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，在肿瘤内形成低血管化和缺氧区域。癌症 由于化疗药物的输送不良和缺乏反应性的细胞， 辐照后的氧物种。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主要调节因子。HIF-1 α蛋白是一种 HIF-1亚基的最佳特征是缺氧稳定，并与HIF-1 α形成二聚体， 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， 共同促进癌症进展和转移。事实上，HIF水平的增加表明， 癌症患者的预后。因此，了解HIFs的功能和调节对于改善HIFs的功能和功能具有重要意义。 癌症治疗首席研究员的小组最近发现，脯氨酸之前的一些肽键 被肽基脯氨酰异构酶的FKBP家族之一顺反异构化。这种异构化使 HIF-1通过阻止泛素介导的降解来抑制。根据这些发现，该小组假设， 异构化抑制由GSK 3 β介导的脯氨酸附近的丝氨酸或苏氨酸的HIF-1 α磷酸化， 这是降解的主要步骤。为了验证这一假设，他们提出了以下三个目标。在目标1中， 他们将使用体外肽测定和细胞转染测定来研究异构化如何调节 磷酸化在目标2中，FKBP耗尽和HIF-1耗尽的细胞生物学后果将是 比较了这包括细胞增殖测定、HIF-靶基因的PCR、细胞迁移测定和免疫荧光测定。 血管生成测定。在目的3中，将用ChIP-seq研究HIF-1 α对FKBP的转录激活， 荧光素酶报告基因测定和电泳迁移率变动测定。这一目标将决定一个积极的反馈 FKBP和HIF-1 α之间的环。这些研究有望揭示一种新的分子机制， HIF-1在缺氧条件下的稳定性及肿瘤治疗的潜在药物靶点。