Viral DNA Binding to NLRC3, an Inhibitory Nucleic Acid Sensor, Unleashes STING, a Cyclic Dinucleotide Receptor that Activates Type I Interferon.

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.

High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation.

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses.

Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1(-/-) mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses.

The 3M complex maintains microtubule and genome integrity.

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP.

Assessing ATP binding and hydrolysis by NLR proteins.

Nucleotide-binding and leucine rich repeat domain-containing proteins (NLRs) are central to the formation of many inflammasome complexes. Several inflammasome forming NLR proteins are known to be ATPases, but the nucleotide binding specificity of many remains to be characterized. The oligomerization of NLR proteins and assembly of inflammasomes require the ATP (or other nucleotide) binding activity of the NLR proteins. Quantitative and qualitative studies of the nucleotide binding properties of these proteins are useful tools in studying the regulation of inflammasome activity, and are outlined in this Chapter.

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP.

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.

Altered gene expression by low-dose arsenic exposure in humans and cultured cardiomyocytes: assessment by real-time PCR arrays.

Chronic arsenic exposure results in higher risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. The purpose of this study was to investigate the effects on expression of selected genes in the blood lymphocytes from 159 people exposed chronically to arsenic in their drinking water using a novel RT-PCR TaqMan low-density array (TLDA). We found that expression of tumor necrosis factor-α (TNF-α), which activates both inflammation and NF-κB-dependent survival pathways, was strongly associated with water and urinary arsenic levels. Expression of KCNA5, which encodes a potassium ion channel protein, was positively associated with water and toe nail arsenic levels. Expression of 2 and 11 genes were positively associated with nail and urinary arsenic, respectively. Because arsenic exposure has been reported to be associated with long QT intervals and vascular disease in humans, we also used this TLDA for analysis of gene expression in human cardiomyocytes exposed to arsenic in vitro. Expression of the ion-channel genes CACNA1, KCNH2, KCNQ1 and KCNE1 were down-regulated by 1-µM arsenic. Alteration of some common pathways, including those involved in oxidative stress, inflammatory signaling, and ion-channel function, may underlay the seemingly disparate array of arsenic-associated diseases, such as cancer, cardiovascular disease, and diabetes.

Global gene expression profiling of hyperkeratotic skin lesions from inner Mongolians chronically exposed to arsenic.

The skin is an organ that is highly sensitive to chronic arsenic (As) exposure. Skin lesions such as hyperkeratoses (HKs) are common early manifestations of arsenicosis in humans. HKs can be precursor lesions of nonmelanoma skin cancers (NMSCs), but the driving forces behind their formation and how they may ultimately progress to NMSCs are unknown. The goal of this study was to examine the global gene expression profiles of As-related HKs in an effort to better understand gene expression changes that are potentially associated with early stages of As carcinogenesis. HK biopsies were removed from individuals living in an arsenicosis-endemic region in Inner Mongolia who had been exposed to high As levels in their drinking water for >20 years. Gene expression profiling was performed on RNA isolated from 7 individuals in this group and from 4 lesion-free skin samples from healthy individuals. Consistent with the pathological characteristics of the HK lesions, major functional categories and known canonical pathways represented by altered transcripts include those involved in development, differentiation, apoptosis, proliferation, and stress response. The results of this study may help define a signature profile of gene expression changes associated with long-term As exposure in the skin.

Elevated ERCC1 gene expression in blood cells associated with exposure to arsenic from drinking water in Inner Mongolia.

BACKGROUND: Chronic arsenic exposure has been associated with human cancer. The objective of this study was to investigate the effects of arsenic exposure on a DNA nucleotide excision repair gene, ERCC1, expression in human blood cells. PATIENTS AND METHODS: Water and toenail samples were collected from a total of 327 Inner Mongolian residents for arsenic analysis. Blood samples were collected to determine mRNA expression levels by real-time PCR. RESULTS: The mRNA levels of ERCC1 expression were positively associated with water arsenic concentration (slope=0.313, p=0.0043) and nail arsenic concentration (slope=0.474, p=0.0073). mRNA levels of ERCC1 expression were significantly correlated with those of OGG1, a base pair excision repair gene (r=0.275, p<0.0001). CONCLUSION: The results showed that mRNA levels of ERCC1 expression were significantly associated with arsenic concentrations in drinking water, implicating the DNA repair response was induced by arsenic exposure.

