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Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex.

Garcia-Santisteban I, Zorroza K, Rodriguez JA - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
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.

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Related in: MedlinePlus

Mapping the UAF1-binding site in USP1.A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site. The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (−) nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and (420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap, showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the (420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay. The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence VERIV, which resembles a UAF1-interacting motif in HPV E1 protein [17], is boxed.
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pone-0038570-g004: Mapping the UAF1-binding site in USP1.A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site. The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (−) nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and (420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap, showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the (420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay. The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence VERIV, which resembles a UAF1-interacting motif in HPV E1 protein [17], is boxed.

Mentions: We generated a series of seven YFP- or GFP-tagged USP1 deletion mutants lacking different amino-terminal, carboxy-terminal, or interstitial amino acid segments (Figure 4A). The NLS1 sequence was added to the amino-terminal end of those USP1 fragments lacking the NLSs (420–785, 450–785 and 420–520) in order to force their nuclear localization. The expression and size of each USP1 mutant was confirmed by immunoblot (Figure 4B). Xpress-UAF1 was co-expressed with each of the USP1 deletion mutants, and the localization of both proteins was examined in transfected 293T cells. The results are summarized in Figure 4A, and illustrated in Figure 4C. As expected, all the USP1 mutants were localized in the nucleus. The fragment 1–672, mimicking USP1 autocatalytic cleavage product [10], and the fragment 1–520 induced nuclear relocation of UAF1. In contrast, UAF1 was cytoplasmic when co-expressed with the USP1 fragment 1–500, suggesting that this mutant was unable to bind UAF1. On the other hand, the USP1 fragment 420–785, but not the fragment 450–785, was able to relocate UAF1 to the nucleus. The 100 amino acid fragment 420–520 readily induced nuclear accumulation of UAF1, whereas deletion of this fragment in the mutant Del(420–520) abolished nuclear relocation of co-expressed UAF1. Combined, these results indicate that USP1 region 420–520 mediates its interaction with UAF1. In fact, a semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution (Figure 4D) showed that the 420–520 fragment was even more efficient than full-length USP1 in promoting nuclear accumulation of UAF1. This observation raises the possibility that UAF1 interaction may be negatively regulated by USP1 sequences outside the UAF1-binding domain.


Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex.

Garcia-Santisteban I, Zorroza K, Rodriguez JA - PLoS ONE (2012)

Mapping the UAF1-binding site in USP1.A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site. The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (−) nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and (420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap, showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the (420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay. The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence VERIV, which resembles a UAF1-interacting motif in HPV E1 protein [17], is boxed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368879&req=5

pone-0038570-g004: Mapping the UAF1-binding site in USP1.A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site. The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (−) nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and (420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap, showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the (420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay. The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence VERIV, which resembles a UAF1-interacting motif in HPV E1 protein [17], is boxed.
Mentions: We generated a series of seven YFP- or GFP-tagged USP1 deletion mutants lacking different amino-terminal, carboxy-terminal, or interstitial amino acid segments (Figure 4A). The NLS1 sequence was added to the amino-terminal end of those USP1 fragments lacking the NLSs (420–785, 450–785 and 420–520) in order to force their nuclear localization. The expression and size of each USP1 mutant was confirmed by immunoblot (Figure 4B). Xpress-UAF1 was co-expressed with each of the USP1 deletion mutants, and the localization of both proteins was examined in transfected 293T cells. The results are summarized in Figure 4A, and illustrated in Figure 4C. As expected, all the USP1 mutants were localized in the nucleus. The fragment 1–672, mimicking USP1 autocatalytic cleavage product [10], and the fragment 1–520 induced nuclear relocation of UAF1. In contrast, UAF1 was cytoplasmic when co-expressed with the USP1 fragment 1–500, suggesting that this mutant was unable to bind UAF1. On the other hand, the USP1 fragment 420–785, but not the fragment 450–785, was able to relocate UAF1 to the nucleus. The 100 amino acid fragment 420–520 readily induced nuclear accumulation of UAF1, whereas deletion of this fragment in the mutant Del(420–520) abolished nuclear relocation of co-expressed UAF1. Combined, these results indicate that USP1 region 420–520 mediates its interaction with UAF1. In fact, a semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution (Figure 4D) showed that the 420–520 fragment was even more efficient than full-length USP1 in promoting nuclear accumulation of UAF1. This observation raises the possibility that UAF1 interaction may be negatively regulated by USP1 sequences outside the UAF1-binding domain.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus