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: 10926314
• 负责人: Chengkai Dai
• 金额: $ 103.65万
• 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/10926314
• 关键词: ADD-1 protein; AKT1 gene; Animals; Body fat; Brain; Cancer Biology; Cell physiology; Cessation of life; Cholesterol; DNA Binding; Data; Gene Expression; Genes; Genetic Transcription; Goals; Growth; 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; PIK3CG gene; 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; cancer addiction; cancer cell; cancer therapy; constitutive expression; 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.

AIM1：检查HSF1在由致癌PI3K/AKT信号传导驱动的组织过度生长中的作用。我们的初步结果表明，PI3K/AKT信号传导级联对于通过MEF中的热休克（HS）和恶性细胞中的组成型HSF1激活需要激活HSR/PSR。重要的是，AKT与HSF1物理相互作用。此外，AKT在Ser230处磷酸化HSF1，并表达组成性活跃的AKT1或肿瘤抑制剂PTEN的丢失足以激活HSF1。相比之下，AKT抑制剂阻断了HSF1 SER230磷酸化及其与HSP基因启动子的DNA结合。此外，组成性活跃的PI3K/AKT信号传导在小鼠中会导致大脑和肝脏的过度生长或扩大，分别类似于大脑脑和人类的肝肿大，导致产后快速死亡。重要的是，在两种组织中同时缺失HSF1会阻碍过度生长并延长动物的存活。此外，HSF1缺失还显着阻碍了缺乏PTEN的小鼠的肝脏过度生长，PTEN抑制肿瘤对PI3K活性负面调节，延长了其生存率。我们的结果进一步表明，组成性活跃的PI3K/AKT会破坏蛋白质的毒性并诱导蛋白质毒性应激，而HSF1缺乏症显着增强。基于这些初步结果，我们计划审问：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信号的组成型激活引起的蛋白毒性应激，从而促进体内组织过度生长； 3）在组织过长的组织中破坏蛋白质的分子机制。目标2：检查HSF1在促进脂质代谢和蛋白质脂化中的作用。我们先前的研究表明，HSF1是AMPK的生理底物，AMPK是一种钥匙细胞代谢传感器，并且AMPK介导的Ser121磷酸化对HSF1激活负面调节。现在，我们使用HSF1缺失构建体缺乏转录活性的初步结果表明，就像野生型HSF1一样，它们与AMPK相互作用并抑制AMPK THR172 THR172磷酸化，这是其激活的修改键，表明HSF1的转录无关机制。相反，HSF1缺乏会导致AMPK激活，该AMPK被AMPK抑制剂阻止。有趣的是，我们的结果表明，HSF1可以与AMPK和LKB1共沉淀，揭示了LKB1-AMPK-HSF1蛋白质复合物。此外，在人类肾脏和乳腺癌样品中，较高的HSF1 mRNA水平与AMPK THR172磷酸化成反比，这与我们的机械研究结果一致。我们的初步数据表明，HSF1缺乏症和增强的HSF1表达会导致细胞脂质含量减少和增强，这表明HSF1促进了脂肪生成以支持恶性肿瘤。令人惊讶的是，HSF1缺陷型小鼠显示出明显降低的全身脂肪量。重要的是，AMPK抑制剂或siRNA介导的AMPK敲低可以明显挽救HSF1对细胞脂质含量和体内脂肪量的影响，这表明HSF1的脂肪生成作用在很大程度上是通过AMPK抑制介导的。在分子水平上，HSF1缺乏会导致SREBP1C失活，SREBP1C是控制脂肪生成基因表达的关键转录因子，此外ACC的失活。胆固醇是一种重要的脂质，与许多关键细胞过程有关，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，我们的结果表明，由HSF1缺乏症引起的细胞胆固醇水平明显降低，这是由AMPK抑制作用挽救的。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是否促进声波刺猬（SHH）蛋白的胆固醇化并支持SHH信号传导； 3）HSF1是否促进异种人黑色素瘤模型中的脂质代谢和SHH胆固醇化。

项目成果

Heat shock factor 1 is a direct anti-amyloid factor: connecting neurodegeneration and uncontrolled growth.

• DOI: 10.4103/1673-5374.320983
• 发表时间: 2022-03
• 期刊: Neural regeneration research
• 影响因子: 6.1
• 作者: Tang Z;Dai C
• 通讯作者: Dai C

mTORC1 senses stresses: Coupling stress to proteostasis.

• DOI: 10.1002/bies.201600268
• 发表时间: 2017-05
• 期刊: BioEssays : news and reviews in molecular, cellular and developmental biology
• 作者: Su KH;Dai C
• 通讯作者: Dai C