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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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Three candidate nuclear localization signals in human USP1.A. onfocal microscopy images showing nuclear localization of GFP-(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs (cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES. Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
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pone-0038570-g001: Three candidate nuclear localization signals in human USP1.A. onfocal microscopy images showing nuclear localization of GFP-(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs (cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES. Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to show the nuclei (DNA panels).

Mentions: Human USP1 is a 785 amino acid protein which, consistent with previous reports [8] localizes to the cell nucleus when expressed as a GFP-tagged protein in 293T cells (Figure 1A, left panels). In addition, immunofluorescence analysis with an anti-USP1 antibody revealed a predominanlty nuclear staining in 293T cells (Figure 1A, right panels) and HeLa cells (data not shown). We have recently reported the presence of an NES in the amino acid sequence of human USP1 [14] but, given the steady state nuclear localization of the protein, any potential nuclear export mediated by the NES appears to be effectively counteracted by rapid nuclear import. However, the USP1 sequences that mediate its import into the nucleus have not been functionally mapped. Using two different web-based NLS prediction programs, PSORT [23] and cNLS mapper [24], we examined USP1 primary amino acid sequence searching for motifs that may constitute nuclear localization signals (NLSs). Three sequences containing short stretches of basic residues were identified by the programs as candidate NLSs (cNLSs) in human USP1 (Figure 1B).


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

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

Three candidate nuclear localization signals in human USP1.A. onfocal microscopy images showing nuclear localization of GFP-(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs (cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES. Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368879&req=5

pone-0038570-g001: Three candidate nuclear localization signals in human USP1.A. onfocal microscopy images showing nuclear localization of GFP-(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs (cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES. Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
Mentions: Human USP1 is a 785 amino acid protein which, consistent with previous reports [8] localizes to the cell nucleus when expressed as a GFP-tagged protein in 293T cells (Figure 1A, left panels). In addition, immunofluorescence analysis with an anti-USP1 antibody revealed a predominanlty nuclear staining in 293T cells (Figure 1A, right panels) and HeLa cells (data not shown). We have recently reported the presence of an NES in the amino acid sequence of human USP1 [14] but, given the steady state nuclear localization of the protein, any potential nuclear export mediated by the NES appears to be effectively counteracted by rapid nuclear import. However, the USP1 sequences that mediate its import into the nucleus have not been functionally mapped. Using two different web-based NLS prediction programs, PSORT [23] and cNLS mapper [24], we examined USP1 primary amino acid sequence searching for motifs that may constitute nuclear localization signals (NLSs). Three sequences containing short stretches of basic residues were identified by the programs as candidate NLSs (cNLSs) in human USP1 (Figure 1B).

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