Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here ...we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS-one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a biotin tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of ...LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.
Triple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based ...tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target.
MS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival.
High FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare.
These results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract.
Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral ...cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors.
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•Vaccinia virus core activation involves reduction of disulfide-bonded core proteins•Poxvirus lateral bodies (LBs) are delivery containers for viral enzymes•LB disassembly is regulated by proteasome activity•Dephosphorylation of STAT1 by LB-resident VH1 requires LB disassembly
Poxviruses, including the devastating variola virus, deliver lateral bodies (LBs) into the host cell cytoplasm during entry. However, the composition and function of these viral structures remains enigmatic. Using vaccinia virus, Mercer and colleagues now identify bona fide LB proteins. They demonstrate that LBs deliver the immunomodulatory phosphatase VH1 into host cells. Proteasome-mediated LB disassembly is required for release and activation of VH1, which then acts on its cellular target, STAT1, prior to viral gene expression.
Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), ...which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.
Abstract
Precision oncology requires a deep understanding of the molecular mechanism in cancer biology. The predominant approach today focuses on genome structure and gene expression data, which have ...become available with the rise of next-gen sequencing technology. On a phenotypic level, however, protein expression and activation are arguably more directly related to cellular function. The availability of combined genome and proteome level data from the same tumor is therefore expected to provide a much more complete picture of a tumor in a particular state. Until recently, proteomics technology could not match the scale of next-gen sequencing and consequently, precision medicine research has almost exclusively been based on gene-level data. Here we show the first truly large-scale data set for protein expression and protein phosphorylation for a large collection of biospecimens derived from the IndivuType cohort of Indivumed, Germany. Matching fresh-frozen tumor and adjacent normal tissue samples from thousands of patients including various cancer entities were obtained from Indivumed's network of partner hospitals Enabled by the novel data-independent acquisition (DIA) workflow, a mass spectrometric method that obtains peptide fragmentation data in a highly parallelized way with high reproducibility and sensitivity, more than 7,000 proteins in the whole proteome (WP) and 20,000 phospho-peptides in the phospho-proteome (PP) workflow were analyzed. Sample processing from 5 mg of tissue per sample was performed on 96-well plates with the help of a liquid handling robot. Phospho-peptide enrichment was carried out using a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads (ReSyn Biosciences). Data-independent acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (WP) and 60 min (PP) gradients. With a throughput of 850 WP and 650 PP samples per month, thousands of samples were analyzed to date. The resulting proteome data is integrated into Indivumed's IndivuType multi-omics database, supporting the identification and validation of new molecular cancer drug targets and biomarkers.
Citation Format: Jakob Vowinckel, Karel Novy, Thomas Corwin, Tobias Treiber, Roland Bruderer, Lukas Reiter, Eike von Leitner, Oliver Rinner, Claudia Escher. Proteomics for precision oncology: Profiling the proteome of matching tumor and adjacent normal tissue using data-independent acquisition abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4266.
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e15672
Background: The rise of precision oncology therapeutics requires deep understanding of all molecular mechanisms involved in cancer biology. IndivuType offers the world’s first ...multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics (WGS), transcriptomics, proteomics, and clinical outcome information. Indivumed is committed to the quality of the IndivuType ecosystem starting with stringent SOP-driven sample collection combined with thorough validation of clinical information and data integrity. The availability of multi-omics data from the same tumor can provide a comprehensive molecular picture of cancer for a given patient. Protein expression and activation are directly related to cellular function and hence provide actionable information about druggable targets. Until recently, the proteomics technology could not match the scale of next-gen sequencing and consequently precision medicine has almost exclusively been based on gene level data. Here we present the first large-scale data set for protein expression and phosphorylation. Enabled by the data independent acquisition (DIA) workflow, a mass spectrometric method provided by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome (WP) and 20,000 phospho-peptides in the phospho-proteome (PP) workflow were profiled. Methods: Sample processing from 5 mg of tissue per sample was performed using liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. DIA LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (WP) and 60 min (PP) gradients. Results: Several thousands of high-quality patient samples of various cancer types have been analyzed to date. The resulting proteome and phospho-proteome data has been integrated into the IndivuType database, thereby providing a solid foundation to advance our understanding of cancer. Conclusions: With the ongoing addition of more samples and associated deep and rich data, the platform could unravel key molecular events and is expected to transform knowledge into actionable treatments and personalized therapies.
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e15536
Background: The rise of precision oncology therapeutics requires deep understanding of the molecular mechanisms implicated in cancer biology. Colorectal cancer (CRC) is one of ...the first solid tumors to be molecularly characterized by defined genes and pathways. Advances in tumor profiling have revealed a profound molecular heterogeneity in CRC leading to the definition of several consensus molecular subtypes (CMS). However, this molecular heterogeneity is still largely defined on the genomic and transcriptomics level. To complement the understanding of genetically defined molecular subgroups, we performed large-scale deep proteomic and phospho-proteomic profiling of CRC patient biopsies and adjacent healthy control tissue, which has enabled to explore the phenotype and obtain more functional insights in cancer biology. Methods: Sample processing from 5-10 mg of tissue per sample was performed using a liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. Data-Independent Acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (whole proteome) and 60 min (phospho-proteome) gradients. Results: Indivumed has built IndivuType, the world’s first multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics, transcriptomics, proteomics, and clinical outcome information. Enabled by the DIA technology, a mass spectrometric method developed by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome and 20,000 phospho-peptides in the phospho-proteome workflow were profiled across more than 900 resected tissue samples of various CMS of CRC. The resulting proteome and phospho-proteome data were integrated into the IndivuType database and cross-analyzed with genomic and transcriptomic markers. Through this combined analysis, novel insights in clinically relevant signaling pathways in CRC subtypes were revealed. Conclusions: The deep phenotypic profiling of cancer samples, using next generation proteomics and phospho-proteomics, has enabled us to go beyond the genomic level in the characterization of tumor molecular heterogeneity. This multi-omics approach provides a solid foundation to advance the understanding of cancer biology, unravel key molecular events, and support the identification of novel therapeutic targets for precision medicine in CRC.
Abstract
Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming ...toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.
Host surfaceome remodeling modulates Listeria infection
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents ...with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
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