced genomes. Highly specific stringent searches (MASCOT) were applied as a first layer screen to identify either known (i.e. present in a database) proteins, or unknown proteins sharing identical peptides with related database sequences. Once the confidently matched spectra were removed, the remainder was filtered against a nonannotated library of background spectra that cleaned up the dataset from spectra of common protein and chemical contaminants. The rectified spectral dataset was further subjected to rapid batch de novo interpretation by PepNovo software, followed by the MS BLAST sequence-similarity search that used multiple redundant and partially accurate candidate peptide sequences. Importantly, a single dataset was acquired at the uncompromised sensitivity with no need of manual selection of MS/MS spectra for subsequent de novo interpretation. This approach enabled a completely automated identification of novel proteins that were, otherwise, missed by conventional database searches.

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