The general goal of this proposal is to investigate mechanisms of cell damage caused by intermittent hypoxia (IH) such as that exhibited in Sleep Disordered Breathing (SDB). IH occurring during SDB and resulting oxidative stress are responsible for serious neuronal loss and loss of neurocognitive functions in animals and humans. Here, we will characterize biochemically and functionally the effects of IH/oxidative stress on interaction between von HippeI-Lindau (VHL) protein complex with E3 ubiquitin ligase activity and Rpb1, the largest, actively elongating subunit of RNA Polymerase II (the primary enzyme responsible for transcription of most mRNAs), in the context of neuronal damage. Our preliminary results demonstrate that VHL is an E3 ligase for Rpb1, causing Rpb1 ubiquitination in response to oxidative stress. This ubiquitination involves hydroxylation of proline within Rpb1 VHL-binding motif. This interaction and Rpb1 ubiquitination are also dramatically induced in parts of brain (cortex) that are sensitive but not in the parts of brain which are resistant to the effects of IH (brainstem) of animals exposed to chronic IH. We hypothesize that VHL-mediated ubiquitiantion of Rpb1 is involved in regulation of DNA damage (and repair) in response to IH and oxidative stress.
The specific aims are following: (1) To characterize biochemical interaction between VHL and Rpb1 and Rpb1 ubiquitination in response to IH in brain tissues from exposed animals, and in PC12 cells exposed in vitro to IH. We will also determine if these interactions are mediated by oxidative stress. (2) We will characterize effects of VHL expression on IH/oxidative stress induced oxidative DNA damage and cell death by apoptosis in PC12 cells expressing different levels of VHL. (3) We will determine the role of proline hydroxylation in Rpb1 ubiquitination by VHL and measure the effects of IH and oxidative stress on proline hydroxylation in brain extracts from exposed animals, and in PC12 cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL066312-05
Application #
6824849
Study Section
Neurodegeneration and Biology of Glia Study Section (NDBG)
Program Officer
Twery, Michael
Project Start
2000-09-30
Project End
2008-05-31
Budget Start
2004-09-01
Budget End
2005-05-31
Support Year
5
Fiscal Year
2004
Total Cost
$307,000
Indirect Cost
Name
University of Cincinnati
Department
Physiology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Ignacak, M L; Harbaugh, S V; Dayyat, E et al. (2009) Intermittent hypoxia regulates RNA polymerase II in hippocampus and prefrontal cortex. Neuroscience 158:1436-45
Haffey, Wendy D; Mikhaylova, Olga; Meller, Jarek et al. (2009) iTRAQ proteomic identification of pVHL-dependent and -independent targets of Egln1 prolyl hydroxylase knockdown in renal carcinoma cells. Adv Enzyme Regul 49:121-32
Mikhaylova, Olga; Ignacak, Monika L; Barankiewicz, Teresa J et al. (2008) The von Hippel-Lindau tumor suppressor protein and Egl-9-Type proline hydroxylases regulate the large subunit of RNA polymerase II in response to oxidative stress. Mol Cell Biol 28:2701-17
Hui, Anna S; Bauer, Amy L; Striet, Justin B et al. (2006) Calcium signaling stimulates translation of HIF-alpha during hypoxia. FASEB J 20:466-75
Zakrzewska, Adriana; Schnell, Phillip O; Striet, Justin B et al. (2005) Hypoxia-activated metabolic pathway stimulates phosphorylation of p300 and CBP in oxygen-sensitive cells. J Neurochem 94:1288-96
Lu, Gang; Seta, Karen A; Millhorn, David E (2005) Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes. J Biol Chem 280:21731-8
Seta, Karen A; Ferguson, Tsuneo K; Millhorn, David E (2004) Discovery of oxygen-responsive genes in pheochromocytoma cells. Methods Enzymol 381:449-64
Seta, Karen A; Millhorn, David E (2004) Functional genomics approach to hypoxia signaling. J Appl Physiol 96:765-73
Czyzyk-Krzeska, Maria F; Meller, Jaroslaw (2004) von Hippel-Lindau tumor suppressor: not only HIF's executioner. Trends Mol Med 10:146-9
Schnell, Phillip O; Ignacak, Monika L; Bauer, Amy L et al. (2003) Regulation of tyrosine hydroxylase promoter activity by the von Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription factors. J Neurochem 85:483-91

Showing the most recent 10 out of 17 publications