RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis.

Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.

A Lifetime of Adventures in Glycobiology.

My initial research experience involved studying how bacteria synthesize nucleotide sugars, the donors for the formation of cell wall polysaccharides. During this time, I became aware that mammalian cells also have a surface coat of sugars and was intrigued as to whether these sugars might be arranged in specific sequences that function as information molecules in biologic processes. Thus began a long journey that has taken me from glycan structural analysis and determination of plant lectin-binding preferences to the biosynthesis of Asn-linked oligosaccharides and the mannose 6-phosphate (Man-6-P) lysosomal enzyme targeting pathway. The Man-6-P system represents an early example of a glycan serving as an information molecule in a fundamental cellular function. The remarkable advances in the field of glycobiology since I entered have uncovered scores of additional examples of oligosaccharide-lectin interactions mediating critical biologic processes. It has been a rewarding experience to participate in the efforts that have established a central role for glycans in biology.

The RIPK1 death domain restrains ZBP1- and TRIF-mediated cell death and inflammation.

RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.

A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits.

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.

UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome.

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.

Autocrine-paracrine prostaglandin E2 signaling restricts TLR4 internalization and TRIF signaling.

The unique cell biology of Toll-like receptor 4 (TLR4) allows it to initiate two signal-transduction cascades: a signal dependent on the adaptors TIRAP (Mal) and MyD88 that begins at the cell surface and regulates proinflammatory cytokines, and a signal dependent on the adaptors TRAM and TRIF that begins in the endosomes and drives the production of type I interferons. Negative feedback circuits to limit TLR4 signals from both locations are necessary to balance the inflammatory response. We describe a negative feedback loop driven by autocrine-paracrine prostaglandin E2 (PGE2) and the PGE2 receptor EP4 that restricted TRIF-dependent signals and the induction of interferon-ß through the regulation of TLR4 trafficking. Inhibition of PGE2 production or antagonism of EP4 increased the rate at which TLR4 translocated to endosomes and amplified TRIF-dependent activation of the transcription factor IRF3 and caspase-8. This PGE2-driven mechanism restricted TLR4-TRIF signaling in vitro after infection of macrophages by the Gram-negative pathogens Escherichia coli or Citrobacter rodentium and protected mice against mortality induced by Salmonella enteritidis serovar Typhimurium. Thus, PGE2 restricted TLR4-TRIF signaling specifically in response to lipopolysaccharide.

Selective autophagy of the adaptor TRIF regulates innate inflammatory signaling.

Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-ß and IL-1ß. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-ß and IL-1ß. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.

S100-alarmin-induced innate immune programming protects newborn infants from sepsis.

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

Metabolic control of the scaffold protein TKS5 in tissue-invasive, proinflammatory T cells.

Pathogenic T cells in individuals with rheumatoid arthritis (RA) infiltrate non-lymphoid tissue sites, maneuver through extracellular matrix and form lasting inflammatory microstructures. Here we found that RA T cells abundantly express the podosome scaffolding protein TKS5, which enables them to form tissue-invasive membrane structures. TKS5 overexpression was regulated by the intracellular metabolic environment of RA T cells-specifically, by reduced glycolytic flux that led to deficiencies in ATP and pyruvate. ATPlopyruvatelo conditions triggered fatty acid biosynthesis and the formation of cytoplasmic lipid droplets. Restoration of pyruvate production or inhibition of fatty acid synthesis corrected the tissue-invasiveness of RA T cells in vivo and reversed their proarthritogenic behavior. Thus, metabolic control of T cell locomotion provides new opportunities to interfere with T cell invasion into specific tissue sites.

Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase.

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. Here, we report a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. We establish the mitochondrial motor-adaptor protein Milton as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that the GlcNAcylation state of Milton is altered by extracellular glucose and that OGT alters mitochondrial motility in vivo. Our findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.

RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.

Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.

Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase.

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.

Structure of dynein-dynactin on microtubules shows tandem adaptor binding.

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4-6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9-13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

Protein sorting at the trans-Golgi network.

The trans-Golgi network (TGN) is an important cargo sorting station within the cell where newly synthesized proteins are packaged into distinct transport carriers that are targeted to various destinations. To maintain the fidelity of protein transport, elaborate protein sorting machinery is employed to mediate sorting of specific cargo proteins into distinct transport carriers. Protein sorting requires assembly of the cytosolic sorting machinery onto the TGN membrane and capture of cargo proteins. We review the cytosolic and transmembrane sorting machinery that function at the TGN and describe molecular interactions and regulatory mechanisms that enable accurate protein sorting. In addition, we highlight the importance of TGN sorting in physiology and disease.

The Family of LPS Signal Transducers Increases: the Arrival of Chanzymes.

After LPS recognition, the MyD88-dependent and the TRIF-dependent pathways are consecutively activated in macrophages. Schappe et al. (2018) show that the chanzyme TRPM7 is required for an efficient LPS receptor complex endosomal relocation and the activation of the TRIF pathway.

Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses.

Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1ß (IL-1ß), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-ß and IL-1ß dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.

The conserved protein adaptors CALM/AP180 and FCHo1/2 cooperatively recruit Eps15 to promote the initiation of clathrin-mediated endocytosis in yeast.

Clathrin-mediated endocytosis (CME) is a critical trafficking process that begins when an elaborate endocytic protein network is established at the plasma membrane. Interaction of early endocytic proteins with anionic phospholipids and/or cargo has been suggested to trigger CME initiation. However, the exact mechanism by which CME sites are initiated has not been fully elucidated. In the budding yeast Saccharomyces cerevisiae, higher levels of anionic phospholipids and cargo molecules exist in the newly formed daughter cell compared to the levels in the mother cell during polarized growth. Taking advantage of this asymmetry, we quantitatively compared CME proteins in S. cerevisiae mother versus daughter cells, observing differences in the dynamics and composition of key endocytic proteins. Our results show that CME site initiation occurs preferentially on regions of the plasma membrane with a relatively higher density of endocytic cargo and/or acidic phospholipids. Furthermore, our combined live cell-imaging and yeast genetics analysis provided evidence for a molecular mechanism in which CME sites are initiated when Yap1801 and Yap1802 (yeast CALM/AP180) and Syp1 (yeast FCHo1/2) coordinate with anionic phospholipids and cargo molecules to trigger Ede1 (yeast Eps15)-centric CME initiation complex assembly at the plasma membrane.

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains.

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and ß2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.

TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria.

Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the "sepsis syndrome," a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.