Preservation of Proteomic Stability and Promotion of Protein Lipidation by HSF1

HSF1 保持蛋白质组稳定性并促进蛋白质脂化

• 批准号: 10702662
• 负责人: Chengkai Dai
• 金额: $ 126.37万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702662
• 关键词: AKT1 gene; Animals; Body fat; Brain; Cancer Biology; Cell physiology; Cessation of life; Cholesterol; DNA Binding; Data; Gene Expression; Genes; Genetic Transcription; Goals; HSF1; Heat-Shock Response; Hepatomegaly; Human; Investigation; Laboratories; Lipids; Liver; Malignant Neoplasms; Mediating; Megalencephaly; Membrane; Messenger RNA; Metabolic; Modeling; Modification; Molecular; Mus; Oncogenic; Outcome; PI3K/AKT; PTEN gene; Phosphorylation; Physiological; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Renal carcinoma; Research; Research Project Grants; Role; SHH gene; STK11 gene; Sampling; Signal Transduction; Small Interfering RNA; Stress; Tissues; Tumor Suppressor Proteins; Xenograft procedure; amyloidogenesis; base; cancer addiction; cancer cell; cancer therapy; experimental study; in vivo; inhibitor; knock-down; lipid biosynthesis; lipid metabolism; malignant breast neoplasm; melanoma; novel; novel therapeutic intervention; postnatal; preservation; promoter; protein complex; proteostasis; proteotoxicity; sensor; smoothened signaling pathway; steroid hormone; transcription factor; tumor

Aim1: To examine the role of HSF1 in sustaining tissue overgrowth driven by oncogenic PI3K/AKT signaling. Our preliminary results show that the PI3K/AKT signaling cascade is required for activation of the HSR/PSR by heat shock (HS) in MEFs and for constitutive HSF1 activation in malignant cells. Importantly, AKT physically interacts with HSF1. Furthermore, AKT phosphorylates HSF1 at Ser230, and expression of the constitutively active AKT1 or loss of the tumor suppressor PTEN is sufficient to activate HSF1. By contrast, AKT inhibitors block HSF1 Ser230 phosphorylation and its DNA binding to HSP gene promoters. Furthermore, constitutively active PI3K/AKT signaling causes overgrowth or enlargement of both brains and livers in mice, conditions similar to megalencephaly and hepatomegaly in humans respectively, leading to rapid postnatal death. Importantly, simultaneous deletion of Hsf1 in both tissues impedes overgrowth and prolongs animal survival. Moreover, Hsf1 deletion also markedly impedes the liver overgrowth in mice deficient for Pten, a tumor suppressor negatively regulating PI3K activity, prolonging their survival. Our results further show that constitutively active PI3K/AKT disrupts proteostasis and induces proteotoxic stress, which is markedly heightened by Hsf1 deficiency. Based on these preliminary results, we plan to interrogate: 1) whether HSF1 is a new physiological substrate for AKT; 2) whether and how HSF1 suppresses proteotoxic stress induced by constitutive activation of PI3K/AKT signaling and thereby promotes tissue overgrowth in vivo; and 3) the molecular mechanisms underlying disrupted proteostasis in overgrown tissues. Aim 2: To examine the role of HSF1 in promoting lipid metabolism and protein lipidation. Our previous studies revealed that HSF1 is a physiological substrate for AMPK, a key cellular metabolic sensor, and that the AMPK-mediated Ser121 phosphorylation negatively regulates HSF1 activation. Now, our preliminary results using HSF1 deletion constructs deficient for transcriptional activity show that, just like the wild-type HSF1, they interact with AMPK and suppress AMPK Thr172 phosphorylation, a modification key to its activation, indicating a transcription-independent mechanism of action of HSF1. Conversely, Hsf1 deficiency causes AMPK activation, which is blocked by the AMPK inhibitor. Interestingly, our results show that HSF1 can be co-precipitated with both AMPK and LKB1, revealing a LKB1-AMPK-HSF1 protein complex. Furthermore, in human kidney and breast cancer samples higher HSF1 mRNA levels are inversely correlated with AMPK Thr172 phosphorylation, congruent with the results of our mechanistic studies. Our preliminary data show that Hsf1 deficiency and enhanced HSF1 expression result in diminished and heightened cellular lipid content, respectively, suggesting that HSF1 promotes lipogenesis to support malignancy. Strikingly, Hsf1-deficient mice display markedly reduced whole-body fat mass. Importantly, these effects of HSF1 on cellular lipid content and body fat mass can be markedly rescued by either AMPK inhibitors or siRNA-mediated AMPK knockdown, suggesting that the lipogenic effect of HSF1 is largely mediated via AMPK suppression. At the molecular level, HSF1 deficiency causes inactivation of SREBP1c, a key transcription factor controlling lipogenic gene expression, in addition to inactivation of ACC. Cholesterol is an important lipid implicated in many key cellular processes, including membrane composition, signaling transduction, and synthesis of steroid hormones. Congruent with diminished cellular lipid content, our results reveal a markedly reduced cellular cholesterol level caused by HSF1 deficiency, which is rescued by AMPK inhibition. Based on these preliminary results, we plan to investigate: 1) the molecular mechanisms underlying AMPK suppression by HSF1; 2) whether HSF1 promotes cholesteroylation of sonic hedgehog (SHH) proteins and supports SHH signaling; and 3) whether HSF1 promotes lipid metabolism and SHH cholesteroylation in xenografted human melanoma models.

