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

泛素(Ub)和泛素样蛋白(UBL)的蛋白质修饰会影响多效性的生物学功能，并被Ub/Ubl酶所抵消。相扑修饰法是目前研究最多的UBL修饰法之一。在真核生物中，用SUMO/前哨蛋白水解酶(SENP1-7)最具特性的是SUMO去结合，小鼠K.O.研究表明，SERPs在低氧、造血、胚胎发生和炎症等生理过程中起着关键作用。最初人们认为，相扑解决术完全是由Senps来调解的。然而，USPl1最近被鉴定为一种非SenP相扑专一性的异肽酶，也具有非酶功能。然而，人们对USPL1的功能研究很少，只有少数几篇论文被PUBMED收录，其生理相关性也完全没有定义。我们现在已经产生了Upl1fl/fl条件性和Upl1C224A敲入小鼠，表达了一种失活的酶。我们未发表的初步工作表明：(1)缺乏USPL1会导致胚胎早期死亡和胚胎干细胞的凋亡。(Ii)在成年Upl1fl/fl MxCre小鼠中诱导缺失会导致致死性贫血。经骨髓移植修复后，出现致死性肝脂肪变性。(Iii)对Upl1fl/flCD4Cre小鼠的分析表明，USPL1在T细胞发育中起着关键作用。相比之下，仅选择性灭活Upl1C224A小鼠的酶活性并不影响存活或导致戏剧性表型。我们现在将继续进行生理分析，深入了解USPL1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USPL1，并通过(I)评估USPL1在胚胎干细胞和胚胎发生中的生物学和分子功能来表征USPL1在体内的生理和分子功能。(Ii)分析USPL1在肝脏、终末分化细胞和不同器官中的生理和分子功能。(3)确定USPL1在造血室中的关键分子和酶功能。鉴定USPL1的生理和分子功能以及脱糖活性的底物，并确定潜在的补偿机制。为此，我们将(I)对Upl1C224A小鼠进行深入的病理和组织学分析，并跟踪观察到的T/B细胞组成的变化。(Ii)分析其他相扑蛋白水解酶的潜在补偿性上调，以及如果适用，通过产生双k.o小鼠而产生的地址冗余。为了规避潜在的适应机制，我们将Upl1f1/f1与Upl1C224A等位基因结合起来，仅允许及时控制USPL1的蛋白酶活性失活。我们有信心阐明USPL1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来分析这种探索较弱的相扑酶。

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 Our unpublished preliminary work shows that (i) Lack of USPL1 causes early embryonic lethality and apoptosis in embryonic stem cells.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.