De-SUMOylation and the Hypoxic Response

描述（由申请人提供）：SUMO （Small Ubiquitin-like Modifier）是一种可以共价修饰大量细胞蛋白的泛素样蛋白。sumo酰化是一个动态过程，通过激活（E1）、偶联（E2）和连接（E3）酶介导，并容易被sumo特异性蛋白酶家族（SENP）逆转。我们发现，SENP1基因失活导致小鼠妊娠中期胚胎死亡，这是由于红细胞生成素（Epo）产生不足引起的严重胎儿贫血。SENP1通过调节缺氧诱导因子11 （HIF11）在缺氧条件下的稳定性来控制Epo的产生。在常氧条件下，HIF11在两个关键脯氨酸残基上被氧感应酶家族羟基化。脯氨酸羟基化对于HIF11结合其泛素连接酶复合物von Hippel-Lindau蛋白VHL/长链蛋白B/C复合物，导致泛素化和蛋白酶体降解是重要的。我们发现，缺氧诱导HIF11的快速sumo化，使其以不依赖于羟基脯氨酸的方式与VHL结合，也导致泛素化和蛋白酶体降解。SENP1逆转HIF11的sumo化，减少与VHL的结合，从而稳定HIF11。因此，SENP1在缺氧时调控HIF11的稳定性中起着关键作用。由于HIF11调节多种关键下游基因，如Epo和VEGF，因此它具有调节红细胞生成和血管生成的潜力。在初步结果中，我们发现summoylated HIF11的积累在暴露于缺氧后4小时达到峰值，随后下降。这对应于summoylation的早期阶段，最有可能是SUMO E3活性的增加，以及去summoylation的后期阶段，最有可能是由SENP1活性的增加介导的。在本研究中，我们将研究SUMOylation和去SUMOylation在缺氧对HIF11调控中的作用及其下游效应，重点是血管生成。目的1：确定缺氧诱导HIF11 SUMOylation的E3。目的2：研究缺氧对SENP1的调控作用。AIM3：确定SENP1在发育过程和肿瘤环境中是否调控血管生成。这些研究应该为去sumoylation途径如何调节缺氧反应提供新的见解。公共卫生相关性：我们发现了一种叫做SENP1的蛋白质，它可以从SUMO修饰的蛋白质中去除SUMO（小泛素样修饰物）。小鼠中SENP1的缺失会导致胚胎因严重贫血而过早死亡，这是因为在缺氧环境中需要SENP1维持缺氧诱导因子11 （HIF11）的稳定性。我们将研究缺氧如何调节SENP1， SUMO如何修饰HIF11，以及SENP1如何调节正常和异常血管的发育。

DESCRIPTION (provided by applicant): SUMO (Small Ubiquitin-like Modifier) is a ubiquitin-like protein that can covalently modify a large number of cellular proteins. SUMOylation is a dynamic process that is mediated by activating (E1), conjugating (E2), and ligating (E3) enzymes and readily reversed by a family of SUMO-specific proteases (SENP). We showed that inactivation of the SENP1 gene causes embryonal lethality in mid- gestation in mice as a result of severe fetal anemia stemming from deficient erythropoietin (Epo) production. SENP1 controls Epo production by regulating the stability of hypoxia-inducible factor 11 (HIF11) in hypoxic condition. During normoxia, HIF11 is hydroxylated in two critical proline residues by a family of oxygen sensing enzymes. Proline hydroxylation is important for HIF11 to binds to its ubiquitin ligase complex, von Hippel-Lindau protein VHL/elongin B/C complex, leading to ubiquitination and proteasomal degradation. We showed that hypoxia induced rapid SUMOylation of HIF11, which allowed it to bind to VHL in a hydroxyl-proline-independent manner, also leading to ubiquitination and proteasomal degradation. SENP1 reverses SUMOylation of HIF11, reduces binding to VHL, and consequently stabilizes HIF11. Thus, SENP1 plays a critical role in regulating HIF11 stability during hypoxia. Since HIF11 regulates multiple, critical downstream genes, such as Epo and VEGF, it has the potential to regulate erythropoiesis and angiogenesis. In preliminary results, we showed that accumulation of SUMOylated HIF11 peaked at four hours after exposure to hypoxia and later declined. This corresponded to an early phase of SUMOylation, most likely by an increase in SUMO E3 activity and a later phase of de-SUMOylation, most likely mediated by an increase in SENP1 activity. In this proposal, we will examine the role of SUMOylation and de-SUMOylation in HIF11 regulation by hypoxia and its downstream effects, focusing on angiogenesis. AIM 1: To identify the E3 responsible for hypoxia-induced SUMOylation of HIF11. AIM 2: To study how SENP1 is regulated by hypoxia. AIM3: To determine whether SENP1 regulates angiogenesis during development and in the tumor environment. These studies should provide novel insights into how the de-SUMOylation pathway regulates the hypoxic response. PUBLIC HEALTH RELEVANCE: We discovered a protein called SENP1 that can remove SUMO (Small Ubiquitin-like modifier) from SUMO- modified proteins. Deletion of SENP1 in mouse causes the embryo to die early due to severe anemia because SENP1 is required to maintain the stability of hypoxia-inducible factor 11 (HIF11) in the hypoxic environment. We will study how SENP1 is regulated by hypoxia, how HIF11 is modified by SUMO, and how SENP1 can regulate the development of normal and abnormal blood vessels.