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

26S蛋白酶体通过附着泛素链或泛素样修饰因子FAT10来降解蛋白质。直到最近，人们一直认为蛋白酶体降解主要是通过泛素或FAT10与其底物的酶连锁来调节的。然而，现在发现26S蛋白酶体本身的活性是通过磷酸化或泛素化来调节的。此外，泛素化底物结合抑制性蛋白酶体相关的去泛素化酶USP14，并通过这种方式激活26S蛋白酶体。NEDD8最终破坏因子1 （NUB1）强烈加速了fat10介导的蛋白酶体降解。值得注意的是，我们现在已经发现NUB1和FAT10一起（而不是单独）在体外激活26S蛋白酶体的能力与泛素偶联物一样强。此外，我们发现NUB1与USP14结合在26S蛋白酶体亚基RPN1中的相同位点，并且NUB1取代了26S蛋白酶体中的USP14。在这个项目中，我们将研究NUB1激活26S蛋白酶体的机制和功能后果。我们将确定这种激活是否需要NUB1的泛素样（UBL）结构域或泛素相关（UBA）结构域和/或FAT10的N端或c端UBL结构域。我们将测试NUB1是否与USP14竞争26S蛋白酶体结合，NUB1是否直接与USP14结合，以及NUB1/FAT10介导的蛋白酶体激活是否需要USP14。由于NUB1还与FAT10以外的松散折叠蛋白结合，我们将测试这些蛋白是否也会与NUB1合作激活蛋白酶体。重要的是，我们将研究NUB1/FAT10是否激活了26S蛋白酶体对泛素偶联物的降解。通过低温电镜，我们将研究NUB1/FAT10是否构象激活26S蛋白酶体，以及这种激活是否依赖于20S蛋白酶体的门打开。使用我们最近生成的NUB1-/-小鼠，我们将测试来自该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NUB1缺陷的HeLa细胞中所显示的那样。我们将把NUB1-/-小鼠与帕金森病和亨廷顿病的转基因小鼠模型配对，以检验缺乏NUB1是否会加剧疾病症状和神经元中蛋白质聚集的形成。病毒感染的细胞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NUB1-/-小鼠，并测量病毒复制、细胞毒性T细胞反应和未折叠蛋白对感染的反应。最后，我们将确定NUB1-/-小鼠是否更能抵抗AOM/DSS化学诱导的结肠癌形成，或者与APCMin/+小鼠杂交。综上所述，本项目有可能发现26S蛋白酶体活化的新机制，并进一步确立NUB1和/或FAT10作为治疗神经系统蛋白聚集性疾病和癌症的药理靶点。

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.