Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH-Alliance, Heidelberg, Germany.
Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH-Alliance, Heidelberg, Germany.
J Biol Chem. 2021 Jan-Jun;296:100324. doi: 10.1016/j.jbc.2021.100324. Epub 2021 Jan 23.
The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.
热休克反应是生物体应对蛋白质平衡失调的一种转录程序。它在所有真核细胞中由热休克转录因子 1（Hsf1）协调。尽管进行了非常深入的研究，但 Hsf1 激活-衰减循环的复杂性在分子水平上仍然难以捉摸。翻译后修饰属于拟议的适应个别细胞 Hsf1 活性的关键机制之一，Hsf1 多个位点的磷酸化引起了广泛关注。根据细胞生物学和蛋白质组学数据，Hsf1 还在多个位点被小泛素样修饰物（SUMO）修饰。SUMOylation 如何在分子水平上影响 Hsf1 活性仍不清楚。在这里，我们使用纯化的成分在体外分析 Hsf1 SUMOylation，以解决在细胞培养模型中无法回答的问题。在体外，Hsf1 主要在赖氨酸 298 处与单个 SUMO 结合，但我们确实在其他位点检测到低水平的 SUMOylation。不同的 SUMO E3 连接酶，如激活 STAT4 的蛋白抑制剂，增强了体外修饰的效率，但没有改变 SUMO 位点偏好。我们提供的证据表明，Hsf1 三聚体化和丝氨酸 303 和 307 的磷酸化增加了 SUMOylation 效率，表明 Hsf1 在其激活状态下被 SUMOylation。Hsf1 可以在结合 DNA 时被 SUMOylation，并且 Hsf1 的 SUMOylation既不改变 DNA 结合亲和力，也不影响热休克同源 71kDa 蛋白（HSPA8）+DnaJ 同源亚家族 B 成员 1 介导的 Hsf1 三聚体单体化和随之从 DNA 上的脱位。我们提出 SUMOylation 作用于热休克反应的转录水平。
