Showing papers in "Science Signaling in 2018"
TL;DR: The data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate, and suggest that tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.
Abstract: Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3ζ or 4-1BB/CD3ζ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3ζ and 4-1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell-like phenotype and function. In contrast, 4-1BB/CD3ζ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.
297 citations
TL;DR: It is suggested that cells alter their tRNA abundance to selectively affect the translation rates of specific transcripts to increase the amounts of required proteins under diverse stress conditions.
Abstract: Decoding the information in mRNA during protein synthesis relies on tRNA adaptors, the abundance of which can affect the decoding rate and translation efficiency To determine whether cells alter tRNA abundance to selectively regulate protein expression, we quantified changes in the abundance of individual tRNAs at different time points in response to diverse stress conditions in Saccharomyces cerevisiae We found that the tRNA pool was dynamic and rearranged in a manner that facilitated selective translation of stress-related transcripts Through genomic analysis of multiple data sets, stochastic simulations, and experiments with designed sequences of proteins with identical amino acids but altered codon usage, we showed that changes in tRNA abundance affected protein expression independently of factors such as mRNA abundance We suggest that cells alter their tRNA abundance to selectively affect the translation rates of specific transcripts to increase the amounts of required proteins under diverse stress conditions
177 citations
TL;DR: This review discusses the current understanding of TNF ligand-receptor conformations and how this informs signaling and the design of current and prospective therapeutics, and reviews the structure and geometry of T NF superfamily ligands, receptors, and their interactions.
Abstract: Gloss Tumor necrosis factor (TNF) superfamily pathways regulate cell proliferation, cell death, and morphogenesis. TNF superfamily receptors mediate various cancers and immune-mediated diseases. Understanding of the mechanisms of TNF superfamily signaling and the structure and geometry of ligands, receptors, and their interactions is essential to designing effective agonist and antagonist drugs. In this review, consisting of four figures, one table, and 96 references, we discuss our current understanding of TNF ligand-receptor conformations and how this informs signaling and the design of current and prospective therapeutics.
177 citations
TL;DR: The data suggest that β-arrestins function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr 1/2- or G protein–deleted cells to GPCR behavior in native systems.
Abstract: G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.
156 citations
TL;DR: Results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism, suggesting that it serves as a failsafe to protect plants even if the immune response is compromised.
Abstract: Cell walls surround all plant cells, and their composition and structure are modified in a tightly controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications, as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana CWD induced by cell wall-degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for the activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane-localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for the loss of PTI signaling elements.
140 citations
TL;DR: It is found that different leaves within the canopy of an Arabidopsis thaliana plant, including leaves not directly exposed to light, coordinated stomatal closure in response to light stress by sending and receiving rapid systemic signals.
Abstract: The plant canopy functions as an aerial array of light-harvesting antennas. To achieve maximal yield, each leaf within this array and the array as a whole need to rapidly adjust to naturally occurring fluctuations in light intensity and quality. Excessive light stress triggers the closing of pores in leaves called stomata to minimize moisture loss. We found that different leaves within the canopy of an Arabidopsis thaliana plant, including leaves not directly exposed to light, coordinated stomatal closure in response to light stress by sending and receiving rapid systemic signals. This response required the plant hormones abscisic acid and jasmonic acid and was mediated by a rapid autopropagating wave of reactive oxygen species (ROS) production. Furthermore, this response depended on the function of genes encoding the ROS-generating NADPH oxidase RBOHD and various stomatal regulators, such as the anion channel SLAC1, GHR1 (guard cell hydrogen peroxide resistant 1), and lipoxygenase 1 (LOX1). Our findings reveal that plants function as highly dynamic and coordinated organisms, optimizing the overall response of their canopies to fluctuating light intensities.
133 citations
TL;DR: The mechanisms by which only a handful of mitotic protein kinases control various mitotic events in distinct spatiotemporal contexts are discussed and important implications for the understanding of the mechanisms ofMitotic regulation are understood and may advance the development of novel antimitotic drugs.
