Oxygen-derived free radicals occur as spontaneous metabolic by-products in aerobic organisms, or at high levels as cytotoxic weapons of the immune system. Nitric oxide (NO) is another free radical with dual roles: at low levels in intercellular signaling, and at high levels during inflammatory responses. These agents elicit specific genetic responses; we wish to understand how these free radicals trigger gene expression. An important model is the E. coli SoxR protein, an unusual transcription factor with [2Fe-2S] clusters; the oxidation state of the metal centers controls SoxR transcriptional activity. SoxR is also activated by direct reaction with NO. Key issues include: (i) the cellular reactions that switch SoxR on during oxidative stress and off when the stress is removed; (ii) the nature of NO-activated SoxR and the mechanisms that reverse the activation; (iii) structural changes in oxidized or NO-modified SoxR that activate transcription.
NO also activates resistance pathways in human cells, but the regulatory proteins remain to be identified. We have shown that increased NO resistance in mammalian cells involves the inducible enzyme heme oxygenase 1, and this induction is controlled by dramatically increased stability of the corresponding mRNA. Key questions include: (i) the mechanism of NO-mediated stabilization of the heme oxygenase 1 message; (ii) the identification of the NO-sensitive component of the signaling pathway; (iii) the role of the enzyme in induced NO resistance; (iv) the nature of other activities involved in inducible NO resistance in mammalian cells.
A third area of interest in our group concerns the mechanisms and roles of human enzymes that repair oxidative (free radical) damage in DNA. We have focused on the main enzyme that lies at the intersection of several pathways, the human abasic endonuclease Ape1. This enzyme processes the products of various DNA glycosylases, and it acts directly on many oxidative products in DNA. Our studies of Ape1 include: (i) the recognition and cleavage mechanism of the protein for specific DNA damages; (ii) the interaction of Ape1 protein with other repair components; (iii) the regulation of Ape1 expression in response to cytotoxic agents and during the normal cell cycle.
Selected Publications:
E. Hidalgo, H. Ding and B. Demple. 1997. Redox Signal Transduction:
Mutations Shifting [2Fe-2S] Centers of the SoxR Sensor-Regulator to the
Oxidized Form. Cell 88: 121-129.
J.C. Marquis and B. Demple. 1998. Complex Genetic Response of Human Cells to Sublethal Levels of Pure Nitric Oxide. Cancer Res. 58: 3435-3440.
Y. Masuda, R.A.O. Bennett and B. Demple. 1998. Dynamics of the Interaction
of Human Apurinic Endonuclease (Ape1) with Its Substrate and Product.
J. Biol. Chem. 273: 30352-30359.