Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.

Analysis and Interpretation of Protein Post-Translational Modification Site Stoichiometry.

Proteins are decorated with a diverse array of post-translational modifications (PTMs) that regulate their spatial and temporal functions. Recent mass spectrometry (MS)-based studies have identified hundreds of thousands of PTM sites in mammalian proteomes. However, the signaling cues and enzymes regulating individual sites are often not known and their functional roles remain uncharacterized. Quantification of PTM site stoichiometry can help in prioritizing sites for functional analyses and is important for constructing mechanistic models of PTM-dependent protein regulation. Here, we review the concept of PTM site stoichiometry, critically evaluate the merits and drawbacks of different MS-based methods used for quantifying PTM site stoichiometry, and discuss the usefulness and limitations of stoichiometry in informing on the biological function of modified sites.

Proteome Analysis of PC12 Cells Reveals Alterations in Translation Regulation and Actin Signaling Induced by Clozapine.

Although antipsychotics are routinely used in the treatment of schizophrenia for the last decades, their precise mechanism of action is still unclear. In this study, we investigated changes in the PC12 cells' proteome under the influence of clozapine, risperidone, and haloperidol to identify protein pathways regulated by antipsychotics. Analysis of the protein profiles in two time points: after 12 and 24 h of incubation with drugs revealed significant alterations in 510 proteins. Further canonical pathway analysis revealed an inhibition of ciliary trophic factor signaling after treatment with haloperidol and showed a decrease in acute phase response signaling in the risperidone group. Interestingly, all tested drugs have caused changes in PC12 proteome which correspond to inhibition of cytokines: tumor necrosis factor (TNF) and transforming growth factor beta 1 (TGF-ß1). We also found that the 12-h incubation with clozapine caused up-regulation of protein kinase A signaling and translation machinery. After 24 h of treatment with clozapine, the inhibition of the actin cytoskeleton signaling and Rho proteins signaling was revealed. The obtained results suggest that the mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) play a central role in the signal transduction of clozapine.

Homogenous Phase Enrichment of Cysteine-Containing Peptides for Improved Proteome Coverage.

We describe a proteomic reactor-based homogeneous phase enrichment of cysteine-containing peptides in a filter aided sample preparation (FASP) format. In this approach thiol-reduced proteins are derivatized with thiol-activated polyethylene glycol (TAPEG) before protein cleavage. Consecutive digestion with endoproteinase LysC and trypsin allows isolation of two fractions of nonderivatized peptides. After reduction of disulfide bonds between cysteine-containing peptides and the polyethylene glycol moieties, a third fraction of peptides is collected. LC-MS/MS analyses revealed that on average this fraction consists of 95% cysteine-containing peptides. Since 85-93% of all peptides are unique to a single subfraction, the combination of TAPEG and FASP offers an efficient peptide separation strategy. Analysis of whole cell lysates of mouse brain, liver, red muscle fibers, and CaCo-2 cells using the TAPEG FASP approach allowed identification of 6,900, 5,800, 4,200 and 7,900 proteins, 10-30% more than were identified using two-step digestion without isolation of Cys-containing peptides. The fractionation also increased the protein sequence coverage by 10-30%.

Impact of imipramine on proteome of rat primary glial cells.

Microglia and astrocytes, two types of glial cells are known to be important targets for antidepressant drugs. Here we used a comprehensive proteomic analysis to examine the effect of imipramine on rat primary mixed glial culture. The two-dimensional differential gel electrophoresis method allowed us to identify 62 proteins that were altered by imipramine. Functional analysis revealed that imipramine influenced the level of proteins involved in oxidative stress; in particular, it elevated the level of glutathione transferases. Imipramine upregulated proteins related to glycolysis but down-regulated many mitochondrial proteins including enzymes involved in oxidative phosphorylation. Mitochondrial dysfunction, especially decrease of mitochondrial membrane potential can be counted as a side effect triggered by imipramine. Imipramine induced lowering of chaperone level and alterations suggesting impaired protein synthesis could be associated with increased apoptosis. One of the most pronounced effect of imipramine is the reduction of vimentin level, this protein is engaged in majority of biological processes which were found to be affected by imipramine. Many imipramine regulated proteins, including chaperones, cathepsins and annexins are involved in immune responses. Additionally, imipramine influenced proteins associated with phagocytosis and cell migration. Overall these findings indicate that imipramine produces complex effect on glial cells, primarily on microglia and suggest their transition towards a more quiescent, metabolically less demanding phenotype.