Donna D Zhang, PhD
Assistant Professor
Department:
Pharmacology and Toxicology
(520) 626-9918
Fax:
(520) 626-2466
E-mail:
Location:
Pharmacy 421
Bio / Research:
The research projects in my laboratory are focused on (1) Mechanistic studies of the Nrf2/Keap1 signaling pathway that is activated by oxidative stress and chemopreventive compounds, (2) the protective role of Nrf2 in arsenic-induced toxicity and carcinogenicity (this project is funded by NIEHS R01 award), and (3) regulation of gene expression by the ubiquitination and proteasomal degradation pathway.
Mechanistic studies of the Nrf2/Keap1 signaling pathway
Oxidative stress, an imbalance between production and removal of reactive oxygen species, can damage biological macromolecules including DNAs, proteins and lipids (1). Oxidative damage to biological macromolecules can have profound effects on cellular functions and has been implicated in cancer, inflammation, neurodegenerative diseases, cardiovascular diseases and aging. Eukaryotic cells have evolved anti-oxidant defense mechanisms to neutralize reactive oxygen species (ROS) and maintain cellular redox homeostasis. One of the most important cellular defense mechanisms against ROS and electrophilic intermediates is mediated through the ARE (antioxidant responsive element, or electrophile responsive elements) sequence in the promoter regions of genes. The ARE-dependent cellular defense system is controlled by the transcription factor Nrf2. Under basal condition, Nrf2-dependent transcription is down-regulated by the negative regulator Keap1. When cells are exposed to chemopreventive agents or oxidative stress, Nrf2 escapes Keap1-mediated repression and activates ARE-defense response that protects cells from oxidative damage.
We have demonstrated that Nrf2 is not passively sequestered in the cytoplasm by Keap1 as initially proposed by Yamamoto and his coworkers. Rather, Keap1 actively targets Nrf2 for ubiquitination and subsequent degradation by the proteasome. We have identified two critical cysteine residues in Keap1 (C273 and C288) that are required for ubiquitination of Nrf2. Furthermore, tBHQ-induced oxidative stress and a chempreventive agent sulforaphane stabilize Nrf2 and activate Nrf2-dependent gene expression. We have identified a third cysteine residue in Keap1 (C151) that is required both for stabilization of Nrf2 and for a novel post-translational modification to Keap1 when cells are exposed to tBHQ or sulforaphane.
In subsequent work, we have demonstrated that Keap1 functions as a substrate adaptor for a Cul3-based E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. tBHQ and sulforaphane inhibit Keap1-dependent ubiquitination of Nrf2 and this inhibition requires the integrity of C151. Neither quinine-induced oxidative stress nor sulforaphane disrupts association of Keap1 with Nrf2, indicating tBHQ or sulforaphane inhibits the activity of the Keap1:Cul3:Rbx1 complex. The laboratory is currently investigating the detailed mechanisms by which oxidative stress and chemical agents activate the Nrf2-mediated ARE-defense response.
The protective role of Nrf2 in arsenic-induced toxicity and carcinogenicity
Another direction of our research is to understand the molecular mechanisms of toxicity/carcinogenicity of environmental pollutants and the endogenous cellular defense systems to cope with pollutants. Drinking water contaminated with arsenic, a known carcinogen, is a worldwide public health issue. Epidemiology studies have linked arsenic exposure to human cancers, including skin, liver, lung, kidney, prostate, and bladder cancer. Arsenic can also cause cellular damage through generation of reactive oxygen species (ROS) that are involved in the initiation, promotion, and progression of tumors. Although arsenic is a well defined carcinogen, it is not mutagenic and induces malignant transformation possibly by an epigenetic or cell signaling mechanism. For example, arsenic has been reported to activate specific cellular signal transduction pathways, leading to the activation of several oncogenes and excessive cellular proliferation, a prerequisite for cellular transformation.
Activated by compounds possessing anti-cancer properties, the ARE-Nrf2-Keap1 signaling pathway has been clearly demonstrated to have profound effects on tumorigenesis. More significantly, Nrf2 knockout mice display increased sensitivity to chemical toxicants and carcinogens and are refractory to the protective actions of chemopreventive compounds.
