Function and regulation of the ISG15 specific isopeptidase USP18

泛素（Ub）和泛素样蛋白（UBL）如SUMO、NEDD 8、FAT 10或Hub 1对蛋白质的修饰影响多效性生物学功能，并被Ub/UBL解偶联蛋白抵消。干扰素刺激基因15（Interferon stimulated gene 15，ISG 15）是一种UBL，其可以在类似于泛素的过程中与靶底物缀合（ISGylation），并且代表主要的I型干扰素（IFN）效应子系统。ISGylation针对病毒和细菌病原体，但也涉及其他过程，包括DNA修复和肿瘤发生。ISG化被ISG 15特异性异肽酶USP 18逆转。使用敲入小鼠，我们最近表明，USP 18的蛋白酶功能的选择性失活抑制病毒载量，这表明USP 18蛋白酶抑制可能构成一种有前途的抗病毒策略。到目前为止，只有少数与USP 18相互作用的蛋白被鉴定出来，而关于其他蛋白如何影响USP 18或USP 18如何调节结合伴侣的功能知之甚少。使用酵母双杂交筛选，我们现在已经鉴定了几种USP 18相互作用蛋白，其暗示USP 18与NFk-B信号通路和表观遗传调控的串扰。在本提案的范围内，我们将使用基于生物化学和细胞的方法分析这些相互作用伴侣和USP 18的功能关系。不考虑其蛋白酶活性，USP 18代表IFN应答的主要负调节剂。因此，我们和其他人最近的工作表明，USP 18的缺失导致小鼠小胶质细胞自发的异常激活，并导致人类胚胎中致命的干扰素病。尽管具有临床相关性，但不受控制的炎症反应的初始触发因素的性质仍有待确定。我们将通过经典的遗传拯救方法来解决这个问题，使用USP 18的双k.o小鼠，结合危险/病原体信号触发途径（如MAVS、MYD 88、STING等）的关键信号分子的耗竭。到目前为止，功能/表型关系的分析受到USP 18-/-小鼠表型中巨大遗传背景依赖性变异的阻碍。我们实验室中产生的条件性USP 18fl/fl小鼠现在将允许我们研究时空控制的USP 18消耗对充分表征的C57/BL 6菌株的后果。此外，C57/BL 6品系中USP 18-/-小鼠的胚胎致死率（构成了人体USP 18缺乏症后果的良好动物模型）有待于本研究中解决的机制探索。总的来说，我们希望我们的研究结果将增强对USP 18功能，其调节及其对相互作用蛋白质的影响的理解。研究结果还有助于深入了解导致USP 18缺陷型人类胚胎死亡的机制，以及一般有害的炎症反应（例如先天性感染引起的炎症反应）。

Protein modification by ubiquitin (Ub) and ubiquitin like proteins (UBLs) such as SUMO, NEDD8, FAT10 or Hub1 affect pleiotropic biological functions and are counteracted by Ub/UBL deconjugating proteins. Interferon stimulated gene 15 (ISG15) is an UBL, which can be conjugated to target substrates (ISGylation) in a process similar to ubiquitin and represents a major type I interferon (IFN) effector system. ISGylation is directed against viral and bacterial pathogens but is also involved in other processes including DNA repair and tumorigenesis. ISGylation is reversed by the ISG15-specific isopeptidase USP18. Using Knock-In mice, we recently showed that selective inactivation of the protease function of USP18 suppressed viral load suggesting that USP18 protease inhibition could constitute a promising antiviral strategy. So far, only a few proteins interacting with USP18 were identified and little is known about how other proteins influence USP18 or how USP18 regulates the function of binding partners. Using a yeast two hybrid screen, we have now identified several USP18 interacting proteins which hint to a crosstalk of USP18 with the NFk-B signaling pathway and epigenetic regulation. Within the scope of this proposal, we will analyze the functional relationship of these interaction partners and USP18 using biochemical and cell based approaches. Irrespective of its protease activity, USP18 represents a major negative regulator of the IFN response. Consequently, recent work by us and others showed that loss of USP18 results in spontaneous, aberrant activation of microglia in mice and causes fatal interferonopathies in human embryos. Despite the clinical relevance, the nature of the initial trigger for the uncontrolled inflammatory response still remains to be defined. We will address this question by classical genetic rescue approaches using double k.o mice for USP18 in combination with depletion of key signaling molecules of danger/pathogen signal triggered pathways such as MAVS, MYD88, STING etc. Until now, analysis of the function/phenotype relationship was hampered by a great genetic background dependent variability in the phenotype of USP18-/- mice. The conditional USP18fl/fl mice generated in our lab will now allow us to investigate the consequences of spatiotemporal controlled USP18 depletion on the well characterized C57/BL6 strain. Furthermore, embryonic lethality of USP18-/- mice in the C57/BL6 strain, which constitutes a good animal model for the consequences of USP18 deficiency in humans, awaits mechanistic exploration which will be addressed in this study. Collectively we expect that our results will enhance the understanding of USP18 function, its regulation and of its influence on interacting proteins. Results should also help to gain insights into the mechanisms responsible for the death of USP18 deficient human embryos in particular and for detrimental inflammatory responses (e.g. those caused by congenital infections) in general.