ABSTRACT
Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.
Subject(s)
Enhancer Elements, Genetic , Histone Demethylases , Histone Demethylases/metabolism , Histone Demethylases/genetics , Humans , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/chemistry , Transcription Factors/metabolism , Transcription Factors/genetics , Animals , Protein Binding , Mice , Cell Differentiation , Gene SilencingABSTRACT
KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone-lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A-matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.
Subject(s)
Brain Neoplasms , Gene Expression Regulation, Neoplastic , Histone Demethylases , Matrix Metalloproteinase 3 , Triple Negative Breast Neoplasms , Up-Regulation , Animals , Humans , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 3/genetics , Histone Demethylases/metabolism , Histone Demethylases/genetics , Brain Neoplasms/genetics , Brain Neoplasms/secondary , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , Female , Cell Line, Tumor , Mice , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Mice, Knockout , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Epigenesis, Genetic , Myeloid-Lymphoid Leukemia ProteinABSTRACT
Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/pathology , Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/metabolismABSTRACT
Stimulator of interferon genes (STING) is an intracellular sensor of cyclic di-nucleotides involved in the innate immune response against pathogen- or self-derived DNA. STING trafficking is tightly linked to its function, and its dysregulation can lead to disease. Here, we systematically characterize genes regulating STING trafficking and examine their impact on STING-mediated responses. Using proximity-ligation proteomics and genetic screens, we demonstrate that an endosomal sorting complex required for transport (ESCRT) complex containing HGS, VPS37A and UBAP1 promotes STING degradation, thereby terminating STING-mediated signaling. Mechanistically, STING oligomerization increases its ubiquitination by UBE2N, forming a platform for ESCRT recruitment at the endosome that terminates STING signaling via sorting in the lysosome. Finally, we show that expression of a UBAP1 mutant identified in patients with hereditary spastic paraplegia and associated with disrupted ESCRT function, increases steady-state STING-dependent type I IFN responses in healthy primary monocyte-derived dendritic cells and fibroblasts. Based on these findings, we propose that STING is subject to a tonic degradative flux and that the ESCRT complex acts as a homeostatic regulator of STING signaling.
Subject(s)
Membrane Proteins , Nucleotides, Cyclic , Humans , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Immunity, Innate , Membrane Proteins/metabolism , Nucleotides, Cyclic/pharmacologyABSTRACT
Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.
Subject(s)
Breast Neoplasms , Inflammatory Breast Neoplasms , Humans , Female , Inflammatory Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Signal Transduction , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Stem Cells/metabolism , STAT3 Transcription Factor/metabolismABSTRACT
The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.
Subject(s)
Epidermal Growth Factor , Proteomics , Epidermal Growth Factor/pharmacology , Extracellular Matrix Proteins , Ligands , PhenotypeABSTRACT
Down syndrome (DS) is a genetic disorder driven by the triplication of chromosome 21 (T21) and characterized by a wide range of neurodevelopmental and physical disabilities. Transcriptomic analysis of tissue samples from individuals with DS has revealed that T21 induces a genome-wide transcriptional disruption. However, the consequences of T21 on the nuclear architecture and its interplay with the transcriptome remain unknown. In this study, we find that unlike human induced pluripotent stem cells (iPSCs), iPSC-derived neural progenitor cells (NPCs) exhibit genome-wide "chromosomal introversion," disruption of lamina-associated domains, and global chromatin accessibility changes in response to T21, consistent with the transcriptional and nuclear architecture changes characteristic of senescent cells. Treatment of T21-harboring NPCs with senolytic drugs alleviates the transcriptional, molecular, and cellular dysfunctions associated with DS. Our findings provide a mechanistic link between T21 and global transcriptional disruption and indicate that senescence-associated phenotypes may play a key role in the neurodevelopmental pathogenesis of DS.
Subject(s)
Down Syndrome , Induced Pluripotent Stem Cells , Neural Stem Cells , Gene Expression Profiling , Humans , Transcriptome/geneticsABSTRACT
While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of "connectivity" to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics .
Subject(s)
Antineoplastic Agents/toxicity , Astrocytes/drug effects , Astrocytes/metabolism , Cardiotoxins/toxicity , Protein Kinase Inhibitors/toxicity , Proteomics , Cell Line, Tumor , Humans , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , ProteomeABSTRACT
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Pyridazines/chemistry , Adenosine Monophosphate/chemistry , Calorimetry, Differential Scanning , Catalytic Domain , Cell Survival/drug effects , Cell Survival/genetics , Cryoelectron Microscopy , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Endoribonucleases/chemistry , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kinetics , Mass Spectrometry , Multienzyme Complexes/ultrastructure , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Multimerization , Pyridazines/pharmacology , Recombinant Proteins , Tetrahydroisoquinolines/chemistryABSTRACT
Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.
