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The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway. Science , this issue p. 1177 , p. 1171 Altered ubiquitination of RAS GTPases is implicated in cancer drug resistance. In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 ( LZTR1 ) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization. Maintenance of a mitochondrial NAD+ pool is critical for cellular life, yet the existence and identity of the transporter responsible for mitochondrial NAD+ uptake was unknown until recently. Here, the authors use genomic, genetic, and metabolomic approaches to demonstrate that SLC25A51 controls NAD+ mitochondrial levels and is the functional homolog of the yeast mitochondrial NAD+ transporter.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses 1 – 3 . Here we show that a previously uncharacterized protein encoded by CXorf21— a gene that is associated with systemic lupus erythematosus 4 , 5 —interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease 4 , 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 TRIF 10 , 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 erythematosus 12 – 14 . The interaction between TASL and SLC15A4 links endolysosomal Toll-like receptors to the transcription factor IRF5, providing a mechanistic explanation for the involvement of the complex in systemic lupus erythematosus.

Solute carriers (SLCs) are the largest family of transmembrane transporters in the human genome with more than 400 members. Despite the fact that SLCs mediate critical biological functions and several are important pharmacological targets, a large proportion of them is poorly characterized and present no assigned substrate. A major limitation to systems‐level de‐orphanization campaigns is the absence of a structured, language‐controlled chemical annotation. Here we describe a thorough manual annotation of SLCs based on literature. The annotation of substrates, transport mechanism, coupled ions, and subcellular localization for 446 human SLCs confirmed that ~30% of these were still functionally orphan and lacked known substrates. Application of a substrate‐based ontology to transcriptomic datasets identified SLC‐specific responses to external perturbations, while a machine‐learning approach based on the annotation allowed us to identify potential substrates for several orphan SLCs. The annotation is available at https://opendata.cemm.at/gsflab/slcontology/ . Given the increasing availability of large biological datasets and the growing interest in transporters, we expect that the effort presented here will be critical to provide novel insights into the functions of SLCs. Synopsis A thorough manual literature‐based annotation of human solute carriers (SLCs) is presented. Complemented by a substrate‐based ontology, it provides a comprehensive resource for the scientific community to get novel insights to the functions of SLCs. The study provides a careful manual annotation of substrates, transport mechanism, coupled ions and subcellular localization for 446 human solute carriers. Substrates are organized in a convenient ontological structure, based on ChEBI (Chemical Entities of Biological Interest). Substrate‐based ontology is applied in interpreting large datasets and de‐orphanizing SLCs using a machine learning approach. Graphical Abstract A thorough manual literature‐based annotation of human solute carriers (SLCs) is presented. Complemented by a substrate‐based ontology, it provides a comprehensive resource for the scientific community to get novel insights to the functions of SLCs.

Endosomal nucleic acid sensing by Toll-like receptors (TLRs) is central to antimicrobial immunity and several autoimmune conditions such as systemic lupus erythematosus (SLE). The innate immune adaptor TASL mediates, via the interaction with SLC15A4, the activation of IRF5 downstream of human TLR7, TLR8 and TLR9, but the pathophysiological functions of this axis remain unexplored. Here we show that SLC15A4 deficiency results in a selective block of TLR7/9-induced IRF5 activation, while loss of TASL leads to a strong but incomplete impairment, which depends on the cell type and TLR engaged. This residual IRF5 activity is ascribed to a previously uncharacterized paralogue, Gm6377 , named here TASL2. Double knockout of TASL and TASL2 (TASL DKO ) phenocopies SLC15A4-deficient feeble mice showing comparable impairment of innate and humoral responses. Consequently, TASL DKO mice fail to control chronic LCMV infection, while being protected in a pristane-induced SLE disease model. Our study thus demonstrates the critical pathophysiological role of SLC15A4 and TASL/TASL2 for TLR7/9-driven inflammatory responses, further supporting the therapeutic potential of targeting this complex in SLE and related diseases. TASL is an adaptor molecule bridging Toll-like receptor signalling and transcription activation by IRF5. Here the authors show that TASL deficiency impacts TLR7/9 responses, and that a TASL paralogue, Gm6377/TASL2, also contributes to IRF5 activation, as dual TASL/TASL2 deficiency dampens both protective and pathogenic inflammatory responses in mice.

