Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.
PLoS One. 2012;7(6):e38570. doi: 10.1371/journal.pone.0038570. Epub 2012 Jun 6.
The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.
人类去泛素化酶 USP1 在癌症相关过程中发挥重要作用，例如 DNA 损伤反应和骨肉瘤细胞未分化状态的维持。USP1 去泛素化酶活性受到其与 WD40 重复蛋白 UAF1 相互作用的严格调控。抑制 USP1/UAF1 复合物的功能可使癌细胞对化疗敏感，表明该复合物是一个相关的抗癌靶点。有趣的是，尽管 UAF1 已被报道位于细胞质中，但 USP1 是一种核蛋白，尽管介导其核输入的序列基序尚未进行功能表征。在这里，我们鉴定了 USP1 中的两个核定位信号（NLS），并表明这些 NLS 介导了 USP1/UAF1 复合物的核输入。利用基于这些结果的细胞重定位测定，我们将 UAF1 结合位点映射到 USP1 中高度保守的 100 个氨基酸基序上。我们的数据支持以下模型：USP1 和 UAF1 在细胞质中形成复合物，随后通过 USP1 NLS 介导的输入转移到细胞核。重要的是，我们的发现对开发针对 USP1 的治疗方法具有实际意义。首先，这里鉴定的 USP1 与 UAF1 相互作用区域可能成为以治疗为目的破坏 USP1/UAF1 相互作用的靶点。另一方面，我们描述了一种细胞重定位测定方法，可以很容易地在高通量环境中实施，以寻找可能使 USP1/UAF1 复合物解离的药物。
