Toll-like receptors (TLRs) are highly-conserved pattern recognition receptors that mediate innate immune responses to invading pathogens and endogenous danger signals released from damaged and dying cells. Activation of TLRs trigger downstream signaling cascades, that culminate in the activation of interferon regulatory factors (IRFs), which subsequently leads to type I interferon (IFN) response. In the current study, we sought to expand the scope of gene expression changes in THP1-derived macrophages upon TLR4 activation and to identify interferon-stimulated genes. RNA-seq analysis led to the identification of several known and novel differentially expressed genes, including CMPK2, particularly in association with type I IFN signaling. We performed an in-depth characterization of CMPK2 expression, a nucleoside monophosphate kinase that supplies intracellular UTP/CTP for nucleic acid synthesis in response to type I IFN signaling in macrophages. CMPK2 was significantly induced at both RNA and protein levels upon stimulation with TLR4 ligand-LPS and TLR3 ligand-Poly (I:C). Confocal microscopy and subcellular fractionation indicated CMPK2 localization in both cytoplasm and mitochondria of THP-1 macrophages. Furthermore, neutralizing antibody-based inhibition of IFNAR receptor in THP-1 cells and BMDMs derived from IFNAR KO and IRF3 KO knockout mice further revealed that CMPK2 expression is dependent on LPS/Poly (I:C) mediated IRF3- type I interferon signaling. In summary, our findings suggest that CMPK2 is a potential interferon-stimulated gene in THP-1 macrophages and that CMPK2 may facilitate IRF3- type I IFN-dependent anti-bacterial and anti-viral roles.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

Though the effect of the recently identified mitochondrial NAD+ transporter SLC25A51 on glucose metabolism has been described, its contribution to other NAD+-dependent processes throughout the cell such as ADP-ribosylation remains elusive. Here, we report that absence of SLC25A51 leads to increased NAD+ concentration not only in the cytoplasm and but also in the nucleus. The increase is not associated with upregulation of the salvage pathway, implying an accumulation of constitutively synthesized NAD+ in the cytoplasm and nucleus. This results in an increase of PARP1-mediated nuclear ADP-ribosylation, as well as faster repair of DNA lesions induced by different single-strand DNA damaging agents. Lastly, absence of SLC25A51 reduces both MMS/Olaparib induced PARP1 chromatin retention and the sensitivity of different breast cancer cells to PARP1 inhibition. Together these results provide evidence that SLC25A51 might be a novel target to improve PARP1 inhibitor based therapies by changing subcellular NAD+ redistribution.

We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.

Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction.

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.

On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses.

Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that CD14 (cluster of differentiation 14) constitutively interacts with the MyD88-dependent TLR7 and TLR9. CD14 was necessary for TLR7- and TLR9-dependent induction of proinflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. CD14 associated with TLR9 stimulatory DNA in precipitation experiments and confocal imaging. The absence of CD14 led to reduced nucleic acid uptake in macrophages. Additionally, CD14 played a role in the stimulation of TLRs by viruses. Using various types of vesicular stomatitis virus, we showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses. These data show that CD14 has a dual role in nucleic acid-mediated TLR activation: it promotes the selective uptake of nucleic acids, and it acts as a coreceptor for endosomal TLR activation.

In this study, we report a comparative and quantitative analysis by mass spectrometry of the protein content of aqueous humour from cataract (control) patients. In addition to protein profiling, the approach is layered with quantitative proteomics using the iTRAQ® methodology. Aqueous humour from ten clinically-matched patients was collected and depleted of albumin and immunoglobulin G. Pairs of patient material were pooled and divided into three aliquots for subsequent analysis by alternative proteomic approaches. Excluding keratin, trypsin, residual albumin and immunoglobulins, a total of 198 protein groups were identified across the entire study. Relative protein quantitation with iTRAQ® revealed that 88% of the proteins had a maximal ±2-fold differential regulation between 3 of the 4 labelled samples, indicating minimal variation. The identified proteins were categorised by gene ontology and one third of the proteins were annotated as extracellular. The major molecular functions of the proteins in aqueous humour are binding (protein, metal ion, heparin, and DNA) and inhibition of proteolytic activity. Complementary to molecular function, the predominant biological processes for the proteins in aqueous humour are assigned to inflammatory and immune responses, and transport.

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.

Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell's physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape.

Drug testing in patient biopsy-derived cells can identify potent treatments for patients suffering from relapsed or refractory hematologic cancers. Here we investigate the use of weakly supervised deep learning on cell morphologies (DML) to complement diagnostic marker-based identification of malignant and nonmalignant cells in drug testing. Across 390 biopsies from 289 patients with diverse blood cancers, DML-based drug responses show improved reproducibility and clustering of drugs with the same mode of action. DML does so by adapting to batch effects and by autonomously recognizing disease-associated cell morphologies. In a post hoc analysis of 66 patients, DML-recommended treatments led to improved progression-free survival compared with marker-based recommendations and physician's choice-based treatments. Treatments recommended by both immunofluorescence and DML doubled the fraction of patients achieving exceptional clinical responses. Thus, DML-enhanced ex vivo drug screening is a promising tool in the identification of effective personalized treatments.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.