A structural proteomics approach to quantify protein reorganization during TDP-43 liquid-to-solid phase transitions

Neurodegenerative disorders such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) are characterized by an accumulation of protein aggregates in the affected brain regions. These protein aggregates are thought to arise from biomolecular condensates undergoing a liquid-to-solid phase transition.

Despite the significant achievement in the identification of proteins partitioning into biomolecular condensates and the driving forces behind phase separation and aggregate formation, the molecular interactions regulating condensates/aggregates remains elusive. The primary factors contributing to this knowledge gap include the lack of a defined 3D structure for the flexible proteins involved and the absence of biochemical tools to quantify protein rearrangements during the liquid-to-solid condensate transition.

To gain insight into the relationship between structure and physical properties, we study TDP-43 as a protein model. Although neuropathological studies have linked TDP-43 mis-localization and aggregation to major neurodegenerative diseases (ALS, FTD and AD), the knowledge about TDP-43"s structural organization is still limited to few protein domains/regions and cannot recapitulate the dynamic re-arrangement of the whole protein during the aggregation process. Combining multiple structural proteomics approaches (crosslinking coupled to mass spectrometry, limited proteolysis, and native mass spectrometry) we probed TDP-43 protein conformational states in-vitro using a set of TDP-43 variants characterized by different condensation properties (wild-type, condensation deficient or aggregation-prone TDP-43 variants). The generated data provide insight into intra-molecular structural rearrangements occurring in TDP-43 condensates and their interconnection with biophysical properties. We identified that the increase flexibility in the C-terminal IDR of the protein foster the interaction between the IDR C terminal region and the RRM1 and RRM2 regulating the process of protein rearrangement within liquid and solid-like condensates.

By applying this panel of structural techniques to the field of neurodegeneration and in vitro and in-vivo condensation/aggregation systems, we aim to describe the molecular mechanisms regulating the aggregation processes, pinpointing interactions uniquely identified in pathological states, and offering potential targets for intervention strategies in neurodegenerative diseases.