Regulation of hypoxic response by HIF isomerization

HIF 异构化调节缺氧反应

• 批准号: 9813473
• 负责人: Nobuaki Kikyo
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813473
• 关键词: Antibodies; Biochemical Reaction; Biological; Biological Assay; Biological Process; CDK4 gene; Cancer Biology; Cancer Patient; Cancer Prognosis; Cell Differentiation process; Cell Proliferation; Cells; ChIP-seq; Drug Targeting; Electrophoretic Mobility Shift Assay; Equilibrium; Family; Feedback; Genes; Genetic Transcription; Glycolysis; Growth Factor; Hypoxia; Hypoxia Inducible Factor; In Vitro; Investigation; Luciferases; Mediating; Migration Assay; Molecular; Molecular Conformation; Myoblasts; Neoplasm Metastasis; Peptides; Peptidylprolyl Isomerase; Pharmaceutical Preparations; Phosphorylation; Play; Prevention; Principal Investigator; Proline; Protein Family; Proteins; Reactive Oxygen Species; Regulation; Reporter; Reporting; Resistance; Role; Serine; Signal Transduction; Tacrolimus Binding Proteins; Testing; Therapeutic; Therapeutic Intervention; Threonine; Transcriptional Activation; Transfection; Ubiquitin; angiogenesis; base; cancer cell; cancer therapy; chemotherapy; cis trans isomerization; dimer; hypoxia inducible factor 1; improved; in vivo; innovation; insight; irradiation; knock-down; member; migration; myogenesis; new therapeutic target; novel; novel marker; prevent; protein folding; response; tacrolimus binding protein 4; targeted cancer therapy; therapy resistant; transcription factor; tumor; tumor progression; uptake

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.

项目摘要 当肿瘤变大时，在肿瘤内形成了低血管化和低氧区域。癌症 由于化学疗法药物的递送不良，并且缺乏反应性，该地区的细胞对治疗具有抵抗力 辐照后的氧。此外，癌细胞上调缺氧的转录因子 诱导因子（HIF）家族作为其对缺氧反应的主要调节剂。 HIF-1蛋白，其中之一 最佳特征的HIF亚基通过缺氧稳定，并形成具有HIF-1的二聚体，以激活大于 2,500个基因。靶基因参与血管生成，糖酵解和生长因子信号传导，这些信号传导 共同促进癌症的进展和转移。确实，HIF的水平增加表明差 癌症患者的预后。这是重要的是要了解HIF的功能和调节以改进 癌症治疗。首席研究员小组最近发现，一些胡椒债券先于脯氨酸 是由petidyl prolyl异构酶的FKBP家族之一与异构酶异构化的顺式trans。这种异构化稳定 通过预防泛素介导的降解，HIF-1。基于这些发现，该小组假设 异构化抑制丝氨酸或苏氨酸的HIF-1磷酸化，附近的催化含量是由GSK3介导的 这是退化的主要步骤。为了检验这一假设，他们提出了以下三个目标。在AIM 1中， 他们将使用体外肽分析和细胞转染测定法研究异构化如何调节 磷酸化。在AIM 2中，FKBP耗竭和HIF-1耗竭的细胞生物学后果将是 比较的。这包括细胞增殖分析，HIF靶基因的PCR，细胞迁移测定和 血管生成测定。在AIM 3中，HIF-1对FKBP的转录激活将与芯片seq一起研究， 荧光素酶报告基因测定和电泳迁移率转移评估。这个目标将决定积极的反馈 在FKBP和HIF-1之间循环。这些研究有望揭示一种新的分子机制调节 HIF-1缺氧和潜在的药物靶标的HIF-1稳定性用于癌症治疗。