Synthetic Par polarity induces cytoskeleton asymmetry in unpolarized mammalian cells.

Polarized cells rely on a polarized cytoskeleton to function. Yet, how cortical polarity cues induce cytoskeleton polarization remains elusive. Here, we capitalized on recently established designed 2D protein arrays to ectopically engineer cortical polarity of virtually any protein of interest during mitosis in various cell types. This enables direct manipulation of polarity signaling and the identification of the cortical cues sufficient for cytoskeleton polarization. Using this assay, we dissected the logic of the Par complex pathway, a key regulator of cytoskeleton polarity during asymmetric cell division. We show that cortical clustering of any Par complex subunit is sufficient to trigger complex assembly and that the primary kinetic barrier to complex assembly is the relief of Par6 autoinhibition. Further, we found that inducing cortical Par complex polarity induces two hallmarks of asymmetric cell division in unpolarized mammalian cells: spindle orientation, occurring via Par3, and central spindle asymmetry, depending on aPKC activity.

Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules.

Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death.

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

A single-amino acid substitution in the adaptor LAT accelerates TCR proofreading kinetics and alters T-cell selection, maintenance and function.

Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.

Epithelial Nlrp10 inflammasome mediates protection against intestinal autoinflammation.

Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1ß and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.

Mitochondrial damage activates the NLRP10 inflammasome.

Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits.

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.

The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Metabolic Control of Astrocyte Pathogenic Activity via cPLA2-MAVS.

Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.

Lateral Inhibition in Cell Specification Mediated by Mechanical Signals Modulating TAZ Activity.

Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.

Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS.

RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

Deficiency of CBL and CBLB ubiquitin ligases leads to hyper T follicular helper cell responses and lupus by reducing BCL6 degradation.

Recent evidence reveals hyper T follicular helper (Tfh) cell responses in systemic lupus erythematosus (SLE); however, molecular mechanisms responsible for hyper Tfh cell responses and whether they cause SLE are unclear. We found that SLE patients downregulated both ubiquitin ligases, casitas B-lineage lymphoma (CBL) and CBLB (CBLs), in CD4+ T cells. T cell-specific CBLs-deficient mice developed hyper Tfh cell responses and SLE, whereas blockade of Tfh cell development in the mutant mice was sufficient to prevent SLE. ICOS was upregulated in SLE Tfh cells, whose signaling increased BCL6 by attenuating BCL6 degradation via chaperone-mediated autophagy (CMA). Conversely, CBLs restrained BCL6 expression by ubiquitinating ICOS. Blockade of BCL6 degradation was sufficient to enhance Tfh cell responses. Thus, the compromised expression of CBLs is a prevalent risk trait shared by SLE patients and causative to hyper Tfh cell responses and SLE. The ICOS-CBLs axis may be a target to treat SLE.

Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Regulation of cGAS- and RLR-mediated immunity to nucleic acids.

Pathogen-derived nucleic acids are crucial signals for innate immunity. Despite the structural similarity between those and host nucleic acids, mammalian cells have been able to evolve powerful innate immune signaling pathways that originate from the detection of cytosolic nucleic acid species, one of the most prominent being the cGAS-STING pathway for DNA and the RLR-MAVS pathway for RNA, respectively. Recent advances have revealed a plethora of regulatory mechanisms that are crucial for balancing the activity of nucleic acid sensors for the maintenance of overall cellular homeostasis. Elucidation of the various mechanisms that enable cells to maintain control over the activity of cytosolic nucleic acid sensors has provided new insight into the pathology of human diseases and, at the same time, offers a rich and largely unexplored source for new therapeutic targets. This Review addresses the emerging literature on regulation of the sensing of cytosolic DNA and RNA via cGAS and RLRs.

Sestrins induce natural killer function in senescent-like CD8+ T cells.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.