Subject(s)
Gene Silencing , Histone-Lysine N-Methyltransferase/metabolism , Neoplasms/genetics , Neoplasms/immunology , Animals , Antigens, Viral/immunology , CRISPR-Cas Systems/genetics , Chromatin/genetics , Chromatin/metabolism , DNA Transposable Elements/genetics , Disease Models, Animal , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Mice , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.
Subject(s)
Erythropoiesis , Mitochondria/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Zebrafish Proteins/metabolism , Animals , Citric Acid Cycle , DNA Methylation , Dihydroorotate Dehydrogenase , Electron Transport , Embryo, Nonmammalian/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Histones/metabolism , Leflunomide/pharmacology , Metabolic Networks and Pathways , Methylation , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Oxygen Consumption , Transcription Factors/genetics , Ubiquinone/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Histones/analysis , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Aspergillus/enzymology , Histones/chemistry , Histones/genetics , Histones/metabolism , Humans , Nucleosomes/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The N-terminal regions (tails) of histone proteins are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly because of their inherently disordered nature. Although hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably because of the poor N-terminal coverage provided by pepsin. Inspired from histone-clipping events, we profiled the activity of cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3, and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.
Subject(s)
Cathepsin L/metabolism , Histones/chemistry , Histone Code , Humans , Hydrogen Deuterium Exchange-Mass Spectrometry , Hydrogen-Ion Concentration , Models, Molecular , Protein Conformation , Protein Folding , Proteolysis , Substrate SpecificityABSTRACT
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported â¼89â¯800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (â¼78â¯900 centroids), giving â¼100% coverage, and â¼10â¯900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σxÌ ). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.
Subject(s)
Antibodies, Monoclonal/chemistry , Hydrogen Deuterium Exchange-Mass Spectrometry , Immunoglobulin Fab Fragments/analysisABSTRACT
Members of the KDM5 histone H3 lysine 4 demethylase family are associated with therapeutic resistance, including endocrine resistance in breast cancer, but the underlying mechanism is poorly defined. Here we show that genetic deletion of KDM5A/B or inhibition of KDM5 activity increases sensitivity to anti-estrogens by modulating estrogen receptor (ER) signaling and by decreasing cellular transcriptomic heterogeneity. Higher KDM5B expression levels are associated with higher transcriptomic heterogeneity and poor prognosis in ER+ breast tumors. Single-cell RNA sequencing, cellular barcoding, and mathematical modeling demonstrate that endocrine resistance is due to selection for pre-existing genetically distinct cells, while KDM5 inhibitor resistance is acquired. Our findings highlight the importance of cellular phenotypic heterogeneity in therapeutic resistance and identify KDM5A/B as key regulators of this process.
Subject(s)
Breast Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Retinoblastoma-Binding Protein 2/genetics , Transcriptome/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Estradiol/pharmacology , Estrogen Receptor Modulators/pharmacology , Female , Fulvestrant/pharmacology , Genetic Heterogeneity , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , MCF-7 Cells , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Retinoblastoma-Binding Protein 2/metabolism , Transcriptome/drug effects , Exome Sequencing/methodsABSTRACT
Upon ultraviolet activation, cannabinergic aliphatic azido (N3) ligands covalently label cannabinoid receptors, prominent G-protein-coupled receptor (GPCR) drug targets. We report here the mechanism of covalent attachment to selected substrates of the high-affinity CBR inverse agonist AM1335 and its deuterated analog AM1335(d10), arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6) peptide and an N-acyl-protected cysteine (NAC). The photochemical reaction products of model substrates with AM1335 and AM1335(d10) were analyzed with tandem electrospray ionization mass spectrometry fragmentation and deuterium exchange mass spectrometry. The nitrene initially formed after photoreaction undergoes rearrangement to an imine which then interacts with the cysteine sulfhydryl group, resulting in ligand attachment. Our results demonstrate that covalent probes carrying aliphatic azides behave more selectively than originally thought and can be used to label protein cysteine residues preferentially.
Subject(s)
Azides/chemistry , Cysteine/chemistry , Membrane Proteins/chemistry , Molecular Probes/chemistry , Amino Acids/chemistry , Binding Sites , Deuterium Exchange Measurement , Ligands , Membrane Proteins/metabolism , Peptides/analysis , Peptides/chemistry , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/metabolism , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Ultraviolet RaysSubject(s)
Aniline Compounds/adverse effects , Human Umbilical Vein Endothelial Cells/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Nitriles/adverse effects , Phosphoproteins/metabolism , Protein Kinase Inhibitors/adverse effects , Proteomics , Quinolines/adverse effects , Aniline Compounds/pharmacology , Human Umbilical Vein Endothelial Cells/pathology , Humans , Nitriles/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinolines/pharmacologyABSTRACT
Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs × 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the "connectivity" framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics.