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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.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.

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

A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation.In the upper part, the formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization of Xpress-UAF1.
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pone-0038570-g005: A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation.In the upper part, the formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization of Xpress-UAF1.

Mentions: Importantly, the striking change in the nucleocytoplasmic localization of Xpress-UAF1 that results from GFP-USP1 co-expression provides the basis for a relocation assay to test USP1/UAF1 complex formation in cells. Here, we have used this assay to map the UAF1-binding motif in USP1, but such an assay can also be applied to evaluate the ability of different drug treatments to interfere with the formation of the USP1/UAF1 complex (Figure 5). Furthermore, by genetically tagging UAF1 with a fluorescent protein, such as RFP, and using automated microscopy procedures, the assay can be readily adapted to a high throughput format in order to allow a high content screening for molecules that interfere with USP1/UAF1 complex formation.


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

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

A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation.In the upper part, the formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization of Xpress-UAF1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038570-g005: A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation.In the upper part, the formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization of Xpress-UAF1.
Mentions: Importantly, the striking change in the nucleocytoplasmic localization of Xpress-UAF1 that results from GFP-USP1 co-expression provides the basis for a relocation assay to test USP1/UAF1 complex formation in cells. Here, we have used this assay to map the UAF1-binding motif in USP1, but such an assay can also be applied to evaluate the ability of different drug treatments to interfere with the formation of the USP1/UAF1 complex (Figure 5). Furthermore, by genetically tagging UAF1 with a fluorescent protein, such as RFP, and using automated microscopy procedures, the assay can be readily adapted to a high throughput format in order to allow a high content screening for molecules that interfere with USP1/UAF1 complex formation.

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.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.

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