Enzymatic and non-enzymatic functions of the SUMO isopeptidase USPL1 in vivo

SUMO 肽酶 USPL1 体内的酶促和非酶促功能

• 批准号: 398279577
• 负责人: Privatdozent Dr. Klaus-Peter Knobeloch
• 金额: --
• 依托单位: Institut für Neuropathologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/398279577?language=en
• 关键词: Enzymatic; non; enzymatic; functions; SUMO

Protein modification by ubiquitin (Ub) and ubiquitin like proteins (UBLs) affect pleiotropic biological functions and are counteracted by Ub/UBL proteases. SUMO modification is one of the best studied UBL-modifications. In eukaryotes SUMO deconjugation by SUMO/Sentrin proteases (SenP1-7) is best characterized and mouse k.o. studies showed the critical role of SenPs in physiological processes including hypoxia, hematopoiesis, embryogenesis and inflammation. It was initially thought that SUMO-deconjugation is exclusively mediated by SenPs. However, recently USPl1 was identified as a non-SenP SUMO-specific isopeptidase which also possesses non-enzymatic functions. Yet, the function of USPL1 is poorly explored, only a handful of publications are listed in Pubmed and the physiological relevance is entirely undefined. We have now generated Uspl1fl/fl conditional and Uspl1C224A knock-In mice expressing an inactive enzyme. Our unpublished preliminary work shows that (i) Lack of USPL1 causes early embryonic lethality and apoptosis in embryonic stem cells. (ii) Induced deletion in adult Uspl1fl/fl MxCre mice results in lethal anemia. When the hematopoietic defect was rescued by bone marrow transplantation, lethal hepatic steatosis was observed. (iii) Analysis of Uspl1fl/flCD4Cre mice shows a critical role of USPL1 in T cell development. In contrast, selective inactivation of only the enzymatic activity in Uspl1C224A mice did not affect viability or cause a dramatic phenotype.We will now follow up on the physiological analysis and get insight into the molecular mechanisms underlying enzymatic and non-enzymatic functions of USPL1 in vivo. Therefore we will1. Determine which cell types and pathways require USPL1 and characterize the physiological and molecular functions of USPL1 in vivo by (i) Evaluating The biological and molecular function of USPL1 in ES cells and embryogenesis. (ii) Analyzing physiological and molecular functions of USPL1 in the liver, terminally differentiated cells and different organs. (iii) Define critical molecular- and enzymatic functions of USPL1 within the hematopoietic compartment.2. Identify physiological and molecular functions and substrates of the deSUMOylation activity of USPL1 and determine potential compensatory mechanisms. To this end we will (i) perform an in depth pathological and histological analysis of Uspl1C224A mice and follow up on the observed shift in T/B cell composition. (ii) Analyse potential compensatory upregulation of other SUMO proteases and if applicable address redundancy by the generation of double k.o mice. To circumvent potential adaptive mechanisms we will combine the Uspl1fl/fl with the Uspl1C224A allele allowing timely controlled inactivation of only the protease activity of USPL1. We are confident to shed light on the physiological and molecular function of USPL1 and will equip the scientific community with novel mouse models and tools for the analysis of this weakly explored SUMO-protease.

泛素（UB）和泛素类似蛋白质（UBL）的蛋白质修饰会影响多效性生物学功能，并由UB/UBL蛋白酶抵消。 SUMO修改是研究最佳的UBL修饰之一。在真核生物中，Sumo/Sentrin蛋白酶（SENP1-7）的Sumo DeCongation是最好的特征，并且Mouse K.O.研究表明，SENP在包括缺氧，造血，胚胎发生和炎症在内的生理过程中的关键作用。最初认为，相扑缀合是由森普（Senps）独家介导的。但是，最近将USPL1鉴定为非SENP SUMO特异性异肽酶，它也具有非酶函数。但是，探索了USPL1的功能，仅在PubMed中列出了少数出版物，并且生理相关性完全不确定。现在，我们已经产生了有条件的uspl1fl/fl和表达不活跃酶的uspl1c224a敲入小鼠。我们未发表的初步工作表明，（i）缺乏USPL1会导致胚胎干细胞中的早期胚胎致死性和凋亡。 （ii）成年USPL1FL/FL MXCRE小鼠的诱导缺失导致致命性贫血。当通过骨髓移植营救造血缺陷时，观察到致命的肝脂肪变性。 （iii）对USPL1FL/FLCD4CRE小鼠的分析显示了USPL1在T细胞发育中的关键作用。相反，仅对USPL1C224A小鼠中的酶活性的选择性失活不会影响生存力或引起戏剧性表型。我们现在将跟进生理分析，并深入了解USPL1 InVIVO中酶促和非酶函数的分子机制。因此，我们会。确定哪些细胞类型和途径需要USPL1，并通过（i）评估USPL1在ES细胞中的生物学和分子功能和胚胎发生来表征USPL1在体内的生理和分子功能。 （ii）分析肝脏中USPL1的生理和分子功能，末端分化的细胞和不同的器官。 （iii）定义造血区室内USPL1的临界分子和酶促功能。2。确定uspl1的desumoylation活性的生理和分子功能以及底物，并确定潜在的补偿机制。为此，我们（i）将对USPL1C224A小鼠进行深度的病理和组织学分析，并跟进T/B细胞组成中观察到的变化。 （ii）分析其他相扑蛋白酶的潜在补偿性上调，并通过产生双重K.O小鼠的适用地址冗余。为了避免潜在的自适应机制，我们将将USPL1FL/FL与USPL1C224A等位基因相结合，允许仅及时控制USPL1蛋白酶活性的失活。我们有信心阐明USPL1的生理和分子功能，并将为科学界配备新型的鼠标模型和工具，以分析这种弱化的Sumo蛋白酶。