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Environmental Factor, February 2012

Intramural papers of the month

By Brant Hamel, Anshul Pandya, Jeffrey Stumpf, and Emily Zhou

Catching DNA polymerase beta in the act of mutagenic bypass of 8-oxoG

NIEHS researchers continue a series of reports that capture three-dimensional structures of human DNA polymerase beta (pol beta) performing a potentially mutagenic event. The structures, published in the Proceedings of the National Academy of Sciences, reveal how pol beta binds to an oxidized guanine base, 8-oxoG, in the template DNA and inserts the incorrect dATP nucleotide.

8-oxoG is a common base damage resulting from oxidative stress caused by endogenous sources and environmental agents. Organisms that cannot remove 8-oxoG develop mutations that can ultimately cause tumor formation. The mutations arise from mutagenic dATP incorporation opposite 8-oxoG instead of the correct dCTP incorporation. Of the 15 human DNA polymerases, pol beta is relatively proficient at mutagenic bypass of 8-oxoG, making it ideal for structural studies.

Mutagenic bypass requires an unconventional Hoogsteen base pair between the syn-conformation of 8-oxoG and dATP. Pol beta stabilizes the syn-conformation with hydrogen bonding between the O8 oxygen from the base and the side chain from arginine-283 from the enzyme. The structures also reveal that the base pair requires a dramatic bend of the DNA backbone. The flexibility of pol beta allows mutagenic bypass of oxidized bases, which is necessary for tolerance of potentially toxic base damage.

CitationBatra VK, Shock DD, Beard WA, McKenna CE, Wilson SH. 2012. Binary complex crystal structure of DNA polymerase beta reveals multiple conformations of the templating 8-oxoguanine lesion. Proc Natl Acad Sci U S A 109(1):113-118.

In utero tobacco smoke exposure associated with illnesses in adult women

A recent epidemiological study conducted by NIEHS scientists found an association between the exposure to tobacco smoke in utero with obesity, hypertension, and gestational diabetes mellitus in adult women. This study is the first to examine the relationship of gestational diabetes mellitus to in utero exposure to tobacco smoke.

Carried out in collaboration with scientists from the University of Bergen in Norway, University of North Carolina at Chapel Hill, and the Norwegian Institute of Public Health, this research included 74,023 women volunteers aged 14 to 47. The analysis was based on the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health. MoBa is a cohort based on 108,000 pregnancies from 90,700 women enrolled from 1999 to 2008.

The results suggested that women exposed to tobacco smoke in utero have higher odds of developing obesity, hypertension, and gestational diabetes mellitus compared with unexposed women. The probability of developing type 2 diabetes mellitus was also observed to increase slightly, but this increase was not statistically significant. This work is in agreement with previous studies, which have established that prenatal exposure to tobacco smoke is detrimental to the developmental process of the fetus. This exposure can have a lasting impact on the health of the individual, even during adult life.

CitationCupul-Uicab LA, Skjaerven R, Haug K, Melve KK, Engel SM, Longnecker MP. 2011. In utero exposure to maternal tobacco smoke and subsequent obesity, hypertension, and gestational diabetes among women in the MoBa cohort. Environ Health Perspect; doi:10.1289/ehp.1103789 [Online 29 November 2011].

Crystal structure of a kinase advances research for rational drug design

In a recent publication in Nature Chemical Biology, NIEHS researchers describe, for the first time, the crystal structure for a key inositol pyrophosphate kinase, PPIP5K2. Precursors and products of this kinase regulate the body’s metabolic balance, which is under frequent environmental insult. Breakdown of metabolic control processes is evident in diabetes, cancer, and aging. The rational design of drugs to improve human health is facilitated by the molecular template that is provided by this atomic level description of the structure and reaction mechanism of this enzyme that synthesizes inositol pyrophosphates.

Human PPIP5K2 phosphorylates IP7 to form IP8. This synthesis reaction requires the formation of high-energy phosphoanhydride bonds, in a class of molecules that have the highest degree of phosphate congestion in nature. Therefore, the focus for the authors was to determine how the active site of PPIP5K2 accommodates the phosphates’ steric bulk and intense electronegativity, and yet retains selective substrate specificity. The exact structure of IP8 also emerged from this work.

This structural information will also be useful for developing small-molecule inhibitors of the kinase for possible therapeutic benefit. Knowledge of the structure of IP8 will now permit its chemical synthesis, the deciphering of its molecular interactions with receptors, and the design of IP8 antagonists.

CitationWang H, Falck JR, Hall TM, Shears SB. 2011. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol 8(1):111-116.

Phosphorylation status links cellular stress and GR transcriptional response

NIEHS researchers have discovered a new pathway that links environmentally-induced cellular stress to the transcriptional response mediated by the glucocorticoid receptor (GR). The GR is a nuclear receptor that binds to stress hormones to activate or repress thousands of genes that control pathways ranging from metabolism to the immune response. Glucocorticoids are some of the most widely proscribed drugs in the world and this work suggests that the response of patients to these drugs may be altered by external factors such as inflammation and stress.

Although GR was known to be phosphorylated in response to hormone binding, the authors discovered a novel hormone-independent site at Ser134. This amino acid is phosphorylated by p38 MAPK in response to a variety of extracellular stress-inducing pathways, including glucose starvation, oxidative stress, and UV irradiation. Significantly, the authors found that Ser134 phosphorylation allowed GR to bind 14-3-3zeta proteins and altered the suite of genes expressed by GR. Ser134 phosphorylation is necessary for the induction of many genes involved in metabolic and endocrine disorders, as well as the inflammatory response.

This particular GR pathway may also explain previous observations that linking chronic glucocorticoid exposure to insulin resistance as Ser134 may act as a metabolic sensor within cells to direct the transcriptional output of the glucocorticoid receptor.

CitationGalliher-Beckley AJ, Williams JG, Cidlowski JA. 2011. Ligand-independent phosphorylation of the glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling. Mol Cell Biol 31(23):4663-4675.

(Brant Hamel Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Laboratory of Signal Transduction Molecular Endocrinology Group. Anshul Pandya, Ph.D., is an IRTA fellow in the NIEHS Laboratory of Neurobiology Ion Channel Physiology Group. Jeffrey Stumpf, Ph.D., is an IRTA Fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group. Emily Zhou, Ph.D., is a research fellow in the NIEHS Laboratory of Signal Transduction Inositol Signaling Group.)

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