碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 100 / During embryonic development, enormous cell proliferation may consume oxygen rapidly and cause local hypoxia environment and activate hypoxia-inducible factors (HIFs) to trigger various downstream target genes. HIF is a hetrodimeric protein complex, which is composed of an oxygen-sensitive α subunit and a constitutively expressed β subunit. In the hypoxia condition, cytoplasmic HIF1α and HIF2α are stabilized and translocated to the nucleus, in which these factors combine with HIF1β (ARNT) to form protein complexes and activate target gene transcription. However, in normoxia environment, HIF1α and HIF2α are hydroxylated by oxygen-sensitive prolyl hydroxylase (PHDs), which in turn promotes HIFα degradation by 26S proteasome according to ubiquitination from the interaction with the von Hippel-Lindau tumor suppressor (VHL) ubiquitin E3 ligase complex.
Knockdown hif1α and hif2α expression abrogates circulation, central nervous system and gastrointestinal tract development in zebrafish embryos. It suggests that HIF1α and HIF2α are stabilized in the embryos. We expect the HIF1α and HIF2α can be stabilized by a local hypoxia environment or dysfunction of PHDs-VHL pathway during early stage of development. Nevertheless, it’s still unclear how the cells produce sufficient amount of HIFα to activate various gene transcription during development. Previously, it was revealed that knockdown of egln3/phd3 or vhl did not stabilize zebrafish HIF2α levels during development. To examine the stability of HIF1α during development, we have established Tol2-drHIF-1αODD- mCherry expression vector and microinjected it into zebrafish embryos at 1-cell stage. It appears that drHIF-1αODD-mCherry is very unstable during development and the fluorescence of drHIF-1αODD-mCherry fusing protein is scarcely detected. Replacing the two putative PHD-targeted proline residues within the drHIF-1αODD domain with alanine residues greatly enhanced the stability of drHIF-1αODD-mCherry, suggesting that the instability of drHIF-1αODD-mCherry is caused by the PHD-mediated protein degradation. Blocking egln2/phd1 and vhl expression slightly increased the fluorescent signaling. Conversely, knockdown egln1/phd2 and egln3/phd3 did not enhance the stability of drHIF-1αODD-mCherry fusing protein. In summary, this study revealed that HIF-1α is very unstable during development due to PHD-VHL-mediated degradation and the vital HIF-1α is probably produced by high efficiency expression with rapid protein turnover rate.
| Identifer | oai:union.ndltd.org:TW/100NTOU5613030 |
| Date | January 2012 |
| Creators | Wei-Lun Chang, 張維倫 |
| Contributors | Chin-Hua Hu, 胡清華 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 81 |
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