-GlcNAc transferase (OGT) modifies serine and threonine residues on nuclear and cytosolic proteins with
-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the ...underlying mechanisms are still enigmatic. We performed a genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferation induced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cells stems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8
T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.
Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) ...response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (
Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is
Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.
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► Integrative strategy for dissecting mammalian signaling networks ► Strategy identifies factors and pathways controlling TLR signals ► Polo-like kinases are key regulators of well-established IFN-inducing pathways ► The kinases activate a signaling branch involving a dozen proteins
An integrative approach combining transcriptional profiling, small-molecule perturbations, and phosphoproteomics shows that Polo-like kinases play an essential role in immune pathways regulated by Toll-like receptors.
Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and ...relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.
Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative ...mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.
Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes' regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine ...CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.
Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of ...non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we ...developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
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•Cell type and temporally resolved cell-surface proteomic profiling in intact brains•Proteome-wide coordinated change of neuronal surface landscape over development•New cell-surface regulators of brain wiring from unexpected molecular families•Cell-autonomous control of dendrite targeting by the lipoprotein receptor LRP1
In situ cell-surface proteomic profiling of developing and mature olfactory projection neurons uncovers the temporal evolution of the neuronal surface landscape in development and reveals many new neural wiring molecules belonging to evolutionarily conserved but previously unexpected molecular families.
Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of ...improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
Peripheral intravenous catheter (PIVC) use in health care is common worldwide. Failure of PIVCs is also common, resulting in premature removal and replacement.
To investigate the characteristics, ...management practices, and outcomes of PIVCs internationally.
Cross-sectional study. Hospitalized patients from rural, regional, and metropolitan areas internationally.
Hospital, device, and inserter characteristics were collected along with assessment of the catheter insertion site. PIVC use in different geographic regions was compared.
We reviewed 40,620 PIVCs in 51 countries. PIVCs were used primarily for intravenous medication (n = 28,571, 70%) and predominantly inserted in general wards (n = 22,167, 55%). Two-thirds of all devices were placed in non-recommended sites such as the hand, wrist, or antecubital veins. Nurses inserted most PIVCs (n = 28,575, 71%); although there was wide regional variation (26% to 97%). The prevalence of iIn this study, we found that many PIVCs were placed in areas of flexion, were symptomatic or idle, had suboptimal dressings, or lacked adequate documentation. This suggests inconsistency between recommended management guidelines for PIVCs and current practice.dle PIVCs was 14% (n = 5,796). Overall, 10% (n = 4,204) of PIVCs were painful to the patient or otherwise symptomatic of phlebitis; a further 10% (n = 3,879) had signs of PIVC malfunction; and 21% of PIVC dressings were suboptimal (n = 8,507). Over one-third of PIVCs (n = 14,787, 36%) had no documented daily site assessment and half (n = 19,768, 49%) had no documented date and time of insertion.
In this study, we found that many PIVCs were placed in areas of flexion, were symptomatic or idle, had suboptimal dressings, or lacked adequate documentation. This suggests inconsistency between recommended management guidelines for PIVCs and current practice.
The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) ...and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.
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