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1.
Immunity ; 48(5): 911-922.e7, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29768176

ABSTRACT

Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.


Subject(s)
Endosomes/immunology , Membrane Transport Proteins/immunology , Molecular Chaperones/immunology , Toll-Like Receptors/immunology , Animals , Dendritic Cells/immunology , Dendritic Cells/metabolism , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Endosomes/metabolism , HEK293 Cells , Humans , Macrophages/immunology , Macrophages/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Stability , Protein Transport/immunology , Signal Transduction/genetics , Signal Transduction/immunology , THP-1 Cells , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism
2.
Mol Cell Proteomics ; 22(6): 100563, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37142057

ABSTRACT

Comprehensive and in-depth identification of the human leukocyte antigen class I (HLA-I) and class II (HLA-II) tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is a powerful technology for direct identification of HLA peptides from patient-derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare and clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high-throughput, sensitive, and single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight MS on the Bruker timsTOF single-cell proteomics system (SCP). We demonstrate greater than twofold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation, and reduces input requirements to as few as 1e6 A375 cells for >800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen and noncanonical proteins. We also apply our optimized single-shot SCP acquisition methods to tumor-derived samples, enabling sensitive, high-throughput, and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.


Subject(s)
Histocompatibility Antigens Class I , Neoplasms , Male , Humans , Histocompatibility Antigens Class I/metabolism , Mass Spectrometry/methods , Neoplasms/metabolism , Peptides/metabolism , Cell Line
3.
Mol Syst Biol ; 17(7): e10125, 2021 07.
Article in English | MEDLINE | ID: mdl-34318608

ABSTRACT

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site-specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.


Subject(s)
Protein Processing, Post-Translational , Proteomics , Antigen-Antibody Complex , Mass Spectrometry , Phenotype
4.
J Immunol ; 193(7): 3257-61, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25187660

ABSTRACT

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.


Subject(s)
Adaptor Protein Complex 1/immunology , Adaptor Protein Complex 2/immunology , B-Lymphocytes/immunology , Membrane Transport Proteins/immunology , Monocytes/immunology , Toll-Like Receptors/immunology , Adaptor Protein Complex 1/genetics , Adaptor Protein Complex 2/genetics , Amino Acid Motifs , B-Lymphocytes/cytology , Cell Line, Tumor , HEK293 Cells , Humans , Membrane Transport Proteins/genetics , Monocytes/cytology , Protein Transport/genetics , Protein Transport/immunology , Toll-Like Receptors/genetics
5.
Cell Rep ; 43(9): 114736, 2024 Sep 24.
Article in English | MEDLINE | ID: mdl-39277863

ABSTRACT

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1ß release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.


Subject(s)
Butyrates , Inflammasomes , Macrophages , NLR Family, Pyrin Domain-Containing 3 Protein , Propionates , Toll-Like Receptors , Humans , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Inflammasomes/metabolism , Propionates/pharmacology , Butyrates/pharmacology , Macrophages/metabolism , Macrophages/drug effects , Toll-Like Receptors/metabolism , Signal Transduction/drug effects , Interleukin-1beta/metabolism , Interleukin-10/metabolism
6.
bioRxiv ; 2023 Mar 18.
Article in English | MEDLINE | ID: mdl-36993564

ABSTRACT

Comprehensive, in-depth identification of the human leukocyte antigen HLA-I and HLA-II tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is powerful technology for direct identification of HLA peptides from patient derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare, clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high throughput, sensitive, single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight mass spectrometry on the Bruker timsTOF SCP. We demonstrate >2-fold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation and reduces input requirements to as few as 1e6 A375 cells for > 800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen, and novel/unannotated open reading frames. We also apply our optimized single-shot SCP acquisition methods to tumor derived samples, enabling sensitive, high throughput and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

7.
Nat Commun ; 14(1): 1851, 2023 04 03.
Article in English | MEDLINE | ID: mdl-37012232

ABSTRACT

Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.


