Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[(18)O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.

Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

Treatment of complex diseases such as cancer, cardiovascular disease, diabetes or neurological disorders frequently warrants the utilization of drug combinations for therapeutic intervention. In fact, the most successful example is the current standard of care for HIV patients. However, identification of successful drug cocktails is not a simple task and is hampered by lack of standardization in terminology, experimental protocols and models as well as data analysis. Here we discuss the most recent developments in combinatorial drug screening by covering technological advancements in screening strategies, cellular model systems as well as novel drug classes. We believe the research progress being made provides promising basis to build on and identify, develop and optimize efficacious clinically relevant combinatorial drug treatments.

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.

Various commercial anti-Spike SARS-CoV-2 antibody tests are used for studies and in clinical settings after vaccination. An international standard for SARS-CoV-2 antibodies has been established to achieve comparability of such tests, allowing conversions to BAU/mL. This study aimed to investigate the comparability of antibody tests regarding the timing of blood collection after vaccination. For this prospective observational study, antibody levels of 50 participants with homologous AZD1222 vaccination were evaluated at 3 and 11 weeks after the first dose and 3 weeks after the second dose using two commercial anti-Spike binding antibody assays (Roche and Abbott) and a surrogate neutralization assay. The correlation between Roche and Abbott changed significantly depending on the time point studied. Although Abbott provided values three times higher than Roche 3 weeks after the first dose, the values for Roche were twice as high as for Abbott 11 weeks after the first dose and 5 to 6 times higher at 3 weeks after the second dose. The comparability of quantitative anti-Spike SARS-CoV-2 antibody tests was highly dependent on the timing of blood collection after vaccination. Therefore, standardization of the timing of blood collection might be necessary for the comparability of different quantitative SARS-COV-2 antibody assays. IMPORTANCE This work showed that the comparability of apparently standardized SARS-CoV-2 antibody assays (Roche, Abbott; both given in BAU/mL) after vaccination depends on the time of blood withdrawal. Initially (3 weeks after the first dose AZD1222), there were 3 times higher values in the Abbott assay, but this relationship inversed before boosting (11 weeks after the first dose) with Roche 2 times greater than Abbott. After the booster, Roche quantified ca. 5 times higher levels than Abbott. This must be considered by clinicians when interpreting SARS-CoV-2 antibody levels.

Patients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited.

Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase-substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.