Sugiyama A, Minami M, Ugajin K
… +16 more, Inaba-Inoue S, Yabuno N, Takekawa Y, Xiaomei S, Takei S, Sasaki M, Nomai T, Jiang X, Kita S, Maenaka K, Hirose M, Yao M, Gooley PR, Moseley GW, Sugita Y, Ose T
Signal transducer and activator of transcription (STAT) family members mediate signaling in the Janus kinase (JAK)-STAT pathway and are activated by phosphorylation at a conserved tyrosine residue, resulting in dimerizat...Signal transducer and activator of transcription (STAT) family members mediate signaling in the Janus kinase (JAK)-STAT pathway and are activated by phosphorylation at a conserved tyrosine residue, resulting in dimerization through reciprocal interactions between the phosphotyrosine and a Src homology 2 (SH2) domain. Tyrosine-phosphorylated STAT (pY-STAT) then translocates to the nucleus to induce the expression of genes encoding antiviral proteins. Although the active and functional forms of STATs are conventionally considered to be dimers, STATs can undergo higher-order oligomerization, which is implicated in regulating transcriptional activity. We present the cryo-electron microscopy (cryo-EM) structure of the tetrameric form of intact pY-STAT1 in complex with DNA, which indicates that interactions between the amino-terminal domains (NTDs) of STAT1 induce oligomerization. The tetrameric structure revealed a compact conformation with a previously uncharacterized binding interface: Two DNA-bound dimers are twofold symmetrically aligned to transform into a tandem DNA-binding model without NTD dimer separation. Moreover, biochemical analyses indicated that the rabies virus P-protein selectively targeted tetrameric pY-STAT1. Combined with data showing which regions contribute to the interaction between pY-STAT1 and the P-protein, we constructed a binding model explaining how P recognizes the pY-STAT1 tetramer. These data provide insight into how pathogenic viruses target signaling pathways that mediate the host immune response.
Enzalutamide, a second-generation androgen receptor inhibitor (also known as an antiandrogen), is used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Tumors often acquire resistance to enz...Enzalutamide, a second-generation androgen receptor inhibitor (also known as an antiandrogen), is used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Tumors often acquire resistance to enzalutamide. Tumor progression and enzalutamide resistance are associated with decreased abundance of the tumor suppressor PDCD4. In normal dividing cells, PDCD4 abundance is low when that of the kinase PLK1 is high. In this study, we found that PLK1 acted on PDCD4 to promote enzalutamide resistance in CRPC cells in culture and in mice via a mechanism that revealed an effective combination therapy. PLK1 phosphorylated PDCD4 at Ser, leading to its degradation and consequently inducing the transcriptional activation of Hedgehog (Hh) signaling by c-MYC. Hh signaling supports tumor cell proliferation and stemness by inducing the enzyme UDP-glucuronosyltransferase 2B15 (UGT2B15), which promotes the metabolic clearance of drugs and steroid hormones. Thus, this pathway may circumvent androgen receptor dependence, thereby reducing cellular sensitivity to enzalutamide. Knocking down UGT2B15 enhanced enzalutamide-induced cell apoptosis and growth arrest in a PDCD4-dependent manner. Combining enzalutamide with the clinically approved Hh pathway inhibitor vismodegib inhibited cell growth and promoted apoptosis in enzalutamide-resistant cell cultures and xenografts in vivo. Our findings reveal a mechanism of PLK1-mediated enzalutamide resistance and suggest a potential therapeutic strategy to overcome this resistance in prostate cancer.
