The Mechanism of Heat Shock Activation

热激激活机制

• 批准号: 6871612
• 负责人: EVGENY A NUDLER
• 金额: $ 29.65万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-27 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871612
• 关键词: Drosophilidae; RNA; antineoplastics; antisense nucleic acid; apoptosis; athymic mouse; breast neoplasms; cell growth regulation; combination cancer therapy; disease /disorder model; drug design /synthesis /production; drug screening /evaluation; gene environment interaction; gene expression; genetic regulation; genetic regulatory element; heat shock proteins; intermolecular interaction; laboratory rat; neoplasm /cancer chemotherapy; neoplasm /cancer thermotherapy; nonhuman therapy evaluation; protein localization; transcription factor; translation factor; yeasts

DESCRIPTION (provided by applicant): Transcription of heat shock (hsp) genes is governed by HSFs - constitutively synthesized transcription factors. HSF1 is the major stress responsive HSF in mammalian cells. Preliminary studies demonstrate that stress-induced activation of HSF 1 requires at least two associated factors: a translation elongation factor EF- 1A and novel untranslated RNA (HSR). The long-term objective of this project is to provide a complete structural and mechanistic description of the HSF1 activation mechanism and its control. It is further proposed to design small modulators of heat shock response based on HSR antisense oligonucleotides for applications in cancer therapy. Specific aims are: I. Structure/functional characterization of the HSF - EF-1A - HSR ternary complex. Experiments are proposed to determine major structural determinants of HSR from higher and lower eukaryotic cells that are responsible for thermosensing and HSF activation. A combination of protein-protein and protein-RNA footprinting along with cross-linking approaches will be used to map sites within HSF and/or EF-1A that directly contact HSR as well as regions in HSR that participate in such interaction. II. Understanding cellular mechanisms underlying HSF activation by HSR and EF-1A. Biochemical and immunological approaches will be used to determine subcellular localization of HSR, EF- 1A, and HSF before and after heat shock. Experiments in Drosophila and yeast are proposed to test whether a similar complex mechanism of HSF activation exists in lower eukaryotes. It will be determined if other cellular factors associate with HSF-1-HSR-EF-1A complex before and after heat shock and if other members of HSF family require HSR-like RNA for their activity. III. Design novel pro-apoptotic anti-cancer drugs based on HSR antisense oligonucleotides. Preliminary studies demonstrate that transfection of mammalian cells with short oligos complementary to certain regions of HSR suppresses hsp expression and sensitizes cells to heat. Chemically modified stable HSR antisense oligos will be obtained for transfection of thermotolerant breast cancer cultural cells and nude mice tumors for making them more susceptible to apoptosis and existing therapies.

描述（由申请人提供）：热休克（HSP）基因的转录受HSF的控制 - 组成构成合成的转录因子。 HSF1是哺乳动物细胞中主要的应激反应性HSF。初步研究表明，应力诱导的HSF 1的激活至少需要两个相关因素：翻译伸长因子EF-1A和新型的未翻译RNA（HSR）。该项目的长期目标是提供HSF1激活机制及其控制的完整结构和机械描述。进一步提议设计基于HSR反义寡核苷酸的小型调节剂，以用于癌症治疗。具体目的是：I。HSF -EF -1A -HSR三元复合物的结构/功能表征。提出了实验来确定负责热效和HSF激活的高层和较低真核细胞的HSR的主要结构决定因素。蛋白质 - 蛋白质和蛋白RNA足迹以及交联方法的组合将用于绘制直接接触HSR以及参与这种相互作用的HSR区域的HSF和/或EF-1A中的位点。 ii。了解HSR和EF-1A激活HSF的细胞机制。生化和免疫学方法将用于确定热休克之前和之后HSR，EF-1A和HSF的亚细胞定位。提出了果蝇和酵母中的实验来测试较低真核生物中HSF激活的类似复杂机制。将确定其他细胞因子是否与热休克之前和之后的H​​SF-1-HSR-1A复合物以及HSF家族的其他成员是否需要HSR样RNA进行活性。 iii。设计基于HSR反义寡核苷酸的新型促凋亡抗癌药物。初步研究表明，用短的寡核能互补地转染哺乳动物细胞与某些HSR区域抑制了HSP的表达并使细胞敏感以热。将获得化学修饰的稳定的HSR反义寡寡寡做，用于转染热耐乳腺癌培养细胞和裸小鼠肿瘤，以使其更容易受到细胞凋亡和现有疗法的影响。