WDR20 regulates shuttling of the USP12 deubiquitinase complex between the plasma membrane, cytoplasm and nucleus

Olazabal‐Herrero, Anne; Sendino, Maria; Arganda‐Carreras, Ignacio; Rodríguez, José Antonio

doi:10.1016/j.ejcb.2018.10.003

Cited by 10 publications

(18 citation statements)

References 38 publications

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“…1B, WDR20 residues F262 and W306 are conserved in DMWD, corresponding to positions F326 and W370, respectively. On the other hand, the WDR20 450‐MDGAIASGVSKFATLSLHD‐468 motif has been shown to function as a nuclear export signal (NES) that mediates XPO1‐dependent export of WDR20 to the cytoplasm [23]. Our alignment revealed a partial conservation of this motif in DMWD, with sequence similarity limited to the C‐terminal half of the NES.…”

Section: Resultsmentioning

confidence: 85%

“…Besides the DUB complex partners (USP12, USP46, and WDR48), shared interactors included the phosphatases PHLPP1 and PHLPP2, the adapter protein YWHAH, and the nuclear export receptor XPO1. Of note, we have previously reported that XPO1 mediates the active export of WDR20 to the cytoplasm [23] through a NES motif that is partially conserved in DMWD (Fig. 1B).…”

Section: Resultsmentioning

confidence: 98%

“…Importantly, the regulatory effect of WDR proteins on USP12 and USP46 extends beyond increasing their enzymatic activity. Thus, we have recently shown that WDR20 mediates XPO1‐dependent nuclear export of USP12 and promotes its relocation to the plasma membrane (PM) [23].…”

Section: Introductionmentioning

confidence: 99%

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The dystrophia myotonica WD repeat‐containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase

et al. 2021

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Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of in silico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope‐tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 98%

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The dystrophia myotonica WD repeat‐containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase

et al. 2021

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“…To this end, optical sectioning images (channels with SRV B/A reporters and YFP-CRM1) or conventional fluorescence images (DAPI-stained nuclei) of 10-15 different areas in each sample were taken using a Zeiss Apotome2 microscope and Zen2.6 Blue edition software. Composite images were created using Fiji software (Schindelin et al, 2012), and analyzed using an ad-hoc script developed previously (Olazabal-Herrero et al, 2019) to automatically quantify the fluorescence intensity in nuclear and cytoplasmic regions. Finally, the nuclear to cytoplasmic (N/C) ratios were calculated, plotted in logarithmic base 2, and samples were compared using the Mann-Whitney U test (GraphPad Prism 7 software).…”

Section: Methodsmentioning

confidence: 99%

Using a simple cellular assay to map NES motifs in cancer-related proteins, gain insight into CRM1-mediated NES export, and search for NES-harbouring micropeptides

Sendino

Omaetxebarria

Rodriguez

2020

Preprint

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The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the 'NES-binding groove' of CRM1 has been studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harbouring human micropeptides.

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“…To this end, optical sectioning images (channels with SRV B/A reporters and YFP-CRM1) or conventional fluorescence images (DAPI-stained nuclei) of 10-15 different areas in each sample were taken using a Zeiss Apotome2 microscope and Zen2.6 Blue edition software. Composite images were created using Fiji software [44], and analyzed using an ad-hoc script developed previously [45] to automatically quantify the fluorescence intensity in nuclear and cytoplasmic regions. Finally, the nuclear to cytoplasmic (N/C) ratios were calculated, plotted in logarithmic base 2, and samples were compared using the Mann-Whitney U test (GraphPad Prism software version 7, San Diego, CA, USA).…”

Section: Rev(14)-gfp and Srv B/a Nuclear Export Assaysmentioning

confidence: 99%

Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides

Sendino

Omaetxebarria

Rodríguez

2020

IJMS

Self Cite

View full text Add to dashboard Cite

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.

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WDR20 regulates shuttling of the USP12 deubiquitinase complex between the plasma membrane, cytoplasm and nucleus

Cited by 10 publications

References 38 publications

The dystrophia myotonica WD repeat‐containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase

The dystrophia myotonica WD repeat‐containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase

Using a simple cellular assay to map NES motifs in cancer-related proteins, gain insight into CRM1-mediated NES export, and search for NES-harbouring micropeptides

Using a Simple Cellular Assay to Map NES Motifs in Cancer-Related Proteins, Gain Insight into CRM1-Mediated NES Export, and Search for NES-Harboring Micropeptides

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