The DNA damage repair kinase ATM is phosphorylated by the NF-κB pathway kinase IKKα, resulting in enhanced DNA damage repair through the nonhomologous end-joining pathway. Thus, inhibition of IKKα enhances the efficacy o...The DNA damage repair kinase ATM is phosphorylated by the NF-κB pathway kinase IKKα, resulting in enhanced DNA damage repair through the nonhomologous end-joining pathway. Thus, inhibition of IKKα enhances the efficacy of cancer therapy based on inducing DNA damage. Here, we found a role for the IKK regulatory subunit NEMO in DNA damage repair mediated by ATM and IKKα. Exposure to damaging agents induced the interaction of NEMO with a preformed ATM-IKKα complex, which was required to target active ATM and IKKα to chromatin for efficient DNA damage repair but not for activating ATM. Recognition of damaged DNA by the IKKα-NEMO-ATM complex was facilitated by the interaction between NEMO and histones and depended on the ADP ribosylation of histones by the enzyme PARP1. NEMO-deficient cells showed increased activity of the kinase ATR, and inhibition of ATR potentiated the effect of chemotherapy in cells lacking NEMO or IKKα. Bioinformatic analysis of colorectal cancer datasets demonstrated that the expression of genes encoding IKKα, NEMO, and ATM correlated with poor patient prognosis, suggesting that the mechanism linking these three elements may be clinically relevant.
Brown and beige adipocytes dissipate energy to generate heat through uncoupled respiration, and the hormone norepinephrine plays an important role in stimulating brown fat thermogenesis and beige adipocyte development in...Brown and beige adipocytes dissipate energy to generate heat through uncoupled respiration, and the hormone norepinephrine plays an important role in stimulating brown fat thermogenesis and beige adipocyte development in white adipose depots. Increasing energy expenditure by promoting the function and development of brown and beige fat is a potential approach to treat obesity and diabetes. Here, we investigated the effects of macrophage sirtuin 6 (SIRT6) on the regulation of the norepinephrine content of brown adipose tissue (BAT) and on obesity in mice. Myeloid SIRT6 deficiency impaired the thermogenic function of BAT, thereby decreasing core body temperatures because of reduced norepinephrine concentrations in BAT and subsequently leading to cold sensitivity. In addition, the oxygen consumption rate was reduced, resulting in severe insulin resistance and obesity. Furthermore, macrophage SIRT6 deficiency inhibited BAT thermogenesis after cold exposure or norepinephrine treatment and cold exposure-induced increases in markers of lipid metabolism and thermogenesis in white adipose tissue. Myeloid-specific SIRT6 deficiency promoted H3K9 acetylation in the promoter regions and the expression of genes encoding the norepinephrine-degrading enzyme MAOA and the norepinephrine transporter SLC6A2 in macrophages in BAT, leading to norepinephrine degradation and obesity. Our findings indicate that SIRT6 in macrophages is essential for maintaining norepinephrine concentrations in BAT in mice.
A mechanosensitive ion channel in a sensory neuron population suppresses the function of thermogenic tissues.A mechanosensitive ion channel in a sensory neuron population suppresses the function of thermogenic tissues.
The kinase IKKα phosphorylates the kinase ATM, enhancing its ability to promote DNA repair. In this issue of , Alonso-Marañón demonstrate that in colorectal cancer cells, the IKK regulatory subunit NEMO targets the ATM/...The kinase IKKα phosphorylates the kinase ATM, enhancing its ability to promote DNA repair. In this issue of , Alonso-Marañón demonstrate that in colorectal cancer cells, the IKK regulatory subunit NEMO targets the ATM/IKKα complex to sites of DNA damage, thus enhancing DNA repair and chemotherapy resistance.
Peptidergic nociceptors promote gastric cancer through the release of the neuropeptide CGRP.Peptidergic nociceptors promote gastric cancer through the release of the neuropeptide CGRP.
Neuroinflammation promotes the progression of various neurological and neurodegenerative diseases. Disrupted homeostasis of protein acetylation is implicated in neurodegeneration, and the lysine acetyltransferase GCN5 (a...Neuroinflammation promotes the progression of various neurological and neurodegenerative diseases. Disrupted homeostasis of protein acetylation is implicated in neurodegeneration, and the lysine acetyltransferase GCN5 (also known as KAT2A) is implicated in peripheral inflammation. Here, we investigated whether GCN5 plays a role in neuroinflammation in the brain. Systemic administration of the bacterial molecule LPS in mice to induce peripheral inflammation increased the abundance of GCN5 in various organs, including in the brain and specifically in microglia. In response to LPS, GCN5 mediated the induction of the proinflammatory cytokines TNF-α and IL-6 and the inflammatory mediators COX-2 and iNOS in microglia. Further investigation in cultured microglial cells revealed that GCN5 was activated downstream of the innate immune receptor TLR4 to acetylate Lys in the NF-κB subunit p65, thereby enabling the nuclear translocation and transcriptional activity of NF-κB and the resulting inflammatory response. Thus, targeting GCN5 might be explored further as a strategy to reduce neuroinflammation in the treatment of associated diseases.