Abstract: Mitosis is controlled by reversible protein phosphorylation involving specific kinases and phosphatases. A handful of major mitotic protein kinases, such as the cyclin B-CDK1 complex, the Aurora kinases, and Polo-like kinase 1 (PLK1), cooperatively regulate distinct mitotic processes. Research has identified proteins and mechanisms that integrate these kinases into signaling cascades that guide essential mitotic events. These findings have important implications for our understanding of the mechanisms of mitotic regulation and may advance the development of novel antimitotic drugs. We review collected evidence that in vertebrates, the Aurora kinases serve as catalytic subunits of distinct complexes formed with the four scaffold proteins Bora, CEP192, INCENP, and TPX2, which we deem "core" Aurora cofactors. These complexes and the Aurora-PLK1 cascades organized by Bora, CEP192, and INCENP control crucial aspects of mitosis and all pathways of spindle assembly. We compare the mechanisms of Aurora activation in relation to the different spindle assembly pathways and draw a functional analogy between the CEP192 complex and the chromosomal passenger complex that may reflect the coevolution of centrosomes, kinetochores, and the actomyosin cleavage apparatus. We also analyze the roles and mechanisms of Aurora-PLK1 signaling in the cell and centrosome cycles and in the DNA damage response.
125 citations
TL;DR: The upstream pathway that mediates mitochondrial clearance is investigated and it is reported that Parkin-mediated mitophagy plays a key role in the beige-to-white adipocyte transition, suggesting a physiological mechanism by which external cues control mitochondrial homeostasis in thermogenic fat cells through mitophileagy.
Abstract: Beige adipocytes are an inducible form of mitochondria-enriched thermogenic adipocytes that emerge in response to external stimuli, such as chronic cold exposure. We have previously shown that after the withdrawal of external stimuli, beige adipocytes directly acquire a white fat–like phenotype through autophagy-mediated mitochondrial degradation. We investigated the upstream pathway that mediates mitochondrial clearance and report that Parkin-mediated mitophagy plays a key role in the beige-to-white adipocyte transition. Mice genetically deficient in Park2 showed reduced mitochondrial degradation and retained thermogenic beige adipocytes even after the withdrawal of external stimuli. Norepinephrine signaling through the PKA pathway inhibited the recruitment of Parkin protein to mitochondria in beige adipocytes. However, mitochondrial proton uncoupling by uncoupling protein 1 (UCP1) was dispensable for Parkin recruitment and beige adipocyte maintenance. These results suggest a physiological mechanism by which external cues control mitochondrial homeostasis in thermogenic fat cells through mitophagy.
105 citations
TL;DR: Bioluminescence-based biosensors enable comprehensive profiling of G protein–coupled receptor signaling bias and demonstrate that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector.
Abstract: G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.
97 citations
TL;DR: It is found that ERα deletion in T cells reduced their pathogenic potential in a mouse model of colitis and correlated with transcriptomic changes that affected T cell activation, which demonstrated that ER α-targeted immunotherapies could be used to treat autoimmune disorders.
Abstract: It has long been appreciated that most autoimmune disorders are characterized by increased prevalence in females, suggesting a potential role for sex hormones in the etiology of autoimmunity. To study how estrogen receptor α (ERα) contributes to autoimmune diseases, we generated mice in which ERα was deleted specifically in T lymphocytes. We found that ERα deletion in T cells reduced their pathogenic potential in a mouse model of colitis and correlated with transcriptomic changes that affected T cell activation. ERα deletion in T cells contributed to multiple aspects of T cell function, including reducing T cell activation and proliferation and increasing the expression of Foxp3, which encodes a critical transcription factor for the differentiation and function of regulatory T cells. Thus, these data demonstrate that ERα in T cells plays an important role in inflammation and suggest that ERα-targeted immunotherapies could be used to treat autoimmune disorders.
96 citations
TL;DR: Understanding of how MerTK signaling induces 5-LOX–derived SPM biosynthesis is understood and a therapeutic strategy to boost inflammation resolution in settings where defective resolution promotes disease progression is suggested.
