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 Our unpublished preliminary work shows that (i) Lack of USPL1 causes early embryonic lethality and apoptosis in embryonic stem cells.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）和泛素样蛋白（UBL）对蛋白质的修饰影响多效性生物学功能，并被Ub/UBL蛋白酶抵消。SUMO修饰是UBL修饰中研究最多的修饰之一。在真核生物中，通过SUMO/Sentrin蛋白酶（SenP 1 -7）的SUMO去缀合被最好地表征，并且小鼠k.o.研究表明，SenPs在包括缺氧、造血、胚胎发生和炎症在内的生理过程中起关键作用。最初认为SUMO-去缀合仅由SenPs介导。然而，最近USP 11被鉴定为非SenP SUMO特异性异肽酶，其也具有非酶功能。然而，USPL 1的功能探索得很差，Pubmed中仅列出了少数出版物，其生理相关性完全不确定。我们现在已经产生了表达失活酶的Uspl 1fl/fl条件性和Uspl 1C 224 A敲入小鼠。我们未发表的初步工作表明，（i）USPL 1的缺乏导致胚胎干细胞的早期胚胎死亡和凋亡。(ii)成年Uspl 1fl/fl MxCre小鼠中的诱导缺失导致致死性贫血。当造血缺陷被骨髓移植挽救时，观察到致死性肝脂肪变性。(iii)Uspl 1fl/flCD 4Cre小鼠的分析显示USPL 1在T细胞发育中的关键作用。相比之下，在Uspl 1C 224 A小鼠中仅选择性失活酶活性并不影响存活力或导致显著的表型。我们现在将跟进生理分析，并深入了解USPL 1在体内的酶和非酶功能的分子机制。因此，我们将1。确定哪些细胞类型和途径需要USPL 1，并通过（i）评估USPL 1在ES细胞和胚胎发生中的生物学和分子功能来表征USPL 1在体内的生理学和分子功能。(ii)分析USPL 1在肝脏、终末分化细胞和不同器官中的生理和分子功能。(iii)定义USPL 1在造血区室中的关键分子和酶功能。鉴定USPL 1的去SUMO化活性的生理和分子功能和底物，并确定潜在的补偿机制。为此，我们将（i）对Uspl 1C 224 A小鼠进行深入的病理学和组织学分析，并跟踪观察到的T/B细胞组成的变化。(ii)分析其他SUMO蛋白酶的潜在补偿性上调，并在适用时通过产生双k.o小鼠解决冗余问题。为了规避潜在的适应性机制，我们将联合收割机将Uspl 1fl/fl与Uspl 1C 224 A等位基因组合，允许仅USPL 1的蛋白酶活性的及时控制失活。我们有信心阐明USPL 1的生理和分子功能，并将为科学界提供新的小鼠模型和工具，用于分析这种探索不足的SUMO蛋白酶。