Regulation of hypoxic response by HIF isomerization

项目摘要 当肿瘤变大时，肿瘤内就会形成血管稀少和缺氧区。癌症 由于化疗药物输送不良和缺乏反应性，这些区域的细胞对治疗产生了抵抗力 辐照后的氧物种。此外，癌细胞还上调了缺氧的转录因子- 诱导因子(HIF)家族是它们对低氧反应的主要调节因子。缺氧诱导因子-1蛋白是 最具特征的HIF亚基，受低氧稳定，并与HIF-1形成二聚体，激活超过 2500个基因。靶基因涉及血管生成、糖酵解和生长因子信号转导，其中 共同促进癌症的进展和转移。事实上，HIF水平的升高表明 癌症患者的预后。因此，了解高强度聚焦因子的功能和调控机制对于提高其功能和水平具有重要意义。 癌症治疗。首席研究小组最近发现，一些位于Pro之前的多肽键 是由FKBP家族中的一种肽基-脯氨酰异构酶进行顺反异构化。这种异构化使之稳定。 HIF-1通过阻止泛素介导的降解。基于这些发现，研究小组假设 异构化抑制HIF-1由介导的靠近脯氨酸的丝氨酸或苏氨酸的磷酸化， 这是退化的重要一步。为了验证这一假设，他们提出了以下三个目标。在目标1中， 他们将使用体外多肽分析和细胞转染法来研究异构化如何调节 磷酸化。在目标2中，FKBP枯竭和HIF-1枯竭的细胞生物学后果将是 比较一下。这包括细胞增殖分析，HIF靶基因的聚合酶链式反应，细胞迁移分析，以及 血管生成检测。在目标3中，将用CHIP-SEQ研究HIF-1对FKBP的转录激活， 荧光素酶报告分析和电泳迁移率改变分析。这一目标将决定积极的反馈 在FKBP和HIF-1之间循环。这些研究有望揭开一种新的分子调控机制 低氧诱导因子-1在低氧条件下的稳定性及肿瘤治疗的潜在药物靶点。

Project Summary When a tumor becomes large, hypovascularized and hypoxic regions are formed inside the tumor. The cancer cells in the regions become resistant to therapy due to poor delivery of chemotherapy drugs and a lack of reactive oxygen species after irradiation. In addition, the cancer cells upregulate transcription factors of the hypoxia- inducible factor (HIF) family as master regulators of their response to hypoxia. The HIF-1 protein, one of the best characterized HIF subunits, is stabilized by hypoxia and forms a dimer with HIF-1 to activate more than 2,500 genes. The target genes are involved in angiogenesis, glycolysis, and growth factor signaling, which collectively facilitate cancer progression and metastasis. Indeed, increased levels of HIF indicate a poor prognosis for cancer patients. Thus, it is important to understand the functions and regulation of HIFs to improve cancer therapy. The principal investigator’s group recently found that some peptide bonds preceding prolines are cis-trans isomerized by one of the FKBP family of peptidyl prolyl isomerases. This isomerization stabilizes HIF-1 by preventing ubiquitin-mediated degradation. Based on these findings, the group hypothesized that the isomerization inhibits HIF-1 phosphorylation of serine or threonine near the prolines that is mediated by GSK3, which is a major step for degradation. To test this hypothesis, they proposed the following three aims. In Aim 1, they will use in vitro peptide assays and cell transfection assays to study how isomerization regulates phosphorylation. In Aim 2, cell biological consequences of FKBP depletion and HIF-1 depletion will be compared. This includes cell proliferation assays, PCR of HIF-target genes, cell migration assays, and angiogenesis assays. In Aim 3, transcription activation of the FKBP by HIF-1 will be studied with ChIP-seq, luciferase reporter assays and electrophoretic mobility shift assays. This aim will determine a positive feedback loop between FKBP and HIF-1. These studies are expected to unravel a novel molecular mechanism regulating HIF-1 stability in hypoxia and potential drug targets for cancer therapy.