Abstract: Inflammation resolution counterbalances excessive inflammation and restores tissue homeostasis after injury. Failure of resolution contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated by endogenous specialized proresolving mediators (SPMs), which are derived from long-chain fatty acids by lipoxygenase (LOX) enzymes. 5-LOX plays a critical role in the biosynthesis of two classes of SPMs: lipoxins and resolvins. Cytoplasmic localization of the nonphosphorylated form of 5-LOX is essential for SPM biosynthesis, whereas nuclear localization of phosphorylated 5-LOX promotes proinflammatory leukotriene production. We previously showed that MerTK, an efferocytosis receptor on macrophages, promotes SPM biosynthesis by increasing the abundance of nonphosphorylated, cytoplasmic 5-LOX. We now show that activation of MerTK in human macrophages led to ERK-mediated expression of the gene encoding sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), which decreased the cytosolic Ca2+ concentration and suppressed the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). This, in turn, reduced the activities of the mitogen-activated protein kinase (MAPK) p38 and the kinase MK2, resulting in the increased abundance of the nonphosphorylated, cytoplasmic form of 5-LOX and enhanced SPM biosynthesis. In a zymosan-induced peritonitis model, an inflammatory setting in which macrophage MerTK activation promotes resolution, inhibition of ERK activation delayed resolution, which was characterized by an increased number of neutrophils and decreased amounts of SPMs in tissue exudates. These findings contribute to our understanding of how MerTK signaling induces 5-LOX-derived SPM biosynthesis and suggest a therapeutic strategy to boost inflammation resolution in settings where defective resolution promotes disease progression.
TL;DR: WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca2+ transfer between the ER and mitochondria and rescued mitochondrial dysfunction.
Abstract: Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca2+ signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca2+ transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca2+ exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca2+ transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases.
TL;DR: It is found that IRE1α was critical to maintaining lipid homeostasis in the liver by repressing the biogenesis of microRNAs (miRNAs) that regulate lipid mobilization, a regulatory pathway distinct from the canonical I RE1α-UPR pathway under acute ER stress.
Abstract: Obesity or a high-fat diet represses the endoribonuclease activity of inositol-requiring enzyme 1α (IRE1α), a transducer of the unfolded protein response (UPR) in cells under endoplasmic reticulum (ER) stress. An impaired UPR is associated with hepatic steatosis and nonalcoholic fatty liver disease (NAFLD), which is caused by lipid accumulation in the liver. We found that IRE1α was critical to maintaining lipid homeostasis in the liver by repressing the biogenesis of microRNAs (miRNAs) that regulate lipid mobilization. In mice fed normal chow, the endoribonuclease function of IRE1α processed a subset of precursor miRNAs in the liver, including those of the miR-200 and miR-34 families, such that IRE1α promoted their degradation through the process of regulated IRE1-dependent decay (RIDD). A high-fat diet in mice or hepatic steatosis in patients was associated with the S-nitrosylation of IRE1α and inactivation of its endoribonuclease activity. This resulted in an increased abundance of these miRNA families in the liver and, consequently, a decreased abundance of their targets, which included peroxisome proliferator–activated receptor α (PPARα) and the deacetylase sirtuin 1 (SIRT1), regulators of fatty acid oxidation and triglyceride lipolysis. IRE1α deficiency exacerbated hepatic steatosis in mice. The abundance of the miR-200 and miR-34 families was also increased in cultured, lipid-overloaded hepatocytes and in the livers of patients with hepatic steatosis. Our findings reveal a mechanism by which IRE1α maintains lipid homeostasis through its regulation of miRNAs, a regulatory pathway distinct from the canonical IRE1α-UPR pathway under acute ER stress.
TL;DR: Insight is provided into μ-opioid receptor desensitization, which may aid in the development of synthetic opioids that do not induce tolerance and thus have reduced potential for addiction, and specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR.
Abstract: G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.
TL;DR: It is shown that chemical Ca2+ indicators, but not a genetically encoded Ca2+.
Abstract: Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.
TL;DR: The findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses, which triggered their proteasome-mediated degradation.
Abstract: Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus.
TL;DR: The data suggest that the spleen not only served as a leukocyte reservoir but also was the site where SPMs were actively generated after coronary ligation in mice, and it is found that macrophages were necessary for SPM generation.
