The Mechanism of Heat Shock Activation

热激激活机制

• 批准号: 7117645
• 负责人: EVGENY A NUDLER
• 金额: $ 29.71万
• 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/7117645
• 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）基因的转录由HSF（组成型合成的转录因子）控制。 HSF1 是哺乳动物细胞中主要的应激反应 HSF。初步研究表明，应激诱导的 HSF 1 激活至少需要两个相关因子：翻译延伸因子 EF-1A 和新型非翻译 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制描述。进一步提出基于HSR反义寡核苷酸设计热休克反应的小型调节剂用于癌症治疗。具体目标是： I. HSF - EF-1A - HSR 三元复合物的结构/功能表征。提议进行实验以确定负责热传感和 HSF 激活的高等和低等真核细胞 HSR 的主要结构决定因素。蛋白质-蛋白质和蛋白质-RNA足迹与交联方法的组合将用于绘制HSF和/或EF-1A内直接接触HSR的位点以及HSR中参与这种相互作用的区域。二.了解 HSR 和 EF-1A 激活 HSF 的细胞机制。将使用生化和免疫学方法来确定热休克前后 HSR、EF-1A 和 HSF 的亚细胞定位。建议在果蝇和酵母中进行实验，以测试低等真核生物中是否存在类似的 HSF 激活复杂机制。将确定热休克前后是否有其他细胞因子与 HSF-1-HSR-EF-1A 复合物相关，以及 HSF 家族的其他成员是否需要 HSR 样 RNA 来发挥其活性。三．基于 HSR 反义寡核苷酸设计新型促凋亡抗癌药物。初步研究表明，用与 HSR 某些区域互补的短寡核苷酸转染哺乳动物细胞可抑制 hsp 表达并使细胞对热敏感。将获得化学修饰的稳定HSR反义寡核苷酸，用于转染耐热乳腺癌培养细胞和裸鼠肿瘤，使它们更容易受到细胞凋亡和现有疗法的影响。