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Intramural Papers of the Month

By Robin Arnette and Jeffrey Stumpf
August 2010

The Structural Elucidation of the 8odGTP-adenine-pol beta Ternary Complex

NIEHS researchers have solved the first crystal structure of human DNA polymerase beta (pol beta) with 8-oxo-7,8-dihydro-2'-dGTP (8odGTP) paired with the template base adenine. The structure, published in Nature Structural & Molecular Biology, revealed that the syn conformation of 8odGTP binds to the anti conformation of adenine, causing a planar arrangement that mimics nonmutagenic base pairs.

The structure confirms that the syn conformation is stabilized by three interactions: a Hoogsteen hydrogen bond between 8odGTP and adenine, a hydrogen bond between 8odGTP and a pol beta asparagine that is crucial for the incorporation of 8odGTP, and the stabilization of the entire complex through an intramolecular hydrogen bond within 8odGTP.

Incorporating 8odGTP opposite cytosine is nonmutagenic, but it is much less efficient than incorporation opposite adenine. The researchers modeled the nonmutagenic incorporation of 8odGTP opposite cytosine and showed that this would result in a steric clash between the anti conformation of 8odGTP and the sugar phosphate backbone of the incoming nucleotide.

The elucidation of the incoming 8odGTP-template adenine-pol beta complex is important because 8odGTP, a highly mutagenic lesion produced by oxidative stress, may contribute to carcinogenesis and aging. 8odGTP preferably binds with adenine during replication and, without repair, results in an A-to-C transversion.

Citation: Batra VK, Beard WA, Hou EW, Pedersen LC, Prasad R, Wilson SH Exit NIEHS ( 2010. Mutagenic conformation of 8-oxo-7,8-dihydro-2'-dGTP in the confines of a DNA polymerase active site. Nat Struct Mol Biol 17(7):889-890.

Cholesterol Trafficking Linked to Inflammatory Response

Investigators from the NIEHS Laboratory of Respiratory Biology report that Myeloid Differentiation Primary Response Protein 88 (MyD88), an adaptor protein in innate immunity signaling pathways, is required for cholesterol export from cells and couples cholesterol export to inflammation. They found that MyD88-dependent inflammatory signals are elicited in macrophages by apolipoprotein A-I (ApoA-I), the major protein component of high-density lipoprotein (HDL) particles.

The model presented in this study holds that ApoA-I induces signals in the macrophage that require the presence of Toll-like Receptors (TLRs), cell surface receptors that normally recognize bacterial-derived lipid ligands. TLR2 and TLR4 then recruit MyD88, activating the transcription factor nuclear factor-kappaB and inducing pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Notably, the authors find that MyD88 is also required for the macrophage to export cholesterol to apoA-I. This action occurs, at least in part, through a feedback effect of MyD88-derived TNF-alpha upon the macrophage.

This work supports a new paradigm in which the innate immune response acts as a physiologic signal in cholesterol homeostasis. Whereas inflammation is generally thought to promote atherosclerosis, the authors provide evidence that immune pathways may also be required for removal of cholesterol from vessel walls. This work may provide future insights in the connection between inflammation and diet.

Citation: Smoak KA, Aloor JJ, Madenspacher J, Merrick BA, Collins JB, Zhu X, et al Exit NIEHS ( 2010. Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation. Cell Metab 11(6):493-502.

Ozone and TLR4 Lead to Asthma

Epidemiologic evidence suggests that high levels of ozone, a key component of air pollution, contribute to the increased prevalence of asthma. Investigators from NIEHS and Duke University Medical Center have recently found that ozone activates pulmonary dendritic cells through a Toll-like receptor 4 (TLR4)-dependent mechanism. The investigators propose that this activation can lead to allergic sensitization and ultimately to asthma.

Members of the research team studied the effect of ozone exposure on allergic sensitization to an inhaled protein, ovalbumin (OVA). They found that mice exposed to OVA alone did not become sensitized to it and were unresponsive to subsequent exposures to this protein. However, mice exposed to both ozone and OVA became sensitized because upon subsequent exposure to OVA, they developed allergic pulmonary inflammation and produced high levels of inflammatory cytokines. The authors further showed that ozone could activate pulmonary dendritic cells, which are critical cells in the initiation of allergic responses.

When tlr4-deficient mice were used in the same type of experiment, they displayed very low levels of airway inflammation and cytokine production. This finding suggests that ozone-mediated allergic sensitization through the airway occurs through a TLR4-dependent mechanism.

Citation: Hollingsworth JW, Free ME, Li Z, Andrews LN, Nakano H, Cook DN Exit NIEHS ( 2010. Ozone activates pulmonary dendritic cells and promotes allergic sensitization through a Toll-like receptor 4-dependent mechanism. J Allergy Clin Immunol 125(5):1167-1170.

C. elegans Genes Increase Lifespan and Resistance to Cadmium

A new study by NIEHS investigators reports that nuclear localized metal responsive (numr)-1 and numr-2, two novel Caenorhabditis elegans genes associated with resistance to metal toxicity, are also involved in increasing the lifespan of the organism. Their research further suggests that the expression of these genes is regulated by the insulin-IGF-like signaling pathway.

The investigators determined that both genes exhibited nearly identical nucleotide sequences and that numr-1/-2 mRNA levels experienced a sevenfold increase following exposure to cadmium. They also discovered that the cellular pattern of numr-1 and numr-2 expression was both tissue specific and dependent on the type of metal exposure. In the absence of metal, numr-1/-2 was expressed in a subset of neurons in the C. elegans head, tail and vulva. Exposure to cadmium increased the expression of numr-1/-2 in the intestine, while exposure to copper increased expression in the pharynx.

A knockdown of numr-1/-2 increased the nematode's sensitivity to cadmium, while over-expression increased the worm's resistance to metals and its lifespan, both in the presence and absence of metal. Although the upstream regulators of numr-1/-2 have not been identified, the two genes have binding sites for DAF-16 and SKN-1, two transcription factors that are involved in the insulin-IGF-like signaling pathway and that regulate the expression of several stress-response genes.

Citation: Tvermoes BE, Boyd WA, Freedman JH Exit NIEHS ( 2010. Molecular characterization of numr-1 and numr-2: genes that increase both resistance to metal-induced stress and lifespan in Caenorhabditis elegans. J Cell Sci 123(Pt12):2124-2134.

(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.) 

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