Abstract: Inflammation promotes healing in myocardial infarction but, if unresolved, leads to heart failure. To define the inflammatory and resolving responses, we quantified leukocyte trafficking and specialized proresolving mediators (SPMs) in the infarcted left ventricle and spleen after myocardial infarction, with the goal of distinguishing inflammation from its resolution. Our data suggest that the spleen not only served as a leukocyte reservoir but also was the site where SPMs were actively generated after coronary ligation in mice. Before myocardial infarction, SPMs were more abundant in the spleen than in the left ventricle. At day 1 after coronary ligation, the spleen was depleted of leukocytes, a phenomenon that was associated with greater numbers of leukocytes in the infarcted left ventricle and increased generation of SPMs at the same site, particularly resolvins, maresin, lipoxins, and protectin. In addition, the infarcted left ventricle showed increased expression of genes encoding lipoxygenases and enhanced production of SPMs generated by these enzymes. We found that macrophages were necessary for SPM generation. The abundance of SPMs in the spleen before myocardial infarction and increased SPM concentrations in the infarcted left ventricle within 24 hours after myocardial infarction were temporally correlated with the resolution of inflammation. Thus, the acute inflammatory response coincided with the active resolving phase in post–myocardial infarction and suggests that further investigation into macrophage-derived SPMs in heart failure is warranted.
TL;DR: It is reported that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI and suggests a potential repurposing of cilnidipine.
Abstract: Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding–dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.
TL;DR: Findings expand the roles of ATM and suggest that the loss of ATM function, such as in the neurodegenerative disease ataxia telangiectasia (A-T), causes broader cellular stress than that limited to a defective DNA damage response.
Abstract: The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage–related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis.
TL;DR: It is found that treating rodents with an oligonucleotide mimicking the microRNA miR-7 either before or within 30 min (but not 2 hours) after focal cerebral ischemia reduced the amount of brain damage and improved motor recovery and quality of life after a stroke.
Abstract: Ischemic stroke, which is caused by a clot that blocks blood flow to the brain, can be severely disabling and sometimes fatal. We previously showed that transient focal ischemia in a rat model induces extensive temporal changes in the expression of cerebral microRNAs, with a sustained decrease in the abundance of miR-7a-5p (miR-7). Here, we evaluated the therapeutic efficacy of a miR-7 mimic oligonucleotide after cerebral ischemia in rodents according to the Stroke Treatment Academic Industry Roundtable (STAIR) criteria. Rodents were injected locally or systemically with miR-7 mimic before or after transient middle cerebral artery occlusion. Decreased miR-7 expression was observed in both young and aged rats of both sexes after cerebral ischemia. Pre- or postischemic treatment with miR-7 mimic decreased the lesion volume in both sexes and ages studied. Furthermore, systemic injection of miR-7 mimic into mice at 30 min (but not 2 hours) after cerebral ischemia substantially decreased the lesion volume and improved motor and cognitive functional recovery with minimal peripheral toxicity. The miR-7 mimic treatment substantially reduced the postischemic induction of α-synuclein (α-Syn), a protein that induces mitochondrial fragmentation, oxidative stress, and autophagy that promote neuronal cell death. Deletion of the gene encoding α-Syn abolished miR-7 mimic-dependent neuroprotection and functional recovery in young male mice. Further analysis confirmed that the transcript encoding α-Syn was bound and repressed by miR-7. Our findings suggest that miR-7 mimics may therapeutically minimize stroke-induced brain damage and disability.
TL;DR: Members of the FAM83 family of proteins were identified as partners of CK1 in cells and the interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM 83 family.
Abstract: Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and e isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
TL;DR: The mechanisms that generate pacemaking activity in human sinoatrial nodal cells are revealed and it is demonstrated that spontaneous rhythmic local Ca2+ releases were coupled to electrogenic surface membrane molecules to trigger rhythmic action potentials, and that Ca2–cAMP–protein kinase A (PKA) signaling regulated clock coupling.
Abstract: The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+-cAMP-protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating "arrested" human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.
TL;DR: It is proposed that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein–coupled receptors that converge on Gαs and PKA.
Abstract: The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαs and PKA.
TL;DR: The findings reveal that MEG3-4 dynamically modulates pulmonary inflammatory responses through transcriptional regulation of immune response genes, extending the decoy and sponge mechanism associated with lncRNAs to antibacterial immunity, which affects both response and disease progression.
Abstract: Long noncoding RNAs (lncRNAs) regulate gene expression. We investigated the role of lncRNAs in the inflammatory response to bacterial infection in the lungs. We identified the lncRNA MEG3 as a tissue-specific modulator of inflammatory responses during bacterial infection. Among the 10 transcript isoforms of MEG3, transcript 4 (referred to as MEG3-4) encodes the isoform with the lowest abundance in mouse lungs. Nonetheless, we found that MEG3-4 bound to the microRNA miR-138 in a competitive manner with mRNA encoding the proinflammatory cytokine interleukin-1β (IL-1β), thereby increasing IL-1β abundance and intensifying inflammatory responses to bacterial infection in alveolar macrophages and lung epithelial cells in culture and in lung tissue in mice. MEG3-4-mediated sponging of miR-138 in the cytoplasm increased the autocrine activity of IL-1β that subsequently induced a negative feedback mechanism mediated by nuclear factor κB that decreased MEG3-4 abundance and inflammatory cytokine production. This timely reduction in MEG3-4 abundance tempered proinflammatory responses in mice with pulmonary bacterial infection, preventing the progression to sepsis. Together, these findings reveal that MEG3-4 dynamically modulates pulmonary inflammatory responses through transcriptional regulation of immune response genes, extending the decoy and sponge mechanism associated with lncRNAs to antibacterial immunity, which affects both response and disease progression.
TL;DR: The results suggest that agonist activation of ERRα could be used to improve mitochondrial quality in metabolic or neurodegenerative diseases or aging and show that co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy.
Abstract: Thyroid hormone receptor β1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and β-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.
TL;DR: The data support a sequential cytokine model for pathogenic TH2 cell differentiation and provide a mechanistic basis for the therapeutic targeting of TSLP signaling in human allergic diseases.
Abstract: Pathogenic T helper 2 (T H 2) cells, which produce increased amounts of the cytokines interleukin-5 (IL-5) and IL-13, promote allergic disorders, including asthma. Thymic stromal lymphopoietin (TSLP), a cytokine secreted by epithelial and innate immune cells, stimulates such pathogenic T H 2 cell responses. We found that TSLP signaling in mouse CD4 + T cells initiated transcriptional changes associated with T H 2 cell programming. IL-4 signaling amplified and stabilized the genomic response of T cells to TSLP, which increased the frequency of T cells producing IL-4, IL-5, and IL-13. Furthermore, the TSLP- and IL-4–programmed T H 2 cells had a pathogenic phenotype, producing greater amounts of IL-5 and IL-13 and other proinflammatory cytokines than did T H 2 cells stimulated with IL-4 alone. TSLP-mediated T H 2 cell induction involved distinct molecular pathways, including activation of the transcription factor STAT5 through the kinase JAK2 and repression of the transcription factor BCL6. Mice that received wild-type CD4 + T cells had exacerbated pathogenic T H 2 cell responses upon exposure to house dust mites compared to mice that received TSLP receptor–deficient CD4 + T cells. Transient TSLP signaling stably programmed pathogenic potential in memory T H 2 cells. In human CD4 + T cells, TSLP and IL-4 promoted the generation of T H 2 cells that produced greater amounts of IL-5 and IL-13. Compared to healthy controls, asthmatic children showed enhancement of such T cell responses in peripheral blood. Our data support a sequential cytokine model for pathogenic T H 2 cell differentiation and provide a mechanistic basis for the therapeutic targeting of TSLP signaling in human allergic diseases.
TL;DR: The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response, however, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers, which proposes that loss of the mitochondrial ROS-s sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.
Abstract: Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing-deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.
TL;DR: Findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles in type II taste cells.
Abstract: Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-μm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca 2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.
TL;DR: It is shown that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein–coupling selectivity.
Abstract: G protein-coupled receptors (GPCRs) are major drug targets Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein-coupling selectivity
TL;DR: It is found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members.
Abstract: A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.