Regulation of 26S proteasome activity and coping with protein aggregation diseases by NUB1

蛋白质被26 S蛋白酶体通过连接泛素链或泛素样修饰剂FAT 10靶向降解。直到最近，人们认为蛋白酶体降解主要是由泛素或FAT 10与其底物的酶促连接调节的。然而，现在出现的是，26 S蛋白酶体本身的活性受到例如磷酸化或泛素化的调节。此外，泛素化底物结合抑制性蛋白酶体相关的去泛素化酶USP 14，并通过这种方式构象激活26 S蛋白酶体。FAT 10介导的蛋白酶体降解被NEDD 8终极终结者1（NUB 1）强烈加速。值得注意的是，我们现在已经发现NUB 1和FAT 10一起但不是单独地在体外激活26 S蛋白酶体，与泛素缀合物一样有效。此外，我们发现NUB 1与26 S蛋白酶体亚基RPN 1中的USP 14结合相同的位点，并且NUB 1取代了26 S蛋白酶体中的USP 14。在这个项目中，我们将研究NUB 1激活26 S蛋白酶体的机制和功能后果。我们将确定NUB 1的泛素样（UBL）结构域或泛素相关（乌巴）结构域和/或FAT 10的N-或C-末端UBL结构域是否是这种激活所必需的。我们将测试NUB 1是否与USP 14竞争26 S蛋白酶体结合，NUB 1是否直接与USP 14结合，以及NUB 1/FAT 10介导的蛋白酶体激活是否需要USP 14。由于NUB 1也与FAT 10以外的松散折叠蛋白质结合，我们将测试这些蛋白质是否同样与NUB 1合作激活蛋白酶体。重要的是，我们将研究NUB 1/FAT 10是否激活26 S蛋白酶体对泛素缀合物的降解。通过冷冻电子显微镜，我们将研究NUB 1/FAT 10是否构象激活26 S蛋白酶体，以及这种激活是否依赖于20 S蛋白酶体门打开。使用我们最近产生的NUB 1-/-小鼠，我们将测试这种小鼠的细胞是否对蛋白质错误折叠诱导药物过敏，正如我们在NUB 1缺陷的HeLa细胞中所显示的那样。我们将NUB 1-/-小鼠与帕金森病和亨廷顿病的转基因小鼠模型配对，以检查NUB 1的缺乏是否会加剧疾病症状和神经元中蛋白质聚集体的形成。病毒感染或肿瘤细胞需要更高的蛋白酶体活性。因此，我们将用淋巴细胞性脉络丛脑膜炎病毒感染NUB 1-/-小鼠，并测量病毒复制、细胞毒性T细胞应答和对感染的未折叠蛋白应答。最后，我们将确定NUB 1-/-小鼠是否对AOM/DSS化学诱导的结肠癌形成或与APCMin/+小鼠交配诱导的结肠癌形成更具抗性。 总之，该项目有可能确定26 S蛋白酶体激活的新机制，并进一步确定NUB 1和/或FAT 10作为治疗神经蛋白聚集疾病和癌症的药理学靶点。

Proteins are targeted for degradation by the 26S proteasome by attachment of ubiquitin chains or the ubiquitin-like modifier FAT10. Until recently it was assumed that proteasomal degradation was mainly regulated by enzymatic linkage of ubiquitin or FAT10 to their substrates. However, it is emerging now that the activity of the 26S proteasome itself is regulated e.g. by phosphorylation or ubiquitylation. Moreover, ubiquitylated substrates bind the inhibitory proteasome-associated deubiquitylating enzyme USP14 and by this means conformationally activate the 26S proteasome. FAT10-mediated proteasomal degradation is strongly accelerated by NEDD8 ultimate buster 1 (NUB1). Remarkably, we have now found that NUB1 and FAT10 together but not alone activated the 26S proteasome in vitro as potently as ubiquitin conjugates. Moreover, we showed that NUB1 binds to the same site in the 26S proteasome subunit RPN1 as USP14 and that NUB1 replaced USP14 from the 26S proteasome. In this project we will investigate the mechanism and functional consequences of 26S proteasome activation by NUB1. We will determine whether the ubiquitin-like (UBL) domain or the ubiquitin associated (UBA) domains of NUB1 and/or the N- or C-terminal UBL domains of FAT10 are required for this activation. We will test if NUB1 competes with USP14 for 26S proteasome binding, whether NUB1 directly binds to USP14 and whether USP14 is required for NUB1/FAT10 mediated proteasome activation. As NUB1 also binds to loosely folded proteins other than FAT10 we will test, whether these will likewise cooperate with NUB1 to activate the proteasome. Importantly, we will investigate if NUB1/FAT10 activate the degradation of ubiquitin conjugates by the 26S proteasome. By cryo electron microscopy we will investigate if NUB1/FAT10 conformationally activate the 26S proteasome and whether this activation relies on 20S proteasome gate opening. Using the NUB1-/- mouse, which we have recently generated, we will test whether cells from this mouse are hypersensitive to protein misfolding-inducing drugs as we have shown in NUB1-deficient HeLa cells. We will pair the NUB1-/- mouse with transgenic mouse models of Parkinson’s and Huntington’s disease to examine whether a lack of NUB1 exacerbates disease symptoms and protein aggregate formation in neurons. Virally infected or tumor cells require higher proteasome activity. Hence we will infect NUB1-/- mice with lymphocytic choriomeningitis virus and measure virus replication, the cytotoxic T cell response and the unfolded protein response to infection. Finally, we will determine whether NUB1-/- mice are more resistant to colon carcinoma formation chemically induced by AOM/DSS or by breeding with APCMin/+ mice. Taken together, this project has the potential to identify a new mechanism of 26S proteasome activation and further establish NUB1 and/or FAT10 as pharmacological targets for the therapy of neurological protein aggregation diseases and cancer.