A gut microbe that is reduced in diabetes produces a metabolite that dulls the appetite for sugar.A gut microbe that is reduced in diabetes produces a metabolite that dulls the appetite for sugar.
Suppressor screens can identify genetic modifiers of biochemical pathways but generally require that the suppressed mutant be viable and fertile. We developed a screening method that obviated this requirement and enabled...Suppressor screens can identify genetic modifiers of biochemical pathways but generally require that the suppressed mutant be viable and fertile. We developed a screening method that obviated this requirement and enabled the identification of mutations that partially suppressed the early developmental arrest and lethality caused by loss of the epidermal growth factor (EGF) receptor ortholog LET-23 in . We chemically mutagenized animals carrying the loss-of-function allele (), recovered () homozygotes that escaped early developmental arrest but were nevertheless inviable, and sequenced their genomes. Testing of candidate causal mutations identified 11 genes that, when mutated, mitigated the early lethality caused by loss of EGF signaling. These included genes encoding homologs of the small guanosine triphosphatase (GTPase) Ras (), which is a downstream effector of LET-23, and of regulators of the small GTPase Rho, including the homolog of the phosphotyrosine-binding protein TENSIN (). We also recovered suppressing mutations in genes encoding nuclear proteins that protect against DNA damage, including the homolog of MutS homolog 4 (). Genetic experiments were consistent with the repression of Rho activity or the activation of the DNA damage response compensating for the loss of EGF signaling. This sequencing-based, whole-animal screening method may be adapted to other organisms to enable the identification of mutations for which the phenotype does not allow the recovery of viable animals.
Sona C, Yeh YT, Li Y
… +15 more, Liu X, Ghosh A, Hinte LC, Ku MC, Rathjen T, Niendorf T, Yu G, Jia S, Kononenko NL, Hermann A, Luo J, Lin J, von Meyenn F, Yan X, Poy MN
Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2)...Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2) mediates microRNA (miRNA)-mediated gene silencing, which is critical for many facets of brain development, including NVU development. Here, we found that in glutamatergic neurons was critical for NVU formation in the developing cortices of mice. Glutamatergic neuron-specific loss of diminished synaptic formation, neuronal-to-endothelial cell contacts, and morphogenesis of the brain vasculature, ultimately compromising the integrity of the blood-brain barrier. Ago2 facilitated miRNA targeting of () mRNA, which encodes a phosphatase that modulates reelin-dependent phosphatidylinositol 3-kinase (PI3K)-Akt signaling within the glutamatergic subpopulation. Conditionally deleting in -deficient neurons restored Akt2 phosphorylation as well as postnatal development and survival. Several mutations in impair small RNA silencing and are associated with Lessel-Kreienkamp syndrome, a neurodevelopmental disorder. When expressed in a neuronal cell line, these human loss-of-function variants failed to suppress PTEN, resulting in attenuated PI3K-Akt signaling, further indicating that dysregulation of Ago2 function may contribute to both impaired development and neurological disorders. Together, these results identify Ago2 as central to the engagement of neurons with blood vessels in the developing brain.
A proximity assay identifies putative interactors and regulators of endogenous GPCRs in cell membranes.A proximity assay identifies putative interactors and regulators of endogenous GPCRs in cell membranes.
