TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins.

Magnitude of Ubiquitination Determines the Fate of Epidermal Growth Factor Receptor Upon Ligand Stimulation.

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials.

MaxQuant.Live Enables Enhanced Selectivity and Identification of Peptides Modified by Endogenous SUMO and Ubiquitin.

Small ubiquitin-like modifiers (SUMO) and ubiquitin are frequent post-translational modifications of proteins that play pivotal roles in all cellular processes. We previously reported mass spectrometry-based proteomics methods that enable profiling of lysines modified by endogenous SUMO or ubiquitin in an unbiased manner, without the need for genetic engineering. Here we investigated the applicability of precursor mass filtering enabled by MaxQuant.

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy.

Spatial Characterization of the Human Centrosome Proteome Opens Up New Horizons for a Small but Versatile Organelle.

After a century of research, the human centrosome continues to fascinate. Based on immunofluorescence and confocal microscopy, an extensive inventory of the protein components of the human centrosome, and the centriolar satellites, with the important contribution of over 300 novel proteins localizing to these compartments is presented. A network of candidate centrosome proteins involved in ubiquitination, including six interaction partners of the Kelch-like protein 21, and an additional network of protein phosphatases, together supporting the suggested role of the centrosome as an interactive hub for cell signaling, is identified.

DNA damage-induced dynamic changes in abundance and cytosol-nuclear translocation of proteins involved in translational processes, metabolism, and autophagy.

Ionizing radiation (IR) causes DNA double-strand breaks (DSBs) and activates a versatile cellular response regulating DNA repair, cell-cycle progression, transcription, DNA replication and other processes. In recent years proteomics has emerged as a powerful tool deepening our understanding of this multifaceted response. In this study we use SILAC-based proteomics to specifically investigate dynamic changes in cytoplasmic protein abundance after ionizing radiation; we present in-depth bioinformatics analysis and show that levels of proteins involved in autophagy (cathepsins and other lysosomal proteins), proteasomal degradation (Ubiquitin-related proteins), energy metabolism (mitochondrial proteins) and particularly translation (ribosomal proteins and translation factors) are regulated after cellular exposure to ionizing radiation.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy.

Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics.

Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking.

A fascinating conundrum in cell signaling is how stimulation of the same receptor tyrosine kinase with distinct ligands generates specific outcomes. To decipher the functional selectivity of EGF and TGF-α, which induce epidermal growth factor receptor (EGFR) degradation and recycling, respectively, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels.

StUbEx: Stable tagged ubiquitin exchange system for the global investigation of cellular ubiquitination.

Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein-protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level.

Analysis of secreted proteins using SILAC.

Secreted proteins serve a crucial role in the communication between cells, tissues, and organs. Proteins released to the extracellular environment exert their function either locally or at distant points of the organism. Proteins are secreted in a highly dynamic fashion by cells and tissues in the body responding to the stimuli and requirements presented by the extracellular milieu.

Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8.

Background: Assembly of primary cilia relies on vesicular trafficking towards the cilium base and intraflagellar transport (IFT) between the base and distal tip of the cilium. Recent studies have identified several key regulators of these processes, including Rab GTPases such as Rab8 and Rab11, the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances, the exact molecular mechanisms by which these proteins interact and target to the basal body to promote ciliogenesis are not fully understood.

Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response.

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs).

Temporal profiling and pulsed SILAC labeling identify novel secreted proteins during ex vivo osteoblast differentiation of human stromal stem cells.

It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC).

Quantitative phosphoproteomics to characterize signaling networks.

Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform for analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks.

Phosphoproteomic analysis of cells treated with longevity-related autophagy inducers.

Macroautophagy is a self-cannibalistic process that enables cells to adapt to various stresses and maintain energy homeostasis. Additionally, autophagy is an important route for turnover of misfolded proteins and damaged organelles, with important implications in cancer, neurodegenerative diseases and aging. Resveratrol and spermidine are able to induce autophagy by affecting deacetylases and acetylases, respectively, and have been found to extend the life-span of model organisms.

PhosphoSiteAnalyzer: a bioinformatic platform for deciphering phospho proteomes using kinase predictions retrieved from NetworKIN.

Phosphoproteomic experiments are routinely conducted in laboratories worldwide, and because of the fast development of mass spectrometric techniques and efficient phosphopeptide enrichment methods, researchers frequently end up having lists with tens of thousands of phosphorylation sites for further interrogation. To answer biologically relevant questions from these complex data sets, it becomes essential to apply computational, statistical, and predictive analytical methods. Here we provide an advanced bioinformatic platform termed "PhosphoSiteAnalyzer" to explore large phosphoproteomic data sets that have been subjected to kinase prediction using the previously published NetworKIN algorithm.

Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics.

Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin or chains of ubiquitin molecules of various types and lengths to targeted proteins is known to alter proteins' lifespan, localization and function and to modulate protein interactions. Despite its central importance in various aspects of cellular life and function there are only a limited number of reports investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination.

GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data.

Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1).

System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.

To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation.

Quantitative analysis of the secretion of the MCP family of chemokines by muscle cells.

The plasticity of skeletal muscle allows the body to adapt to various physiological demands such as growth, exercise and tissue regeneration and repair. The secreted factors from muscle exert their action via auto-, para-, and endocrine mechanisms, thereby influencing the maintenance of total body homeostasis. In addition, the regulation of muscle proliferation, differentiation, and regeneration is often perturbed by inflammatory processes and is dependent on the pattern of expression of pro-inflammatory stimuli.

Mapping protein-protein interactions by quantitative proteomics.

Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners.

Organelle proteomics by label-free and SILAC-based protein correlation profiling.

The ability to purify cell organelles and protein complexes on a large scale, combined with advances in protein identification using mass spectrometry, has provided a wealth of information regarding protein localization and function. A major challenge in these studies has been the ability to identify bona fide organelle components from a background of co-purifying contaminants because none of the available biochemical purification protocols afford pure preparations. Since this situation is unlikely to change alternative strategies have been devised to meet this challenge by making use of the information inherent in the fractionation profile of organelles isolated by density gradient centrifugation.

Proteome-wide quantitation by SILAC.

Ongoing improvements in instrumentation, fractionation techniques, and enrichment procedures have dramatically increased the coverage of the proteome achievable via LC-MS/MS-based methodologies, opening the call for approaches to quantitatively assess differences at a proteome-wide scale. Stable isotope labeling by amino acids in cell culture (SILAC) has emerged as a powerful and versatile approach for proteome-wide quantitation by mass spectrometry. SILAC utilizes the cells' own metabolism to incorporate isotopically labeled amino acids into its proteome which can be mixed with the proteome of unlabeled cells and differences in protein expression can easily be read out by comparing the abundance of the labeled versus unlabeled proteins.

Dynamics of the skeletal muscle secretome during myoblast differentiation.

During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells.

Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response.

To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin.