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.

目的：探讨HSF1在PI3K/AKT致癌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信号级联对于热休克(HS)激活MEF中的HSR/PSR和在恶性细胞中结构性激活HSF1是必需的。重要的是，AKT与HSF1在物理上相互作用。此外，AKT在Ser230处使HSF1磷酸化，AKT1的表达或肿瘤抑制基因PTEN的缺失足以激活HSF1。相反，AKT抑制剂可阻断HSF1 Ser230的磷酸化及其DNA与HSP基因启动子的结合。此外，结构性激活的PI3K/AKT信号导致小鼠大脑和肝脏过度生长或增大，分别类似于人类的大脑和肝肿大，导致出生后迅速死亡。重要的是，在这两个组织中同时缺失HSF1可以防止过度生长并延长动物的存活时间。此外，HSF1缺失还显著阻止Pten缺陷小鼠的肝脏过度生长，Pten是一种负向调节PI3K活性的肿瘤抑制因子，延长了小鼠的生存时间。我们的结果进一步表明，结构性激活的PI3K/AKT破坏了蛋白稳定并诱导了蛋白毒性应激，而HSF1缺乏明显加剧了这种应激。基于这些初步结果，我们计划询问：1)HSF1是否是AKT的新的生理底物；2)HSF1是否以及如何抑制由PI3K/AKT信号的结构性激活而引起的蛋白毒性应激，从而促进体内组织的过度生长；以及3)过度生长的组织中蛋白质平衡被破坏的分子机制。目的：研究热休克蛋白1(HSF1)在促进脂代谢和蛋白质脂化中的作用。我们以前的研究表明，HSF1是AMPK的生理底物，AMPK是一个关键的细胞代谢传感器，AMPK介导的Ser121磷酸化负向调节HSF1的激活。现在，我们使用转录活性不足的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的磷酸化，这是其激活的修饰关键，表明HSF1的作用机制不依赖于转录。相反，HSF1缺乏导致AMPK激活，而AMPK抑制剂则阻止了AMPK的激活。有趣的是，我们的结果表明，HSF1可以与AMPK和LKB1共沉淀，显示出LKB1-AMPK-HSF1蛋白复合体。此外，在人类肾癌和乳腺癌样本中，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。我们的初步数据显示，HSF1缺乏和HSF1表达增强分别导致细胞脂质含量减少和增加，这表明HSF1促进脂肪生成以支持恶性肿瘤。令人惊讶的是，HSF1缺陷小鼠的全身脂肪质量显著减少。重要的是，HSF1对细胞脂质含量和体脂质量的影响可以被AMPK抑制剂或siRNA介导的AMPK基因敲除显著拯救，这表明HSF1的造脂作用主要是通过抑制AMPK来实现的。在分子水平上，除了ACC的失活外，HSF1缺乏还会导致SREBP1c的失活，SREBP1c是控制成脂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一致，我们的结果显示HSF1缺乏引起的细胞胆固醇水平显著降低，这可以通过AMPK抑制来挽救。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Sonic Hedgehog(SHH)蛋白的胆固醇基化并支持SHH信号；以及3)HSF1是否促进移植的人类黑色素瘤模型的脂代谢和SHH胆固醇基化。

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