The worldwide annual frequency and lethality of head and neck squamous cell carcinoma (HNSCC) is not improving, and thus, new therapeutic approaches are needed. Approximately 70% of HNSCC cases have either amplification...The worldwide annual frequency and lethality of head and neck squamous cell carcinoma (HNSCC) is not improving, and thus, new therapeutic approaches are needed. Approximately 70% of HNSCC cases have either amplification or overexpression of , which encodes the kinase LZK. Here, we found that LZK is a therapeutic target in HNSCC and that small-molecule inhibition of its catalytic function decreased the viability of HNSCC cells with amplified . Inhibition of LZK suppressed tumor growth in -amplified xenografts derived from HNSCC patients. LZK stabilized the transcription factor c-MYC through its kinase activity and gain-of-function mutants of p53 in a kinase-independent manner. We designed a proteolysis-targeting chimera (PROTAC) that induced LZK degradation, leading to decreased abundance of both c-MYC and gain-of-function p53, and reduced the viability of HNSCC cells. Our findings demonstrate that LZK-targeted therapeutics, particularly PROTACs, may be effective in treating HNSCCs with amplification.
The tools of modern genetics and neurobiology have propelled a renaissance of research that has advanced our understanding of the pathophysiology of drug addiction. We know that an individual's risk for addiction is dete...The tools of modern genetics and neurobiology have propelled a renaissance of research that has advanced our understanding of the pathophysiology of drug addiction. We know that an individual's risk for addiction is determined by interactions between genetics and environment and that only a minute fraction of chemical agents share the ability to act on this vulnerability to induce a state of addiction. Repeated exposure to these drugs causes addiction through repeated activation of dopaminergic transmission (and many other actions) in the brain, inducing changes at the molecular, cellular, and synaptic levels that, over time, rewire the circuitry throughout the limbic system. In this Review, I discuss how we are gaining a clearer picture of this drug-induced plasticity-some of which is shared by all addictive drugs, whereas other aspects are specific to certain drug classes-and of the ways in which these adaptations mediate the range of behavioral abnormalities that define the addicted state. Despite the challenges, there is reason for optimism in translating this rich biological understanding of addiction into improved treatments for the many individuals burdened by this illness around the world.
G protein-coupled receptor (GPCR) signaling is a dynamic process involving various conformational intermediates in addition to those captured in static three-dimensional structures. Here, we used newly developed G protei...G protein-coupled receptor (GPCR) signaling is a dynamic process involving various conformational intermediates in addition to those captured in static three-dimensional structures. Here, we used newly developed G protein peptidomimetics to characterize the interactions of the ghrelin receptor (GHSR) with G proteins. Coupling to the G protein peptidomimetic not only affected the conformational features of the cytoplasmic regions of the receptor where the G protein binds but also allosterically affected the extracellular ligand-binding pocket. These conformational and allosteric changes increased the affinity of G protein-coupled GHSR for the endogenous agonist ghrelin. In addition, our data identified different complexes along the G protein activation pathway that differed in the engagement of the Gα C-terminal helix. Given that this helix is the main link between the activated receptor and the Gα nucleotide-binding pocket, these findings suggested a stepwise process involving distinct states in GPCR-catalyzed G protein activation. Collectively, our results provide evidence for the dynamic behavior of GPCR-G protein signaling complexes, with such dynamics most likely contributing to signaling selectivity and/or efficacy.
Smooth muscle cells from the gallbladder induce retinoic acid signaling to promote repair of intrahepatic biliary ducts.Smooth muscle cells from the gallbladder induce retinoic acid signaling to promote repair of intrahepatic biliary ducts.
The alarmin cytokine IL-33 stimulates the formation of immune hubs within otherwise "cold" tumors.The alarmin cytokine IL-33 stimulates the formation of immune hubs within otherwise "cold" tumors.