Elevated human telomerase reverse transcriptase gene expression in blood cells associated with chronic arsenic exposure in Inner Mongolia, China.

BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase-containing human telomerase reverse transcriptase (hTERT) can extend telomeres of chromosomes, delay senescence, and promote cell proliferation leading to tumorigenesis. OBJECTIVE: The goal of this study was to investigate the effects of As on hTERT mRNA expression in humans and in vitro. METHOD: A total of 324 Inner Mongolia residents who have been exposed to As via drinking water participated in this study. Water and toenail samples were collected and analyzed for As. Blood samples were quantified for hTERT mRNA expression using real-time polymerase chain reaction. The hTERT mRNA levels were linked to water and nail As concentrations and skin hyperkeratosis. Human epidermal keratinocytes were treated with arsenite to assess effects on cell viability and hTERT expression in vitro. RESULTS: hTERT mRNA expression levels were significantly associated with As concentrations of water (p<0.0001) and nails (p=0.002) and also associated with severity of skin hyperkeratosis (p<0.05), adjusting for age, sex, smoking, and pesticide use. Females showed a higher slope than males (females: 0.126, p=0.0005; males: 0.079, p=0.017). In addition to water and nail As concentrations, age (p<0.0001) and pesticide use (p=0.025) also showed significant associations with hTERT expression. The hTERT expression levels decreased with age. Tobacco smoking did not affect hTERT expression (p=0.13). hTERT expression was significantly correlated with OGG1 and ERCC1 expression. The in vitro results also showed a dose-response relationship between arsenite concentrations and hTERT expression and reached the peak at 1 microM. CONCLUSIONS: hTERT expression was associated with As exposure in vivo and in vitro. The increased hTERT expression may be a cellular response to genomic insults by As and may also indicate that As may function as a tumor promoter in carcinogenesis in humans.

Chronic arsenic exposure and oxidative stress: OGG1 expression and arsenic exposure, nail selenium, and skin hyperkeratosis in Inner Mongolia.

Arsenic, a human carcinogen, is known to induce oxidative damage to DNA. In this study we investigated oxidative stress and As exposure by determining gene expression of OGG1, which codes for an enzyme, 8-oxoguanine DNA glycosylase, involved in removing 8-oxoguanine in As-exposed individuals. Bayingnormen (Ba Men) residents in Inner Mongolia are chronically exposed to As via drinking water. Water, toenail, and blood samples were collected from 299 Ba Men residents exposed to 0.34-826 microg/L As. RNA was isolated from blood, and mRNA levels of OGG1 were determined using real-time polymerase chain reaction. OGG1 expression levels were linked to As concentrations in drinking water and nails, selenium concentrations in nails, and skin hyperkeratosis. OGG1 expression was strongly associated with water As concentrations (p < 0.0001). Addition of the quadratic term significantly improved the fit compared with the linear model (p = 0.05) . The maximal OGG1 response was at the water As concentration of 149 microg/L. OGG1 expression was also significantly associated with toenail As concentrations (p = 0.015) but inversely associated with nail Se concentrations (p = 0.0095) . We found no significant differences in the As-induced OGG1 expression due to sex, smoking, or age even though the oldest group showed the strongest OGG1 response (p = 0.0001) . OGG1 expression showed a dose-dependent increased risk of skin hyperkeratosis in males (trend analysis, p = 0.02) , but the trend was not statistically significant in females. The results from this study provide a linkage between oxidative stress and As exposure in humans. OGG1 expression may be useful as a biomarker for assessing oxidative stress from As exposure.

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling.

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.

Regulation of ALK-1 signaling by the nuclear receptor LXRbeta.

The transforming growth factor beta (TGF-beta) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and another endothelial-specific TGF-beta receptor, endoglin, both bind TGF-beta with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-beta, and the mechanism by which these receptors signal in endothelial cells remains unknown. Here we report the identification of the nuclear receptor liver X receptor beta (LXRbeta) as a modulator/mediator of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to LXRbeta in vitro and in vivo. Expression of activated ALK-1 results in translocation of LXRbeta from the nuclear compartment to the cytoplasmic compartment. The interaction of activated ALK-1 with LXRbeta in the cytoplasmic compartment results in the specific phosphorylation of LXRbeta by ALK-1, primarily on serine residues. LXRbeta subsequently modulates signaling by ALK-1 and the closely related TGF-beta receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRbeta as a potential modulator/mediator of ALK-1/ALK-2 signaling.