Affinity-based antibody-specification for characterization of PKA-isoforms in hyperalgesic signaling in nerve terminals in vitro and in vivo

• Applicants: Professor Dr. Friedrich-Wilhelm Herberg; Professor Dr. Tim Hucho
• Subject Area: Pharmacology ; Cell Biology
• Term: from 2021 to 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 459759629

• Final Report Year: 2025

Final Report Abstract

Antibodies are widely used in life-sciences and medical therapy. However, broadly applied antibody-based methods to localise protein targets in cells or tissues such as immunocyto- or histochemistry lack the possibility to determine antibody specificity. Therefore, we developed a simple-to-use approach to characterize and quantify antibody binding properties in a cellular system of choice. Based on Lagergren’s pseudo first order model, we developed a model of antibody accumulation, which we have used to construct a computational tool (Julia packages) to analyse quantitative image data from antibody dilution series. This tool improves the selection of specific antibodies, optimizes the choice of a dilution to maximize signal-specificity, and expands the scope of antibody-based imaging to detect changes of the subcellular nano-environment. In a next step, we were aiming to apply this tool to select the most specific nanobodies raised against isoforms of the protein kinase A (PKA) and to detect potential activity dependent conformational changes in the tetrameric PKA holoenzyme isoforms. For this, we expressed and purified the 4 different PKA regulatory subunit isoforms (RIα, RIβ, RIIα, RIIβ), immunized alpacas, and generated a phagemid library encoding for the respective nanobodies. PKA-isoform specific nanobodies were selected by phage display and screened by ELISA assay on immobilized R-subunits. After sequencing and clustering of the hits, we selected 10 nanobodies for further characterization in cells by quantitative High content imaging. Four of the nanobodies showed isoform-selective binding to PKA-RIβ in transfected HeLa cells and produced a neuronal staining pattern in primary sensory neurons. None of these RIβ-specific nanobodies showed changes in signal intensity following activation of all PKA isoforms by cAMP stimulation, suggesting that they are not suitable for the isoform-specific visualization of PKA-RIβ activity in cells. However, they influence the interaction between the RIβ- and the catalytic (C)- subunits and therefore affected holoenzyme formation. Pre-incubation of RIβ-subunits with the respective nanobodies increased the number of RIβ-subunits required to inhibit the activity of C-subunits suggesting that all four RIβ-nanobodies interfere with holoenzyme formation. In further studies, we aim to identify the binding sites of the nanobodies and to investigate whether they modulate PKA-RIβ activity or can be used to localize endogenous PKA-RIβ in primary neurons.

Publications

• Drugging the Undruggable: How Isoquinolines and PKA Initiated the Era of Designed Protein Kinase Inhibitor Therapeutics. Biochemistry, 60(46), 3470-3484.
  Lorenz, Robin; Wu, Jian; Herberg, Friedrich W.; Taylor, Susan S. & Engh, Richard A.
  
• Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinase A. IUBMB Life, 75(4), 311-323.
  Taylor, Susan S.; Herberg, Friedrich W.; Veglia, Gianluigi & Wu, Jian
  
• Modelling the Effect of Antibody Depletion on Dose-Response Behavior for Common Immunostaining Protocols
  Tschimmel, Dominik, Steffen Waldherr & Tim Hucho
  
• Protein-Environment-Sensitive Computational Epitope Accessibility Analysis from Antibody Dose-Response Data
  Tschimmel, Dominik, Momina Saeed, Maria Milani, Steffen Waldherr & Tim Hucho