Method to Measure Ubiquitination of NLRs.

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here we describe a method to detect poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins.

TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation.

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT's intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action.

Heterogeneity in extracellular vesicle secretion by single human macrophages revealed by super-resolution microscopy.

The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1- or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1- and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on ∼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2-like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1ß Secretion in Human Macrophages.

Interleukin 1ß (IL-1ß) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1ß release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1ß release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1ß release in human macrophages.

Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles.

Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.

Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

USP7 and USP47 deubiquitinases regulate NLRP3 inflammasome activation.

The assembly and activation of the inflammasomes are tightly regulated by post-translational modifications, including ubiquitin. Deubiquitinases (DUBs) counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How DUBs control the assembly and activation of inflammasomes is unclear. Here, we show that the DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by inhibition of USP7 and USP47. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP-1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether, these data reveal a new post-transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and the release of the pro-inflammatory cytokines IL-1ß and IL-18, and implicate dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.

Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis.

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.

Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.

Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome.

In targeted proteomics it is critical that peptides are not only proteotypic but also accurately represent the level of the protein (quantotypic). Numerous approaches are used to identify proteotypic peptides, but quantotypic properties are rarely assessed. We show that measuring ratios of proteotypic peptides across biological samples can be used to empirically identify peptides with good quantotypic properties. We applied this technique to identify quantotypic peptides for 21% of the human kinome.

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.