目标 1：检查 HSF1 在维持由致癌 PI3K/AKT 信号驱动的组织过度生长中的作用。我们的初步结果表明，PI3K/AKT 信号级联是 MEF 中热休克 (HS) 激活 HSR/PSR 以及恶性细胞中 HSF1 组成型激活所必需的。重要的是，AKT 与 HSF1 发生物理相互作用。此外，AKT 在 Ser230 位点磷酸化 HSF1，并且组成型活性 AKT1 的表达或肿瘤抑制因子 PTEN 的缺失足以激活 HSF1。相比之下，AKT 抑制剂可阻断 HSF1 Ser230 磷酸化及其 DNA 与 HSP 基因启动子的结合。此外，持续活跃的 PI3K/AKT 信号传导会导致小鼠大脑和肝脏过度生长或增大，分别类似于人类巨脑畸形和肝肿大，导致出生后快速死亡。重要的是，同时删除两个组织中的 Hsf1 可阻止过度生长并延长动物存活时间。此外，Hsf1缺失还显着阻止缺乏Pten（一种负调节PI3K活性的肿瘤抑制因子）的小鼠的肝脏过度生长，从而延长其生存期。我们的结果进一步表明，组成型活性 PI3K/AKT 会破坏蛋白质稳态并诱导蛋白质毒性应激，而 Hsf1 缺乏会显着加剧这种应激。基于这些初步结果，我们计划探讨：1）HSF1是否是AKT的新生理底物？ 2) HSF1是否以及如何抑制由PI3K/AKT信号传导的组成性激活诱导的蛋白毒性应激，从而促进体内组织过度生长； 3）过度生长的组织中蛋白质稳态被破坏的分子机制。目标2：研究HSF1在促进脂质代谢和蛋白质脂化中的作用。我们之前的研究表明，HSF1 是 AMPK（一种关键的细胞代谢传感器）的生理底物，并且 AMPK 介导的 Ser121 磷酸化负向调节 HSF1 激活。现在，我们使用转录活性缺陷的HSF1缺失构建体的初步结果表明，就像野生型HSF1一样，它们与AMPK相互作用并抑制AMPK Thr172磷酸化，这是其激活的关键修饰，表明HSF1的转录独立作用机制。相反，Hsf1 缺陷会导致 AMPK 激活，而 AMPK 抑制剂会阻止该激活。有趣的是，我们的结果表明 HSF1 可以与 AMPK 和 LKB1 共沉淀，从而揭示 LKB1-AMPK-HSF1 蛋白复合物。此外，在人类肾脏和乳腺癌样本中，较高的 HSF1 mRNA 水平与 AMPK Thr172 磷酸化呈负相关，这与我们的机制研究结果一致。我们的初步数据表明，Hsf1 缺乏和 HSF1 表达增强分别导致细胞脂质含量减少和升高，这表明 HSF1 促进脂肪生成以支持恶性肿瘤。引人注目的是，Hsf1 缺陷小鼠的全身脂肪量显着减少。重要的是，HSF1 对细胞脂质含量和体脂肪量的这些影响可以通过 AMPK 抑制剂或 siRNA 介导的 AMPK 敲低来显着挽救，这表明 HSF1 的脂肪生成作用很大程度上是通过 AMPK 抑制介导的。在分子水平上，除了 ACC 失活之外，HSF1 缺陷还会导致 SREBP1c 失活，SREBP1c 是控制脂肪生成基因表达的关键转录因子。胆固醇是一种重要的脂质，参与许多关键的细胞过程，包括膜组成、信号转导和类固醇激素的合成。与细胞脂质含量减少相一致，我们的结果表明，HSF1 缺乏导致细胞胆固醇水平显着降低，而 AMPK 抑制可以挽救这一水平。基于这些初步结果，我们计划研究：1）HSF1抑制AMPK的分子机制； 2) HSF1是否促进声刺猬(SHH)蛋白的胆固醇化并支持SHH信号传导； 3) HSF1是否促进异种移植人类黑色素瘤模型中的脂质代谢和SHH胆固醇化。