Dysregulation of pathogen-recognition pathways of the innate immune system is associated with multiple autoimmune disorders. Due to the intricacies of the molecular network involved, the identification of pathway- and disease-specific therapeutics has been challenging. Using a phenotypic assay monitoring the degradation of the immune adapter TASL, we identify feeblin, a chemical entity which inhibits the nucleic acid-sensing TLR7/8 pathway activating IRF5 by disrupting the SLC15A4-TASL adapter module. A high-resolution cryo-EM structure of feeblin with SLC15A4 reveals that the inhibitor binds a lysosomal outward-open conformation incompatible with TASL binding on the cytoplasmic side, leading to degradation of TASL. This mechanism of action exploits a conformational switch and converts a target-binding event into proteostatic regulation of the effector protein TASL, interrupting the TLR7/8-IRF5 signaling pathway and preventing downstream proinflammatory responses. Considering that all components involved have been genetically associated with systemic lupus erythematosus and that feeblin blocks responses in disease-relevant human immune cells from patients, the study represents a proof-of-concept for the development of therapeutics against this disease. The authors identify feeblin, an inhibitory compound of the proinflammatory TLR7/8/9-IRF5 pathway with therapeutic potential, which acts by binding SLC15A4 via an allosteric mechanism mediating degradation of its signaling partner TASL.

Toll-like receptors (TLRs) are a class of proteins that play critical roles in recognizing pathogens and initiating innate immune responses. TASL, a recently identified innate immune adaptor protein for endolysosomal TLR7/8/9 signaling, is recruited by the lysosomal proton-coupled amino-acid transporter SLC15A4, and then activates IRF5, which in turn triggers the transcription of type I interferons and cytokines. Here, we report three cryo-electron microscopy (cryo-EM) structures of human SLC15A4 in the apo monomeric and dimeric state and as a TASL-bound complex. The apo forms are in an outward-facing conformation, with the dimeric form showing an extensive interface involving four cholesterol molecules. The structure of the TASL-bound complex reveals an unprecedented interaction mode with solute carriers. During the recruitment of TASL, SLC15A4 undergoes a conformational change from an outward-facing, lysosomal lumen-exposed state to an inward-facing state to form a binding pocket, allowing the N-terminal helix of TASL to be inserted into. Our findings provide insights into the molecular basis of regulatory switch involving a human solute carrier and offers an important framework for structure-guided drug discovery targeting SLC15A4-TASL-related human autoimmune diseases. Three structures of human SLC15A4 are presented in the outward-facing apo monomeric and dimeric states and the inward-facing TASL-bound state, revealing the molecular mechanism of SLC15A4-mediated TASL recruitment in human endolysosomal TLRs signalling.

Detection of viral nucleic acids is central to antiviral immunity. Recently, DAI/ZBP1 (DNA‐dependent activator of IRFs/Z‐DNA binding protein 1) was identified as a cytoplasmic DNA sensor and shown to activate the interferon regulatory factor (IRF) and nuclear factor‐kappa B (NF‐κB) transcription factors, leading to type‐I interferon production. DAI‐induced IRF activation depends on TANK‐binding kinase 1 (TBK1), whereas signalling pathways and molecular components involved in NF‐κB activation remain elusive. Here, we report the identification of two receptor‐interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI‐induced NF‐κB signals through the recruitment of the RHIM‐containing kinases RIP1 and RIP3. We show that knockdown of not only RIP1, but also RIP3 affects DAI‐induced NF‐κB activation. Importantly, RIP recruitment to DAI is inhibited by the RHIM‐containing murine cytomegalovirus (MCMV) protein M45. These findings delineate the DAI signalling pathway to NF‐κB and suggest a possible new immune modulation strategy of the MCMV.

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.

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of SLCs in response to environmental stresses remain poorly elucidated. To infer functional relationships between SLCs and metabolites, we developed a strategy to identify SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9–based transcriptional activation approach to uncover transporters relieving cells from growth-limiting metabolic bottlenecks. Among the transporters identified, we characterized the cationic amino acid transporter SLC7A3 as a gene that, when up-regulated, overcame low availability of arginine and lysine by increasing their uptake, whereas SLC7A5 was able to sustain cellular fitness upon deprivation of several neutral amino acids. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells uncovered functional transporter-nutrient relationships and revealed that transport activity up-regulation may be sufficient to overcome environmental metabolic restrictions.