Fic-mediated AMPylation tempers the unfolded protein response during physiological stress.

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

cGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus.

Introduction: Type I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms. Methods: This study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis. Results: The Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages. Conclusion: These findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.

Interference on Cytosolic DNA Activation Attenuates Sepsis Severity: Experiments on Cyclic GMP-AMP Synthase (cGAS) Deficient Mice.

Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis-a life-threatening organ dysfunction due to systemic infection-are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1ß), cGAS, IFN-γ and supernatant cyclic GMP-AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response.

The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.

cGAS promotes sepsis in radiotherapy of cancer by up-regulating caspase-11 signaling.

Radiotherapy is the most common strategy in the treatment of cancer. However, radiation-induced acute complications, in particular sepsis, render patients in a life-threatening status or lead to delay of therapy that largely influences patients' overall responses. The understanding of sepsis in radiotherapy is currently scant and effective medicine is not available by far. Here, with WT mice as control, we challenged mice deficient to cGas, Caspase-11, Gsdmd or Asc with cecal ligation and puncture (CLP, a sepsis model) after a treatment of thorax irradiation. We found that radiation robustly upgraded caspase-11 pathway in irradiated region and consequently deteriorated lung injury and mortality in the sepsis model. cGas knockout markedly attenuated radiation-upgraded caspase-11 and restored sepsis. Deficiency of non-canonical inflammasome, caspase-11 and the downstream GSDMD, rather than an AIM2 inflammasome component, ASC, dramatically protected against radiation-promoted injury and mortality in septic mice. The protection may attribute to the inhibition of caspase-11-mediated pyroptosis in endothelial cells of the lung. Thus, blocking cGAS/caspase-11 signaling would be an adjuvant treatment strategy for preventing sepsis in radiotherapy of cancer.

Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins.

The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.

The role of the adaptor molecule STING during Schistosoma mansoni infection.

Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting-/- and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting-/- mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting-/- animals. Surprisingly, Sting-/- mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting-/- neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting-/- mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP.

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.

The GRA15 protein from Toxoplasma gondii enhances host defense responses by activating the interferon stimulator STING.

Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15-/-T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

HldE Is Important for Virulence Phenotypes in Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrheal illness in third world countries and it especially affects children and travelers visiting these regions. ETEC causes disease by adhering tightly to the epithelial cells in a concerted effort by adhesins, flagella, and other virulence-factors. When attached ETEC secretes toxins targeting the small intestine host-cells, which ultimately leads to osmotic diarrhea. HldE is a bifunctional protein that catalyzes the nucleotide-activated heptose precursors used in the biosynthesis of lipopolysaccharide (LPS) and in post-translational protein glycosylation. Both mechanisms have been linked to ETEC virulence: Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane and is needed for transport of heat-labile toxins to the host cells, and ETEC glycoproteins have been shown to play an important role for bacterial adhesion to host epithelia. Here, we report that HldE plays an important role for ETEC virulence. Deletion of hldE resulted in markedly reduced binding to the human intestinal cells due to reduced expression of colonization factor CFA/I on the bacterial surface. Deletion of hldE also affected ETEC motility in a flagella-dependent fashion. Expression of both colonization factors and flagella was inhibited at the level of transcription. In addition, the hldE mutant displayed altered growth, increased biofilm formation and clumping in minimal growth medium. Investigation of an orthogonal LPS-deficient mutant combined with mass spectrometric analysis of protein glycosylation indicated that HldE exerts its role on ETEC virulence both through protein glycosylation and correct LPS configuration. These results place HldE as an attractive target for the development of future antimicrobial therapeutics.

Rev1 contributes to proper mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

Impairment of Hematopoietic Precursor Cell Activation during the Granulopoietic Response to Bacteremia in Mice with Chronic-Plus-Binge Alcohol Administration.

Alcohol abuse impairs immune defense. To study the effect of chronic-plus-binge alcohol exposure on the granulopoietic response, acute alcohol intoxication (intraperitoneal injection of 5 g alcohol/kg body weight) was introduced to mice chronically fed on the Lieber-DeCarli low-fat liquid alcohol diet for 5 weeks. Bacteremia was induced by intravenous injection of Escherichia coli Bacteremia caused a remarkable increase in marrow lin- c-kit+ Sca-1+ cells. Activation of cell proliferation supported the increase in marrow lin- c-kit+ Sca-1+ cells. Alcohol administration inhibited this activation of lin- c-kit+ Sca-1+ cells. The bone marrow of pair-fed control mice receiving intraperitoneal saline stored a large number of mature granulocytes expressing a high level of Gr1 (Gr1hi cells). The proportion of Gr1hi cells and the total number of Gr1+ cells were markedly reduced in the bone marrow, along with an increase in the ratio of Gr1+ granulocytes in peripheral white blood cells following bacteremia. E. coli infection stimulated proliferation of granulopoietic precursor cells, resulting in a marked increase in the ratio of immature Gr1lo cells in the bone marrow. Alcohol administration itself triggered marrow release of Gr1+ cells, resulting in reduction of the marrow granulocyte reserve with an elevation of granulocytes in the circulation. Alcohol also impaired activation of granulopoietic precursor proliferation following bacteremia. Alcohol disrupted lipopolysaccharide (LPS)-TLR4-ERK1/2-cyclin D1 signaling and inhibited upregulation of Sca-1 and C/EBPß expression by lineage-negative marrow cells in response to bacteremia. These results indicate that chronic-plus-binge alcohol exposure inhibits the granulopoietic response by disrupting key cell signaling for hematopoietic precursor cell activation and commitment to granulocyte lineage development.

cGAS is essential for cellular senescence.

Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.

ISPD gene homozygous deletion identified by SNP array confirms prenatal manifestation of Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is a rare form of autosomal recessive, congenital muscular dystrophy that is associated with brain and eye anomalies. Several genes encoding proteins involved in abnormal α-dystroglycan glycosylation have been implicated in the aetiology of WWS, most recently the ISPD gene. Typical WWS brain anomalies, such as cobblestone lissencephaly, hydrocephalus and cerebellar malformations, can be prenatally detected through routine ultrasound examinations. Here, we report two karyotypically normal foetuses with multiple brain anomalies that corresponded to WWS symptoms. Using a SNP-array examination on the amniotic fluid DNA, a homozygous microdeletion was identified at 7p21.2p21.1 within the ISPD gene. Published data and our findings led us to the conclusion that a homozygous segmental intragenic deletion of the ISPD gene causes the most severe phenotype of Walker-Warburg syndrome. Our results also clearly supports the use of chromosomal microarray analysis as a first-line diagnostic test in patients with a foetus with one or more major structural abnormalities identified on ultrasonographic examination.

Rev1 recruits ung to switch regions and enhances du glycosylation for immunoglobulin class switch DNA recombination.

By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1(-/-)Ung(+/+) B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1(+/+)Ung(-/-) B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1(-/-)Msh2(-/-) B cells. Rescue of CSR in Rev1(-/-) B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1's scaffolding function, not its enzymatic function.

Error-prone translesion synthesis mediates acquired chemoresistance.

The development of cancer drug resistance is a persistent clinical problem limiting the successful treatment of disseminated malignancies. However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. Error-prone translesional DNA synthesis (TLS) is known to underlie the mutagenic effects of numerous anticancer agents, but little is known as to whether mutation induced by this process is ultimately relevant to tumor drug resistance. Here, we use a tractable mouse model of B-cell lymphoma to interrogate the role of error-prone translesional DNA synthesis in chemotherapy-induced mutation and resistance to front-line chemotherapy. We find that suppression of Rev1, an essential TLS scaffold protein and dCMP transferase, inhibits both cisplatin- and cyclophosphamide-induced mutagenesis. Additionally, by performing repeated cycles of tumor engraftment and treatment, we show that Rev1 plays a critical role in the development of acquired cyclophosphamide resistance. Thus, chemotherapy not only selects for drug-resistant tumor population but also directly promotes the TLS-mediated acquisition of resistance-causing mutations. These data provide an example of an alteration that prevents the acquisition of drug resistance in tumors in vivo. Because TLS also represents a critical mechanism of DNA synthesis in tumor cells following chemotherapy, these data suggest that TLS inhibition may have dual anticancer effects, sensitizing tumors to therapy as well as preventing the emergence of tumor chemoresistance.

Specific electron transport chain abnormalities in amyotrophic lateral sclerosis.

In an amyotrophic lateral sclerosis (ALS) patient who also had an IgA gammopathy, autopsy studies identified the IgA in the surviving motor neurons. Further, the IgA bound the surface of isolated bovine motor neurons and inhibited neuronal proliferation in culture. To determine the pathologic basis of this IgA interaction with motor neurons, a neuroblastoma cDNA library was generated and screened with the IgA monoclonal antibody. Reactive clones were identified as flavin adenine dinucleotide (FAD) synthetase. To extend this finding to ALS in general, quantitative RT-PCRs were performed on blood samples from 26 ALS and 30 control blood samples to determine mRNA expression levels of FAD synthetase and other electron transport chain proteins, specifically riboflavin kinase (RFK), cytochrome C1 (CYC1), and succinate dehydrogenase complex subunit B (SDHB). All expression levels were measured against a control enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression levels for a non-respiratory chain protein (beta-actin) were also measured. We found that FAD synthetase expression levels were decreased in ALS samples compared to expression levels in controls (P = 0.0151). Expression levels for RFK, CYC1, and SDHB were also significantly decreased in the ALS group (P = 0.0025, P = 0.0002, and P < 0.0001, respectively). As control, expression levels for beta-actin did not show a significant difference between ALS and control groups (P = 0.2118). Our data show that a reduction in electron transport proteins, namely FAD synthetase, RFK, CYC1, and SDHB, is seen in patients with ALS. It is possible that this may have an effect on oxygen-dependent metabolic pathways. Human motor neurons may be particularly susceptible to injury if there is sub-optimal oxidative metabolism.

Cytokine secretion requires phosphatidylcholine synthesis.

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT alpha-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT alpha is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT alpha-deficient cells. Pro-tumor necrosis factor alpha and interleukin-6 remain lodged in the secretory compartment of CCT alpha-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1 beta secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT alpha-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor alpha or interleukin-6. These results identify CCT alpha as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.