The small GTPase R-RAS2 regulates homeostatic proliferation and survival of T and B lymphocytes and, when present in high amounts, drives the development of B cell chronic lymphocytic leukemia. In normal and leukemic lym...The small GTPase R-RAS2 regulates homeostatic proliferation and survival of T and B lymphocytes and, when present in high amounts, drives the development of B cell chronic lymphocytic leukemia. In normal and leukemic lymphocytes, R-RAS2 constitutively binds to antigen receptors through their immunoreceptor tyrosine-based activation motifs (ITAMs) and promotes tonic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Here, we examined the molecular mechanisms underlying this direct interaction and its consequences for R-RAS2 activity. R-RAS2 exhibited direct, high-affinity interactions with ITAM peptides derived from B and T cell receptors through a proline-rich sequence in the hypervariable domain of R-RAS2. In resting T and B cells, the presence of antigen receptors at the plasma membrane was sufficient to promote the activation of R-RAS2 and PI3K, and mutations that abolished the interaction of R-RAS2 with ITAMs reduced R-RAS2 signaling. Binding to ITAMs increased GDP-GTP exchange on R-RAS2 through a mechanism distinct from that by which conventional cytosolic guanosine nucleotide exchange factors (GEFs) activate RAS proteins. These results define antigen receptors as noncanonical GEFs involved in the basal activation state of R-RAS2 in lymphocytes. Such a mechanism may underlie the leukemic transformation of B cells that occurs when wild-type R-RAS2 is present in high amounts.
Activation of the stimulator of interferon genes (STING) pathway by cytosolic DNA leads to the activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB). Although many vi...Activation of the stimulator of interferon genes (STING) pathway by cytosolic DNA leads to the activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB). Although many viruses produce proteins that inhibit IRF3-dependent antiviral responses, some viruses produce proteins that inhibit STING-induced NF-κB activation without blocking IRF3 activation. Here, we found that STING-activated, NF-κB-dependent, and IRF3-independent innate immunity inhibited the replication of the DNA virus herpes simplex virus type 1 (HSV-1), the RNA virus coxsackievirus A16 (CV-A16), and the retrovirus HIV-1. The HIV-1 nonstructural protein Vpu bound to STING and prevented it from interacting with the upstream NF-κB pathway kinase inhibitor of NF-κB subunit β (IKKβ), thus blocking NF-κB signaling. This function of Vpu was conserved among Vpu proteins from diverse HIV-1 and simian immunodeficiency virus strains and was distinct from its action in disrupting other host antiviral pathways. Furthermore, the ORF3a protein from the coronavirus SARS-CoV-2 also promoted viral replication by interacting with STING and blocking STING-induced activity of NF-κB but not of IRF3. These findings demonstrate that diverse viral proteins have convergently evolved to selectively inhibit NF-κB-mediated innate immunity downstream of STING activation, suggesting that targeting this pathway may represent a promising antiviral strategy.
Chronic exposure to manganese (Mn) induces manganism and has been widely implicated as a contributing environmental factor to Parkinson's disease (PD), featuring notable overlaps between the two in motor symptoms and cli...Chronic exposure to manganese (Mn) induces manganism and has been widely implicated as a contributing environmental factor to Parkinson's disease (PD), featuring notable overlaps between the two in motor symptoms and clinical hallmarks. Here, we developed an adult model of Mn toxicity that recapitulated key parkinsonian features, spanning behavioral deficits, neuronal loss, and dysfunctions in lysosomes and mitochondria. Metabolomics analysis of the brain and body tissues of these flies at an early stage of toxicity identified systemic changes in the metabolism of biotin (also known as vitamin B) in Mn-treated groups. Biotinidase-deficient flies showed exacerbated Mn-induced neurotoxicity, parkinsonism, and mitochondrial dysfunction. Supplementing the diet of wild-type flies with biotin ameliorated the pathological phenotypes of concurrent exposure to Mn. Biotin supplementation also ameliorated the pathological phenotypes of three standard fly models of PD. Furthermore, supplementing the culture media of human induced stem cells (iPSCs) differentiated midbrain dopaminergic neurons with biotin protected against Mn-induced mitochondrial dysregulation, cytotoxicity, and neuronal loss. Last, analysis of the expression of genes encoding biotin-related proteins in patients with PD revealed increased amounts of biotin transporters in the substantia nigra compared with healthy controls, suggesting a potential role of altered biotin metabolism in PD. Together, our findings identified changes in biotin metabolism as underlying Mn neurotoxicity and parkinsonian pathology in flies, for which dietary biotin supplementation was preventative.