THE DEVELOPMENT AND CHARACTERIZATION OF NANOBODIES SPECIFIC TO PROTEIN TYROSINE PHOSPHATASE 4A3 (PTP4A3/PRL-3) TO DISSECT AND TARGET ITS ROLE IN CANCER.

Author

Caroline Smith, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0002-1876-7790

Date Available

5-9-2023

Year of Publication

2023

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Jessica Blackburn

Abstract

Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of 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. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only new antibody specific for PRL-3, PRL-3-zumab, is inaccessible to the research community while in clinical trial.

To address the lack of tools available to study PRL-3, I have developed alpaca-derived single-domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics. Their advantages include their small size and lack of light chains, allowing them to reach cavities within active sites that conventional antibodies cannot access. Nanobodies also have high specificity and affinity for their antigens. I hypothesized that PRL-3 nanobodies may be able to differentiate between the PRL family and provide new insights into the role of PRL-3 in cancer progression.

Following synthesis, sequencing, and purification, I identified seven unique nanobodies that bind to PRL-3 with little to no activity towards PRL-1 and PRL-2, making them one of the first tools to selectively target and bind directly to PRL-3 in its native conformation. The nanobodies can be used in immunoprecipitation and immunofluorescence assays. Interestingly, I found that N-terminal tags on PRL-3, such as 3XFLAG or GFP, enhanced PRL-3 localization to the membrane, while untagged PRL-3 is widely distributed throughout the cell. These data may have important implications for previous PRL-3 functional studies that necessarily relied on tagged-PRL-3.

The nanobody binding affinity for PRL-3 is within a KD of 30 - 300 nM, similar to that of commercially available antibodies. Hydrogen-deuterium exchange mass spectrometry showed nanobodies bind near the PRL-3 active site and could reduce PRL-3 phosphatase activity against a generic substrate. The interaction between PRL-3 and the nanobody also showed overlap with the binding site of a known PRL-3 interacting partner, the magnesium transport protein CNNM3. A competition assay showed that the nanobody and CNNM3 can bind PRL-3 simultaneously, but the nanobody partially outcompetes the CNNM3 binding.

The nanobodies can be used immediately as a PRL-3 specific research tool and can be further developed as an inhibitor. They also have a great deal of potential in additional applications. First, we are using these nanobodies to stabilize PRL-3 in X-ray crystallography to develop higher-resolution structures to establish exactly where the nanobodies bind PRL-3. Determining the location of these binding pockets would allow us to better contribute to substrate identification and drug design. Secondly, we are using a fluorescently labeled nanobody to examine the PRL-3:CNNM complex, PRL-3 trafficking and function during cancer processes, such as proliferation, invasion, and stress. Our ultimate goal is to provide new insight into how PRL-3 contributes to cancer progression with the use of this exciting tool.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.159

Funding Information

NCI R37CA227656 (2022)

NIH DP2CA228043 (2020)

GMaP Region 1 North Stimulus Award (2020 - 2021)

Recommended Citation

Smith, Caroline, "THE DEVELOPMENT AND CHARACTERIZATION OF NANOBODIES SPECIFIC TO PROTEIN TYROSINE PHOSPHATASE 4A3 (PTP4A3/PRL-3) TO DISSECT AND TARGET ITS ROLE IN CANCER." (2023). Theses and Dissertations--Molecular and Cellular Biochemistry. 63.
https://uknowledge.uky.edu/biochem_etds/63