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Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2′-5′oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases. Cytosolic DNA arising from intracellular bacterial or viral infections induces type I interferon through activation of the DNA sensor cGAS, which catalyses the synthesis of cyclic dinucleotide which in turn activates STING; here the crystal structures of a carboxy-terminal fragment of cGAS alone and in complex with UTP and DNA–ATP–GTP complex are determined. DNA sensing by cGAS The mechanism of sensing and signalling of cytosolic DNA by the innate immune system is a topic of intense research interest as it is the means by which invading bacteria and viruses are detected. Cytosolic DNA is known to induce type I interferon through activation of the DNA sensor cyclic-GMP-AMP synthetase (cGAS), which catalyses the synthesis of a cyclic dinucleotide which in turn activates a protein known as STING (stimulator of interferon genes). Karl-Peter Hopfner and co-workers present the crystal structures of a C-terminal fragment of cGAS alone, in complex with UTP, and as a DNA–ATP–GTP complex. In a complementary paper [in this issue], Veit Hornung and coworkers show that the product of cGAS is distinct from previously characterized cyclic dinucleotides. Rather it is an unorthodox cyclic dinucleotide with a 2′–5′ linkage between guanosine and adenosine. This two-step synthesis of cGAMP(2′–5′) could be a focus for the development of specific inhibitors for the treatment of autoimmune diseases that engage the cGAS–STING axis.

Apoptosis: how cells become targets Apoptosis occurs in essentially all tissues as part of normal development and homeostasis. Yet even in tissues with high cellular turnover, apoptotic cells are rarely seen; this has been attributed to the ability of apoptotic cells to advertise their presence via release of 'find-me' signals to recruit phagocytes and initiate prompt clearance. It has been unclear, however, what type of find-me signals are released by apoptotic cells and how these are sensed by phagocytes. In this paper apoptotic cells are shown to release ATP and UTP that act as a 'find me ' signal and chemoattractant for phagocytes expressing the P2Y 2 ATP/UTP receptor. The efficient removal of apoptotic cells in vivo is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes. Here, the caspase-dependent release of ATP and UTP during the early stages of apoptosis is demonstrated. ATP and UTP are found to act as chemoattractants in a process mediated through the ATP/UTP receptor P2Y 2 , which is present on monocytes and macrophages. Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo . We then identified the ATP/UTP receptor P2Y 2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y 2 -null mice 3 . The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y 2 -/- (also known as P2ry2 -/- ) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y 2 -/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y 2 -dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo .

Cid1 protein is a crucial component in the RNA interference pathway and abnormal nuclear RNA turnover processes, primarily responsible for adding uridine to the 3′ end of RNA. Cid1 exhibits selective polymerization of UTP over other nucleoside triphosphates. To explore the mechanism of this selectivity, five systems: free-Cid1, Cid1-ATP, Cid1-UTP, Cid1-CTP, and Cid1-GTP with 500 ns Gaussian accelerated molecular dynamics (GaMD) simulations were performed to investigate conformational changes and binding affinities between substrates and Cid1. The results showed that UTP formed stronger and more numerous non-covalent interactions with Cid1 compared to the other three substrates. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) binding energy analysis revealed a substrate preference for Cid1 polymerase in the order of UTP, followed by ATP, CTP, and GTP. These findings provide theoretical insights into the substrate selectivity mechanism of Cid1 and provide theoretical clues for the design and modification of Cid1 polymerase.

N -acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae , a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The K m values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase–primer–template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3′-terminal CMP to generate the dinucleoside tetraphosphate products Ap ₄C, Up ₄C, and Cp ₄C, respectively. Pre–steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3′deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

PurposeCapecitabine is an oral pre-pro-drug of the anti-cancer drug 5-fluorouracil (5-FU). The biological activity of the 5-FU degrading enzyme, dihydropyrimidine dehydrogenase (DPD), and the target enzyme thymidylate synthase (TS), are subject to circadian rhythmicity in healthy volunteers. The aim of this study was to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK) and pharmacodynamics (PD) of capecitabine therapy adapted to this circadian rhythm (chronomodulated therapy).MethodsPatients aged ≥18 years with advanced solid tumours potentially benefitting from capecitabine therapy were enrolled. A classical dose escalation 3 + 3 design was applied. Capecitabine was administered daily without interruptions. The daily dose was divided in morning and evening doses that were administered at 9:00 h and 24:00 h, respectively. The ratio of the morning to the evening dose was 3:5 (morning: evening). PK and PD were examined on treatment days 7 and 8.ResultsA total of 25 patients were enrolled. The MTD of continuous chronomodulated capecitabine therapy was established at 750/1250 mg/m2/day, and was generally well tolerated. Circadian rhythmicity in the plasma PK of capecitabine, dFCR, dFUR and 5-FU was not demonstrated. TS activity was induced and DPD activity demonstrated circadian rhythmicity during capecitabine treatment.ConclusionThe MTD of continuous chronomodulated capecitabine treatment allows for a 20% higher dose intensity compared to the approved regimen (1250 mg/m2 bi-daily on day 1–14 of every 21-day cycle). Chronomodulated treatment with capecitabine is promising and could lead to improved tolerability and efficacy of capecitabine.

Focal and segmental glomerulosclerosis (FSGS) is a kidney disorder of unknown etiology, and up to 20% of patients on dialysis have been diagnosed with it. Here we show that a large family with hereditary FSGS carries a missense mutation in the TRPC6 gene on chromosome 11q, encoding the ion-channel protein transient receptor potential cation channel 6 (TRPC6). The proline-to-glutamine substitution at position 112, which occurs in a highly conserved region of the protein, enhances TRPC6-mediated calcium signals in response to agonists such as angiotensin II and appears to alter the intracellular distribution of TRPC6 protein. Previous work has emphasized the importance of cytoskeletal and structural proteins in proteinuric kidney diseases. Our findings suggest an alternative mechanism for the pathogenesis of glomerular disease.