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.
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•Coregulation between membrane lipid species is organized in a circular network•The lipid network is conserved and reflects metabolism, localization, and adaptation•Sphingolipid metabolism regulates TLR trafficking, signaling, and cytokine release•Network-wide functional lipid annotations predict TLR responses in patient cells
Combining lipidomics with genetic perturbations in immune cells reveals the logic of inter-lipid regulatory structure and enables the functional assignment of lipids to different steps of Toll-like receptor signaling. Moreover, quantitative lipidomics alone can predict the inflammatory response of patient-derived cells.
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 (
) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the
ortholog
resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of
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
disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for
involvement in a variety of inherited and acquired human disorders.
Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann-Pick disease, ...and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann-Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase.
Through their capacity to sense danger signals and to generate active interleukin-1β (IL-1β), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1β, little is known ...about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1β was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.
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► IL-1α is cosecreted with IL-1β upon inflammasome activation ► IL-1α secretion can be inflammasome dependent or independent ► Caspase-1 has a protease activity-independent function that controls IL-1 secretion ► Osmotic stress and TRP channel-activity induce inflammasome activation
Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses
. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene ...that is associated with systemic lupus erythematosus
-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease
. 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
. 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
.
Highlights • Membrane lipid metabolism and TLR signaling are highly connected processes. • Cytokine release is altered in lysosomal lipid storage disorders in mouse and human. • The function of TLRs ...and adaptor proteins depends on membrane lipid association. • TLR-related processes are differently regulated by specific lipid species. • Systematic approaches allow the dissection of lipid function in complex membranes.
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.
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.
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•CRISPR screen identifies SLC4A7 to be required for phagosome acidification•Intracellular bacterial killing by macrophages is impaired in absence of SLC4A7•Bicarbonate transport activity of SLC4A7 is required for phagosome acidification•SLC4A7 regulates phagosome acidification via homeostatic control of cytoplasmic pH
Phagosome acidification is a necessary step in pathogen clearance by macrophages. Sedlyarov et al. identify the bicarbonate transporter SLC4A7 as essential for phagosome acidification. SLC4A7-mediated bicarbonate import ensures homeostatic regulation of cytoplasmic pH. In the absence of SLC4A7, the macrophage cytoplasm acidifies, which perturbs phagosome maturation and impairs bacterial killing by macrophages.
Fundamental insight gained over the last decades led to the discovery of cytokines as pivotal drivers of inflammatory diseases such as rheumatoid arthritis, psoriasis/psoriasis arthritis, ...inflammatory bowel diseases, atopic dermatitis and spondylarthritis. A deeper understanding of the pro-inflammatory and anti-inflammatory effects of various cytokines has prompted new cytokine-targeting therapies, which revolutionised the treatment options in the last years for patients with inflammatory disorders. Disease-associated immune responses typically involve a complex interplay of multiple cytokines. Therefore, blockade of one single cytokine does not necessarily lead to a persistent remission in all patients with inflammatory disorders and fostered new therapeutic strategies targeting intracellular pathways shared by multiple cytokines. By inhibiting JAK-STAT signalling pathways common to families of cytokines, JAK-inhibitors (JAKinibs) have created a new paradigm for the treatment of inflammatory diseases. Multiple agents have been approved for various disorders and more are being investigated for several new indications. Second-generation selective JAKinibs have been devised with the aim to achieve an increased selectivity and a possible reduced risk of side effects. In the current review, we will summarise the current body of evidence of pan versus selective JAKinibs and the most recent insights on new side effects and indications, including COVID-19.
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