Regulation of hypoxic response by HIF isomerization

HIF 异构化调节缺氧反应

基本信息

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
项目类别：
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

项目摘要

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.

项目概要当肿瘤变大时，肿瘤内部会形成血管不足和缺氧区域。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抗药性此外，癌细胞会上调缺氧的转录因子。诱导因子 (HIF) 家族是缺氧反应的主要调节因子，HIF-1α 蛋白是其中之一。最具特征的 HIF 亚基，通过缺氧稳定并与 HIF-1 形成二聚体，以激活超过 2,500 个基因涉及血管生成、糖酵解和生长因子信号传导。事实上，HIF 水平升高表明癌症进展和转移较差。因此，了解 HIF 的功能和调节对于改善癌症患者的预后非常重要。癌症治疗的主要研究小组最近发现一些肽键位于脯氨酸之前。被肽基脯氨酰异构酶 FKBP 家族之一顺反异构化，这种异构化稳定。基于这些发现，该小组通过阻止泛素介导的降解来抑制 HIF-1α。异构化抑制由 GSK3 介导的脯氨酸附近的丝氨酸或苏氨酸的 HIF-1 磷酸化，为了检验这一假设，他们在目标 1 中提出了以下三个目标：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在目标 2 中，FKBP 耗尽和 HIF-1α 耗尽的细胞生物学后果将是这包括细胞增殖测定、HIF 靶基因的 PCR、细胞迁移测定等。在目标 3 中，将使用 ChIP-seq 研究 HIF-1α 对 FKBP 的转录激活，荧光素酶报告基因检测和电泳迁移率变化检测将确定正反馈。 FKBP 和 HIF-1α 之间的环路有望揭示一种新的分子调节机制。 HIF-1α 在缺氧条件下的稳定性和癌症治疗的潜在药物靶点。