Investigating specific binding to neurodegenerative disease-related proteins in cell models towards developing targeted protein degradation tools

BBSRC strategic theme: Bioscience for an integrated understanding of health

Background: Biomolecular condensates (BCs) are discrete membraneless subcellular compartments that provide efficient and reversible spatiotemporal control of cellular processes in an energy-dependent manner. BCs facilitate a plethora of physiological processes due to their propensity to act as reaction crucibles by increasing the local concentrations of substrates and effectors such as enzymes, and their capacity to undergo dynamic exchange with the surrounding intracellular milieu.

In contrast, in dysregulated phase separation (LLPS), BCs lose their dynamic nature such that there is progressive transformation from liquid-like states to gel-like and solid states. There is therefore increasing evidence that aberrant LLPS leads to pathological protein aggregates such as disease-related aggregates seen in neurodegenerative disorders.

Recent evidence has therefore prompted the search for therapeutic interventions aimed at modulating LLPS and restoring normal physiological conditions.

The overarching aim of the PhD project will be to apply designer LLPS-CTPRs to understand the molecular mechanisms by which neurodegenerative proteins behave in physiological LLPS environment and how protein mutations dysregulate the LLPS environment leading to irreversible aggregation. Aim 1: Use designer biomolecular condensates to understand how disease-related proteins behave in an LLPS environment.

This will involve: a. in vitro characterisation of BCs formed from LLPS-CTPRs that can recruit a-synuclein and TDP-43 in condensate formation with the aim of determining the biophysical properties of droplets.

b. in cellulo characterisation of protein droplet formation using SH-SY5Y cells overexpressing fluorescent-tagged a-synuclein and a TDP-43 tagged cell model of ALS developed by the Kumita lab.

Aim 2: Determine how mutations in disease-related proteins accelerate transition from reversible LLPS to irreversible protein aggregation.

In clonal cell lines that express a-synuclein and TDP-43 mutations I will repeat the same characterisation protocols to investigate changes in structural features within the LLPS/aggregate systems. This will allow for comparison of protein recruitment, and droplet formation between physiological and pathological states.

Aim 3: Determine the therapeutic potential of using engineered proteins to enhance the degradation of disease-related proteins.

Key information obtained from in vitro and in cellulo characterisation of droplet properties will direct reconciliation of protein recruitment with efficient targeted degradation. Once disease-related protein recruitment in LLPS formation is better understood, targeted degradation strategies being developed in the Kumita group can be utilised to determine if disease-related BCs can be targeted to the autophagy pathway.

As aberrantly misfolded proteins are a pathological hallmark of neurodegenerative diseases, removal of these species through facilitated protein degradation may prove beneficial for therapeutic intervention.

The results from this study will yield significant advances in understanding the underlying molecular mechanisms that link the beneficial LLPS formation with pathological amyloid formation. This will enable the development of therapeutic intervention strategies, such as targeted degradation of disease-related proteins.