Preservation of Proteomic Stability and Promotion of Protein Lipidation by HSF1

目的1：研究HSF 1在维持由致癌基因驱动的组织过度生长中的作用。 PI 3 K/AKT信号传导。我们的初步研究结果表明，PI 3 K/AKT信号级联反应是一个重要的信号通路。 在MEFs中通过热休克（HS）激活HSR/PSR和组成性HSF 1所需的 激活恶性细胞。重要的是，AKT与HSF 1发生物理相互作用。此外，委员会还认为， AKT在Ser 230磷酸化HSF 1，并且组成型活性AKT 1的表达或HSF 1的缺失， 肿瘤抑制因子PTEN足以激活HSF 1。相比之下，AKT抑制剂阻断了 HSP 1 Ser 230磷酸化及其与HSP基因启动子的DNA结合此外，委员会认为， 组成型活性PI 3 K/AKT信号传导引起大脑过度生长或扩大， 小鼠的肝脏，分别类似于人类的巨脑畸形和肝肿大， 导致产后快速死亡。重要的是，在两种组织中同时缺失Hsf 1 阻碍了过度生长和哺乳动物的生存。此外，Hsf 1缺失也明显阻碍了 Pten缺陷小鼠肝脏过度生长，Pten是一种负性调节PI 3 K的肿瘤抑制因子 活动，延长其生存期。我们的研究结果进一步表明， PI 3 K/AKT破坏蛋白质稳态并诱导蛋白毒性应激， Hsf 1缺乏症。基于这些初步结果，我们计划询问：1）HSF 1是否 是AKT的一种新的生理底物; 2）HSF 1是否以及如何抑制蛋白毒性 由PI 3 K/AKT信号传导的组成性激活诱导的应激，从而促进组织 体内过度生长;和3）破坏蛋白质稳态的分子机制， 过度生长的组织目的2：研究HSF 1促进脂代谢的作用， 蛋白质脂化我们以前的研究表明，HSF 1是一种生理底物， AMPK是一种关键的细胞代谢传感器，AMPK介导的Ser 121磷酸化 负调节HSF 1活化。现在，我们用HSF 1缺失的初步结果 缺乏转录活性的构建体显示，正如野生型HSF 1， 它们与AMPK相互作用并抑制AMPK Thr 172磷酸化，这是其 激活，表明转录独立的作用机制的HSF 1。相反地， Hsf 1缺陷导致AMPK活化，其被AMPK抑制剂阻断。有趣的是， 我们的研究结果表明，HSF 1可以与AMPK和LKB 1共沉淀，揭示了HSF 1与AMPK和LKB 1的共沉淀。 LKB 1-AMPK-HSF 1蛋白复合物。此外，在人类肾癌和乳腺癌样本中， HSF 1 mRNA水平与AMPK Thr 172磷酸化呈负相关，与AMPK的表达一致。 我们的机械研究的结果。我们的初步数据显示，Hsf 1缺乏和 增强的HSF 1表达导致细胞脂质含量的减少和增加， 提示HSF 1促进脂肪生成以支持恶性肿瘤。引人注目的是， hsf 1缺陷小鼠显示全身脂肪量显著减少。重要的是，这些影响 AMPK可显著缓解HSF 1对细胞脂质含量和体脂量的影响， 抑制剂或siRNA介导的AMPK敲低，表明HSF 1的脂肪生成作用是 主要通过AMPK抑制介导。在分子水平上，HSF 1缺乏导致 SREBP 1c是一种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，涉及许多关键的 细胞过程，包括膜组成，信号转导和合成 类固醇激素与细胞脂质含量减少一致，我们的结果揭示了 显著降低由HSF 1缺乏引起的细胞胆固醇水平， AMPK抑制。基于这些初步结果，我们计划研究：1）分子 HSF 1抑制AMPK的潜在机制; 2）HSF 1是否促进AMPK的胆固醇化， 音速刺猬（SHH）蛋白和支持SHH信号传导;和3）是否HSF 1促进脂质 在异种移植的人黑素瘤模型中的代谢和SHH胆固醇化。

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.