Development and characterization of nanobodies specifically targeting the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3)
| Autor: | Min Wei, Jessica S. Blackburn, Yelena Chernyavskaya, K. Martin Chow, Zachary A. Williamson, Konstantin V. Korotkov, Caroline N. Smith, Louis B. Hersh |
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| Rok vydání: | 2020 |
| Předmět: |
endocrine system
endocrine system diseases medicine.diagnostic_test Phosphatase Cancer Protein tyrosine phosphatase Biology medicine.disease Immunofluorescence Metastasis Tumor progression Cancer cell Dual-specificity phosphatase Cancer research medicine biology.protein hormones hormone substitutes and hormone antagonists |
| DOI: | 10.1101/2020.10.02.311787 |
| Popis: | Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis in various solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. In particular, small molecules do not exhibit binding specificity for PRL-3 over highly homologous family members PRL-1 and PRL-2, and antibodies directed against PRL-3 are limited by assay type. 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 PRL-1 and PRL-2. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and co-immunoprecipitation with a known PRL-3 substrate showed the nanobodies bind PRL-3 outside of the active site, meaning they can be used to study PRL-3 interaction with binding partners. The nanobodies were also specific to PRL-3 over other PRLs in immunoprecipitation and immunofluorescence experiments in human cancer cells that overexpressed the PRL family. We found that 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 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. |
| Databáze: | OpenAIRE |
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