# Phosphorylation as a site-specific regulatory mechanism of the liquid-liquid phase separation of TDP-43 low complexity domain.

> **NIH NIH F30** · CASE WESTERN RESERVE UNIVERSITY · 2022 · $51,273

## Abstract

Project Summary
 The TAR DNA-binding protein of 43 kDa (TDP-43) is a nucleic-acid binding protein whose fragments have
been detected in pathological amyloid inclusions in the brains of patients with age-related neurodegenerative
disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration.
These fragments contain a region of TDP-43 called the low complexity domain (LCD), which has low amino acid
diversity and a propensity to form amyloid aggregates. The LCD also drives TDP-43 liquid-liquid phase
separation (LLPS), a phenomenon in which the protein self-associates into a reversible, dynamic, droplet-like
phase. LLPS is a physiologic process that mediates several functions of TDP-43, including its involvement in the
cellular stress response, but prolonged LLPS has been shown to promote pathological aggregation,
underscoring the need to understand how this process is regulated in vivo.
 Phosphorylation of TDP-43 LCD has been well-documented in Alzheimer’s disease and other age-related
neurodegenerative disorders. Though the role of this modification remains largely unexplored for TDP-43, it does
influence LLPS in similar RNA-binding proteins such as FUS. To determine if LCD phosphorylation can regulate
TDP-43 LLPS, phosphomimetic variants of the LCD with Ser->Asp substitutions at known phosphorylation sites
were prepared. Phase separation of variants was studied using light-scattering and microscopy, and it was found
the phosphomimetic substitutions dramatically modulated LLPS in a site-specific manner. LCD variants with
phosphomimetic substitutions in the α-helical region, a part of the protein involved in protein-protein contacts,
displayed reduced LLPS-propensity; conversely, LCD variants with phosphomimetic substitutions in the C-
terminal region (CTR), which is known to be phosphorylated in disease, displayed enhanced LLPS-propensity.
 To elucidate the mechanisms behind this site-specific modulation of LLPS, we propose a three-pronged
approach using nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR)
spectroscopy and fluorescence resonance energy transfer (FRET). NMR will be employed to determine local
secondary structure of the phosphomimetic α-helical variant. As the α-helical region is known to be altered by
oligomerization, EPR will be used concurrently to see if the α-helical phosphomimetic substitution changes the
oligomerization behavior of this variant. Preliminary data suggest the CTR variants have higher LLPS-
propensities because of an intermolecular electrostatic interaction created by the phosphomimetic substitution
at the CTR. Our second aim will thus use FRET to verify the presence of this hypothesized intermolecular
interaction and probe which residues contribute to it. Furthermore, fluorescence recovery after photobleaching
(FRAP) will be employed to determine how these phosphomimetic substitutions alter the material properties of
TDP-43 LCD drop...

## Key facts

- **NIH application ID:** 10401259
- **Project number:** 5F30AG071339-02
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** Raza Haider
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $51,273
- **Award type:** 5
- **Project period:** 2021-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10401259

## Citation

> US National Institutes of Health, RePORTER application 10401259, Phosphorylation as a site-specific regulatory mechanism of the liquid-liquid phase separation of TDP-43 low complexity domain. (5F30AG071339-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10401259. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*