Abstract
BDPIC (bromoTAG-dTAG heterobifunctional chimera) is a novel heterobifunctional molecule which recruits two known protein tags, dTAG and bromoTAG. It can recruit together any protein of interest(POI) and post-translational modification (PTM)-inducing enzyme where one is tagged with dTAG and the other bromoTAG, forming a ternary complex. This would induce proximity between the PTM-inducing enzyme and the POI, allowing its activity to target the POI specifically. This allows any PTM, such as dephosphorylation, phosphorylation, ubiquitination, and others to be induced and investigated. This is a useful proof of concept tool and enables investigation of the potential downstream effects of inducing any PTM as a therapeutic modality.Hyperphosphorylation of TAU is a key stage in the formation of TAU plaques and tangles in tauopathies, including Alzheimer's disease and many others. The concept of designing drugs for targeted dephosphorylation of hyperphosphorylated TAU in Alzheimer’s disease has been poorly explored. Developing a heterobifunctional molecule that recruits phosphatases to hyperphosphorylated TAU has potential as a novel therapeutic treatment and may potentially reverse TAU pathologies. BDPIC was used as a proof-of concept to demonstrate that a heterobifunctional molecule proximity-induction method can recruit dTAG-PPP2CA (protein phosphatase 2 catalytic subunit alpha), a phosphatase with known tau dephosphorylation activity, to induce targeted dephosphorylation of TAU-bromoTAG.
In this thesis, it was demonstrated that in CRISPR-Cas9 knockin SK-N-MC cells expressing TAU-bromoTAG and dTAG-PPP2CA at the endogenous level, BDPIC treatment led to a decrease in phosphorylation levels of TAU. The optimal concentrations and timescales for induced dephosphorylation were evaluated. Further experiments were conducted to confirm ternary complex formation by BDPIC, and that the decrease in phosphorylation was indeed due to the dTAG-PPP2CA activity within the ternary complex.
| Date of Award | 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Gopal Sapkota (Supervisor) |