Search

STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis- piv SATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.

Protein-protein interactions are essential biologic processes that occur at inter- and intracellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein-protein interaction networks in living cells. The combination of proximity-dependent labelling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein-protein interactions in mitochondrial subcellular compartments., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

The thymic function to produce self-protective and self-tolerant T cells is chiefly mediated by cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs). Recent studies including single-cell transcriptomic analyses have highlighted a rich diversity in functional mTEC subpopulations. Because of their limited cellularity, however, the biochemical characterization of TECs, including the proteomic profiling of cTECs and mTECs, has remained unestablished. Utilizing genetically modified mice that carry enlarged but functional thymuses, here we show a combination of proteomic and transcriptomic profiles for cTECs and mTECs, which identified signature molecules that characterize a developmental and functional contrast between cTECs and mTECs. Our results reveal a highly specific impact of the thymoproteasome on proteasome subunit composition in cTECs and provide an integrated trans-omics platform for further exploration of thymus biology., (Published by Elsevier Inc.)

Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.

Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.

The p38 MAPK cascade is activated by various stresses or cytokines. Downstream of p38 MAPKs, there are diversification and extensive branching of signaling pathways. Fluorescent two-dimensional difference gel electrophoresis of phosphoprotein-enriched samples from HeLa cells in which p38 MAPK activity was either suppressed or activated enabled us to detect approximately 90 candidate spots for factors involved in p38-dependent pathways. Among these candidates, here we identified four proteins including Bcl-2-associated athanogene 2 (BAG2) by peptide mass fingerprintings. BAG family proteins are highly conserved throughout eukaryotes and regulate Hsc/Hsp70-mediated molecular chaperone activities and apoptosis. The results of two-dimensional immunoblots suggested that the phosphorylation of BAG2 was specifically controlled in a p38 MAPK-dependent manner. Furthermore, BAG2 was directly phosphorylated at serine 20 in vitro by MAPK-activated protein kinase 2 (MAPKAP kinase 2), which is known as a primary substrate of p38 MAPK and mediates several p38 MAPK-dependent processes. We confirmed that MAPKAP kinase 2 is also required for phosphorylation of BAG2 in vivo. Thus, p38 MAPK-MAPKAP kinase 2-BAG2 phosphorylation cascade may be a novel signaling pathway for response to extracellular stresses.