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Environmental Factor, May 2012

Intramural Papers of the Month

By Anshul Pandya, Ian Thomas, and Darshini Trivedi

Determining specificity for enzymes used in heparin and heparan sulfate production

NIEHS scientists have successfully crystallized an oligosaccharide substrate synthesized via an innovative chemoenzymatic approach, with the enzyme 3-O-sulfotransferase isoform 1 (3-OST-1).  Sulfation by 3-OST-1 is the last step involved in the production of anticoagulant heparin and heparan sulfate. Heparin is an anticoagulant traditionally used to prevent the occurrence of blood clots, and recently has shown promise as an anticancer and anti-inflammatory drug. Understanding the intricate interactions between sulfotransferases and their heparan sulfate oligosaccharide substrates provides a molecular foundation that can aid researchers in specifically engineering heparin for a variety of other purposes. Heparin derivatives would ideally be synthesized to function more efficiently, using lower dosages, and with fewer side effects. 

Guided by multiple crystal structures of 3-O-sulfotransferases, multiple mutations were generated and analyzed for sulfotransferase activity and substrate binding specificity.  Researchers discovered that in spite of structural similarities between the different 3-OST isoforms, these enzymes bind their substrates in different conformations. This work was performed in collaboration with investigators Jian Liu, Ph.D., from the University of North Carolina at Chapel Hill and Robert Linhardt, Ph.D., from the Rensselaer Polytechnic Institute. (AP)

CitationMoon AF, Xu Y, Woody SM, Krahn JM, Linhardt RJ, Liu J, Pedersen LC. 2012. Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin. Proc Natl Acad U S A 109(14):5265-5270.

Calcium influx is a critical component of embryonic development

Upon fertilization by the sperm, repetitive calcium oscillations occur as a result of the movement of calcium from egg storage or outside the cell, into the egg cytoplasm, and then back into storage or out of the egg. These calcium oscillations are essential for mammalian egg activation and the early stages of embryonic development. NIEHS scientists determined that some of the signaling pathways induced by calcium movements take place directly under the egg’s plasma membrane, rather than entirely inside of the egg. The research has far-reaching implications, since defects in calcium signaling at fertilization can result in a failure of the embryo to implant or develop to term.

When mammalian sperm and egg first interact, a sperm-specific phospholipase, C zeta, contributes to the release of calcium from endoplasmic reticulum stores. Using a technique known as calcium insulation, the investigators induced persistent calcium oscillations in the egg cell while preventing an influx and efflux of calcium. In the absence of calcium influx, the fertilized eggs failed to undergo spindle rotation and emit the second polar body, thus resulting in the formation of three pronuclei, a fertilization abnormality. These studies have important implications for clinically assisted reproduction and fertility preservation technologies. (DT)

CitationMiao YL, Stein P, Jefferson WN, Padilla-Banks E, Williams CJ. 2012. Calcium influx-mediated signaling is required for complete mouse egg activation. Proc Natl Acad Sci U S A 109(11):4169-4174.

Members of the Ccr4-Not complex crucial for embryonic stem cell circuitry

NIEHS researchers have characterized the role of three key members of the Ccr4-Not complex within embryonic stem cell (ESC) self-renewal circuitry. The study demonstrates that Cnot1, Cnot2, and Cnot3, functioning as a protein complex, maintain self-renewal in both mouse and human ESCs, through the possible inhibition of extraembryonic lineage differentiation. The work may provide valuable insight into mammalian embryonic development, and facilitate the use of ESCs in various drug and cellular therapies.

Using gene silencing along with a technique that they developed, the Oct4GiP reporter assay, the investigators showed that inhibition of Cnot1, 2, and 3 expression, not other components of the Ccr4-Not complex, led to significant differentiation of ESCs. The relative expression of Cnot1, 2, and 3, decreased during ESC differentiation and the silencing of these genes induced the expression of several early trophectoderm (TE) transcription factors.

These studies support the hypothesis that the complex maintains self-renewal by inhibiting TE transcription factors, thus preventing TE differentiation in mouse ESCs. Furthermore, these studies have identified that Cnot1, 2 and 3 have conserved functions in both mouse and human ESCs, and are important in the maintenance of both the naive and primed pluripotency state. (DT)

CitationZheng X, Dumitru R, Lackford BL, Freudenberg JM, Singh AP, Archer TK, Jothi R, Hu G. 2012. Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and Inhibit extraembryonic differentiation. Stem Cells 30(5):910-922.

Scientists link impaired lung development to Nrf2 deficiencies in neonatal mice under oxidant stress

A collaborative team, led by scientists at NIEHS, determined that Nrf2, a critical transcription factor involved the body’s protection against oxidant disorders, is essential for cell cycle and DNA repair, immune function, and antioxidant defense during postnatal normal lung maturation in mice. In addition, the researchers found a beneficial role for Nrf2 in hyperoxia-induced injury of undeveloped lung. The work has important implications for a wide range of oxidative stress-associated neonatal disorders, including bronchopulmonary dysplasia (BPD), a chronic lung disorder found in roughly 20 percent of low birth weight infants born each year in the United States. 

Through the novel use of lung transcriptomics and pathway analysis, the researchers characterized complex gene expression networks in the saccular-to-alveolar stage transition during postnatal lung maturation. They also utilized microarray analyses to evaluate Nrf2-dependent defense mechanisms under normoxic and hyperoxic conditions in immature rodent lungs. Their studies found that newborn Nrf2(-/-) mice exposed to hyperoxia exhibited exacerbated pulmonary injury and DNA lesions, and were far more prone to an arrest of alveolarization than Nrf2(+/+) mice, implicating the importance of Nrf2. The results suggest a possible therapeutic role for Nrf2 in the protection of human BPD. (IT)

CitationCho HY, van Houten B, Wang X, Miller-Degraff L, Fostel J, Gladwell W, Perrow L, Panduri V, Kobzik L, Yamamoto M, Bell DA, Kleeberger SR. 2012. Targeted deletion of Nrf2 impairs lung development and oxidant injury in neonatal mice. Antioxid Redox Signal; doi:10.1089/ars.2011.4288 [Online 18 April 2012].

(Anshul Pandya, Ph.D., is an Intramural Research Training Award [IRTA] fellow in the NIEHS Laboratory of Neurobiology. Ian Thomas is a public affairs specialist with the NIEHS Office of Communications and Public Liaison, and a regular contributor to the Environmental Factor. Darshini Trivedi, Ph.D., is an IRTA fellow in the NIEH Laboratory of Toxicology and Pharmacology.)

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