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: 10486963
• 负责人: Chengkai Dai
• 金额: $ 91.36万
• 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/10486963
• 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/antagonist; 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.

目的1：研究HSF 1在维持由致癌PI 3 K/AKT信号转导驱动的组织过度生长中的作用。我们的初步研究结果表明，PI 3 K/AKT信号级联需要热休克（HS）在MEFs中的HSR/PSR的激活和恶性细胞中的组成性HSF 1激活。重要的是，AKT与HSF 1发生物理相互作用。此外，AKT在Ser 230处磷酸化HSF 1，并且组成型活性AKT 1的表达或肿瘤抑制因子PTEN的缺失足以激活HSF 1。相比之下，AKT抑制剂阻断HSF 1 Ser 230磷酸化及其DNA与HSP基因启动子的结合。此外，组成型活性PI 3 K/AKT信号传导导致小鼠大脑和肝脏的过度生长或扩大，分别类似于人类的巨脑畸形和肝肿大，导致出生后快速死亡。重要的是，在两种组织中同时缺失Hsf 1阻碍了过度生长和哺乳动物的存活。此外，Hsf 1缺失还显著阻碍Pten缺陷小鼠的肝脏过度生长，Pten是一种负调节PI 3 K活性的肿瘤抑制因子，延长了它们的生存期。我们的研究结果进一步表明，组成型活性PI 3 K/AKT破坏蛋白质稳态和诱导蛋白毒性应激，这是显着提高Hsf 1缺乏。基于这些初步结果，我们计划询问：1）HSF 1是否是AKT的新生理底物; 2）HSF 1是否以及如何抑制由PI 3 K/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;以及3）过度生长组织中破坏蛋白稳态的分子机制。目的2：研究热休克因子1（HSF 1）促进脂质代谢和蛋白质脂化的作用。我们以前的研究表明，HSF 1是AMPK的生理底物，AMPK是一种关键的细胞代谢传感器，AMPK介导的Ser 121磷酸化负调控HSF 1的激活。现在，我们使用缺乏转录活性的HSF 1缺失构建体的初步结果表明，就像野生型HSF 1一样，它们与AMPK相互作用并抑制AMPK Thr 172磷酸化，这是其激活的修饰关键，表明HSF 1的作用机制不依赖于转录。相反，Hsf 1缺乏导致AMPK激活，这被AMPK抑制剂阻断。有趣的是，我们的结果表明，HSF 1可以与AMPK和LKB 1共沉淀，揭示了LKB 1-AMPK-HSF 1蛋白复合物。此外，在人肾癌和乳腺癌样本中，较高的HSF 1 mRNA水平与AMPK Thr 172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，Hsf 1缺陷和增强的HSF 1表达分别导致细胞脂质含量减少和升高，这表明HSF 1促进脂肪生成以支持恶性肿瘤。引人注目的是，Hsf 1缺陷小鼠显示出明显减少的全身脂肪量。重要的是，HSF 1对细胞脂质含量和体脂量的这些作用可以通过AMPK抑制剂或siRNA介导的AMPK敲低而显著地挽救，这表明HSF 1的脂肪生成作用主要通过AMPK抑制介导。在分子水平上，HSF 1缺陷导致SREBP 1c失活，SREBP 1c是控制脂肪生成基因表达的关键转录因子，除了ACC失活。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一致，我们的研究结果揭示了由HSF 1缺乏引起的细胞胆固醇水平显着降低，这是由AMPK抑制拯救的。基于这些初步结果，我们计划研究：1）HSF 1抑制AMPK的分子机制; 2）HSF 1是否促进音刺猬（SHH）蛋白的胆固醇化并支持SHH信号传导; 3）HSF 1是否促进异种移植人黑素瘤模型中的脂质代谢和SHH胆固醇化。