Fig. 2: Engineering of the duAb architecture using target-specific peptides.

A Instead of using a YFP Nb, which is not a therapeutically relevant binder, target-specific peptides can instead be leveraged for a more programmable method of protein stabilization. Created in BioRender. Hong, L. (2025) https://BioRender.com/v65m595. B β-catenin-sfGFP stabilization in HEK293T cells comparing the four different DUB domain candidates linked to βcat_SnP_7¹⁰ measured by flow cytometric analysis. Cells were transiently transfected in the presence or absence of 4 μM PR-619 DUB inhibitor. Data are the average of independent replicates (n = 3). C β-catenin-sfGFP stabilization by βcat_SnP_7-linked stabilizers, specifically comparing the βcat_SnP_7-L2-OTUB1 fusion with the OTUB1 C91S and OTUB1 ASA mutants. Data are the average of individual replicates (n = 3). D Time-course experiment demonstrates that potent duAb activity can be achieved within three days of treatment. Data was collected by extracting treated HEK293T cells at t = 6, 12, 24, 36, 48, and 72 h post transfection. E TOP-GFP assay for quantifying Wnt signaling in HEK293T cells²¹. Stabilization of endogenous β-catenin results in higher levels of Wnt signaling and increased GFP levels, measured by flow cytometry. Data are the average of independent replicates (n = 3). Created in BioRender. Hong, L. (2025) https://BioRender.com/v65m595. F TOP-GFP signals in HEK293T cells measured by flow cytometric analysis. Cells were transiently transfected in the presence or absence of 4 μM PR-619 DUB inhibitor. Data are the average of independent replicates (n = 3). G TOP-GFP signals in HEK293T cells comparing the βcat_SnP_7-L2-OTUB1 fusion with the OTUB1 C91S and OTUB1 ASA mutants measured by flow cytometric analysis. Cells were transiently transfected, and data are the average of independent replicates (n = 3). For B, G, normalized cell fluorescence was calculated by dividing the percentage of sfGFP+ cells in a sample by that of (-) DUB with no DUB inhibitor for control cells. Statistical analysis was performed using the two-tailed Student’s t-test using GraphPad Prism 10 software, with calculated p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Samples with p value representations above their respective bars reflect comparisons between the control and that sample; all other p value notations compare those specific samples. H Nano LC-MS/MS analysis of total proteins collected from HEK293T cells co-transfected with plasmids encoding β-catenin-sfGFP and either βcat_SnP_7-L2-OTUB1 or polyG-L2-OTUB1. Data was log2-transformed, and a t-test was performed to generate a volcano plot of differentially abundant proteins. Most notably, both exogenous β-catenin-sfGFP (CTNNB1GFP) and endogenous β-catenin (CTNNB1) were among the few proteins that were abundantly present in the β-catenin-stabilizing duAb samples over the non-targeting duAb control. I Overexpressed β-catenin-sfGFP (CTNNB1GFP) abundances comparing non-targeting vs. β-catenin-stabilizing duAb treatment in HEK293T cells. J Endogenous β-catenin (CTNNB1) abundances comparing non-targeting vs. β-catenin-stabilizing duAb treatment in HEK293T cells. For I, J, statistical analysis was performed using the one-tailed Student’s t-test using GraphPad Prism 10 software, with calculated p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Please refer to source data for numeric p values.