Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.
Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by devel...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2023-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0285964 |
| _version_ | 1797802009900875776 |
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| author | Caroline N Smith Kyle Kihn Zachary A Williamson K Martin Chow Louis B Hersh Konstantin V Korotkov Daniel Deredge Jessica S Blackburn |
| author_facet | Caroline N Smith Kyle Kihn Zachary A Williamson K Martin Chow Louis B Hersh Konstantin V Korotkov Daniel Deredge Jessica S Blackburn |
| author_sort | Caroline N Smith |
| collection | DOAJ |
| description | Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression. |
| first_indexed | 2024-03-13T04:59:14Z |
| format | Article |
| id | doaj.art-f688ec8b8a0a4c2b94b6ee1357dbd491 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-03-13T04:59:14Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-f688ec8b8a0a4c2b94b6ee1357dbd4912023-06-17T05:32:01ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-01185e028596410.1371/journal.pone.0285964Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.Caroline N SmithKyle KihnZachary A WilliamsonK Martin ChowLouis B HershKonstantin V KorotkovDaniel DeredgeJessica S BlackburnPhosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.https://doi.org/10.1371/journal.pone.0285964 |
| spellingShingle | Caroline N Smith Kyle Kihn Zachary A Williamson K Martin Chow Louis B Hersh Konstantin V Korotkov Daniel Deredge Jessica S Blackburn Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. PLoS ONE |
| title | Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. |
| title_full | Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. |
| title_fullStr | Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. |
| title_full_unstemmed | Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. |
| title_short | Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3. |
| title_sort | development and characterization of nanobodies that specifically target the oncogenic phosphatase of regenerating liver 3 prl 3 and impact its interaction with a known binding partner cnnm3 |
| url | https://doi.org/10.1371/journal.pone.0285964 |
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