Introduction: Transactive response DNA-binding protein 43 kDa (TDP-43) is a nuclear RNA processing protein whose cytoplasmic aggregates are hallmarks of various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Emerging evidence indicates that the ubiquitin-like N-terminal domain (NTD) does not mediate aggregate formation but may antagonise it by driving the assembly of protective higher-order oligomers (Afroz et al. 2017; Jiang et al. 2017). Probing of binding sites on the NTD has identified a novel ligand (nTRD22) that allosterically modulates RNA binding and increases TDP-43 turnover (Mollasalehi et al. 2020). Such ligands can form the basis of our efforts to synthesise bifunctional molecules which can trigger the degradation of aberrant TDP-43 via the ubiquitin-proteasome pathway.
Aims: This study aims to (a) express and purify the NTD of TDP-43, (b) structurally assess the produced NTD to delineate the dimerisation and aggregation potential, and (c) use native mass spectrometry (MS) to detect secondary complexes of the NTD with known ligands.
Methods, Results and Discussion: We expressed His-SUMO-tagged NTD in BL21 E. coli and purified using immobilised metal affinity and size exclusion chromatography to produce stable and pure (>1 mg/mL) concentrates of non-aggregating NTD. We are presently optimising conditions for native (MS) to visualise secondary complexes formed by protein with both nTRD22 and derived analogues. Preliminary data suggests that our purified NTD replicates the monomer-dimer equilibrium observed in vivo and thus constitutes a clinically relevant protein target for further studies.
Future Directions: Using the NTD, we will perform fragment-based ligand discovery using nuclear magnetic resonance spectroscopy to identify novel ligands and apply native MS to validate ligand binding. As the wider project aims to develop bifunctional drugs that expedite the proteasomal degradation of TDP-43, we will further apply established native MS methods to detect the ternary complexes of NTD, degrader candidates and proteasome-associated E3 ligases.