Therefore, we hypothesize that activation of the ARE-Nrf2-Keap1 pathway acts as an endogenous protective system against arsenic-induced toxicity and carcinogenicity. The following Specific Aims are intended to further elucidate mechanism of Nrf2-activation in protection from arsenic-induced toxicity/tumorigenicity. We will (1) Determine the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and cell transformation using a model cell line UROtsa. (2) Define the molecular mechanisms of activation of the ARE-Nrf2-Keap1 pathway by arsenic, sulforaphane, and tBHQ. (3) Define the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and tumorigenicity using Nrf2 knockout mouse model.
Regulation of gene expression by the ubiquitination and proteasomal degradation pathway
We are also interested in investigating gene regulation through the ubiquitination and proteasomal degradation pathway. In particular, we will focus on the genes that encode substrate proteins of the cullin-containing E-3 ubiquitin enzymes. Once such genes are identified, we will study the functional roles of there genes and the consequence of disruption of the genes.
PhD 1991-1997 Molecular Toxicology/Oncology New York University
PD 1997-1998 DuPont-Haskell Laboratories Newark, DE
Selected Publications
Zhang D. D., S-C Lo, Z. Sun, G. M. Habib, M. W. Lieberman, and M. Hannink. 2005. Ubiquitination of Keap1, a BTB-Kelch Substrate Adaptor Protein for Cul3, Targets Keap1 for Degradation by a Proteosome-Independent Pathway. Journal of Biological Chemistry. 280:30091-30099
Ansell PJ, Lo SC, Newton LG, Espinosa-Nicholas C, Zhang DD, Liu JH, Hannink M, Lubahn DB. 2005. Repression of cancer protective genes by 17beta-estradiol: Ligand-dependent interaction between human Nrf2 and estrogen receptor alpha. Molecualr and Cellular Endocrinology. Sep 27
Li X., D. D. Zhang, M. Hannink and L. J. Beamer. 2004. Crystal Structure of the Kelch Domain of Keap1. Journal of Biological Chemistry. 279: 54750-54758.
Zhang D. D., S-C Lo, J. V. Cross, D. J. Templeton and M. Hannink. 2004. Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex. Molecular and Cellular Biology. 24: 10941-10953.
Li X., D. D. Zhang, M. Hannink and L. J. Beamer. 2004. Crystallization and Initial Crystallographic Analysis of the Kelch Domain from Human Keap1. Acta Crystallographica. Section D, Biological Crystallography. 60: 2346-2348
Zhang D. D, and M. Hannink. 2003. Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress. Molecular and Cellular Biology. 23: 8137-8151.
Cullinan S. B., D. D. Zhang, M. Hannink, E. Arvisais, R. J. Kaufman, and J. A. Diehl. 2003. Nrf2 is a Direct PERK Substrate and Effector of PERK-Dependent Cell Survival. Molecular and Cellular Biology. 23: 7198-7209.
Salnikow K., T. Davidson, T. Kluz, H. Chen, D. Zhou*, and M. Costa. 2003 GeneChip Analysis of Signaling Pathways Effected by Nickel. Journal of Environmental Monitoring. 5: 206-209.
Zhang D. D., M. A. Hartsky, and D. B. Warheit. 2002. Time Course of Quartz and TiO2 Particle-Induced Pulmonary Inflammation and Neutrophil Apoptotic Responses in Rats.
Experimental Lung Research. 28: 641-670.
Sachdev, S. A., S. Bagchi, D. D. Zhang, A. Mings, and M. Hannink. 2000. Nuclear Is Accomplished by a Ran-Independent Transport Pathway.BImport of I Molecular and Cellular Biology. 20: 1571-1582.
Zhou D*., M. A. Hartsky, and D. B. Warheit. 1999. Differential Apoptotic Responses of Pulmonary Inflammatory Cells in Rats Following in vivo Exposures to Quartz or TiO2 Particles. ILSI proceedings.
Salnikow K., D. Zhou*, T. Kluz, C. Wang, and M. Costa. 1999. A New Gene Induced by a Rise in Free Intracellular Ca2+ Following Ni 2+ Exposure. In Metals and Genetics. ed. Sarkar (Plenum, New York). 131-144.
Zhou D*., K. Salnikow, and M. Costa. 1998. Cap 43, a Novel Gene Specifically Induced by Ni2+ Compounds. Cancer Research. 58: 2182-2189.
Costa M., K. Salnikow, Y. Lee, and D. Zhou*. 1997. Molecular Biology of Nickel Carcinogenesis. Journal of Inorganic Biochemistry. 67 (1-4): 372-372.
*Zhou D. is the former name of Donna D. Zhang