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.
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