Subject(s)
Lung Neoplasms , Proteome , Male , Humans , Proteome/metabolism , Workflow , Peptides , Proteomics/methods
8.
Cell Rep ; 34(10): 108826, 2021 03 09.
Article in English | MEDLINE | ID: mdl-33691121

ABSTRACT

A major pathway for proinflammatory protein release by macrophages is inflammasome-mediated pyroptotic cell death. As conventional secretion, unconventional secretion, and cell death are executed simultaneously, however, the cellular mechanisms regulating this complex paracrine program remain incompletely understood. Here, we devise a quantitative proteomics strategy to define the cellular exit route for each protein by pharmacological and genetic dissection of cellular checkpoints regulating protein release. We report the release of hundreds of proteins during pyroptosis, predominantly due to cell lysis. They comprise constitutively expressed and transcriptionally induced proteins derived from the cytoplasm and specific intracellular organelles. Many low-molecular-weight proteins including the cytokine interleukin-1ß, alarmins, and lysosomal-cargo proteins exit cells in the absence of cell lysis. Cytokines and alarmins are released in an endoplasmic reticulum (ER)-Golgi-dependent manner as free proteins rather than by extracellular vesicles. Our work provides an experimental framework for the dissection of cellular exit pathways and a resource for pyroptotic protein release.


Subject(s)
Alarmins/analysis , Cytokines/analysis , Proteomics/methods , Pyroptosis , Adenosine Triphosphate/pharmacology , Alarmins/metabolism , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Cytokines/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Nigericin/pharmacology , Tandem Mass Spectrometry
9.
Cell Rep ; 30(4): 1260-1270.e5, 2020 01 28.
Article in English | MEDLINE | ID: mdl-31995763

ABSTRACT

The inflammatory functions of the cytokine tumor necrosis factor (TNF) rely on its ability to induce cytokine production and to induce cell death. Caspase-dependent and caspase-independent pathways-apoptosis and necroptosis, respectively-regulate immunogenicity by the release of distinct sets of cellular proteins. To obtain an unbiased, systems-level understanding of this important process, we here applied mass spectrometry-based proteomics to dissect protein release during apoptosis and necroptosis. We report hundreds of proteins released from human myeloid cells in time course experiments. Both cell death types induce receptor shedding, but only apoptotic cells released nucleosome components. Conversely, necroptotic cells release lysosomal components by activating lysosomal exocytosis at early stages of necroptosis-induced membrane permeabilization and show reduced release of conventionally secreted cytokines.


Subject(s)
Apoptosis , Caspase 8/metabolism , Cytokines/metabolism , Necroptosis , Pentanoic Acids/pharmacology , Proteome/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Apoptosis/drug effects , Caspase Inhibitors/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Chemokine CCL2/metabolism , Chemokine CCL24/metabolism , Dipeptides/pharmacology , Exocytosis/drug effects , Extracellular Vesicles/drug effects , Extracellular Vesicles/metabolism , HEK293 Cells , Histones/metabolism , Humans , Indoles/pharmacology , Interleukin-8/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Mass Spectrometry , Necroptosis/drug effects
10.
Nat Neurosci ; 21(9): 1196-1208, 2018 09.
Article in English | MEDLINE | ID: mdl-30127427

ABSTRACT

Mononuclear phagocytes are key regulators of both tissue damage and repair in neuroinflammatory conditions such as multiple sclerosis. To examine divergent phagocyte phenotypes in the inflamed CNS, we introduce an in vivo imaging approach that allows us to temporally and spatially resolve the evolution of phagocyte polarization in a murine model of multiple sclerosis. We show that the initial proinflammatory polarization of phagocytes is established after spinal cord entry and critically depends on the compartment they enter. Guided by signals from the CNS environment, individual phagocytes then switch their phenotype as lesions move from expansion to resolution. Our study thus provides a real-time analysis of the temporospatial determinants and regulatory principles of phagocyte specification in the inflamed CNS.


Subject(s)
Leukocytes, Mononuclear/pathology , Multiple Sclerosis/pathology , Phagocytes/pathology , Animals , Astrocytes/pathology , Astrocytes/ultrastructure , Bone Marrow Cells/pathology , Bone Marrow Cells/ultrastructure , Cell Polarity , Computer Systems , Encephalomyelitis, Autoimmune, Experimental/pathology , Humans , Inflammation/pathology , Leukocytes, Mononuclear/ultrastructure , Mice , Mice, Inbred C57BL , Neuroglia/pathology , Neuroglia/ultrastructure , Phagocytes/ultrastructure , Phagocytosis , Phenotype , Sequence Analysis, RNA , Spinal Cord/pathology , Spinal Cord/ultrastructure
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