# Chemically Probing and Regulating Misfolding and Aggregation of Intrinsically Disordered Proteins in Membraneless Organelles

> **NIH NIH R35** · PENNSYLVANIA STATE UNIVERSITY, THE · 2021 · $391,596

## Abstract

PROJECT SUMMARY
Membraneless organelles have important functions in cellular physiology and pathology. Recent studies show
that these organelles are formed through liquid-liquid phase separation of intrinsically disordered proteins (IDPs)
and RNA molecules. IDPs phase separate into liquid droplets in test tubes and form P bodies or stress granules
in stressed cells. Both mutant and wild type forms of several IDPs are found aggregated in neurons and
associated with neurodegenerative disorders. However, very little is known about how IDPs misfold and
aggregate in these organelles and how this process can be regulated. Lack of this knowledge is attributed to the
current method that is used to monitor membraneless organelles in live cells: this process is visualized through
imaging fluorescent protein-tagged IDPs to analyze changes of their location and diffusion rate before and after
organelle formation. Nonetheless, this method does not reveal whether IDPs misfold or aggregate within the
organelle, because the morphology remains unchanged before and after IDPs aggregation. To overcome this
challenge, the PI has developed a novel imaging method, hereinafter named AggTag (aggregation tag), to
enable fluorogenic detection (turn-on fluorescence) of misfolded soluble oligomers both in test tubes and live
cells. In this MIRA proposal, the PI plans to further develop the AggTag method with new probes that can
distinguish soluble oligomers from insoluble aggregates using orthogonal fluorescent signals (Project 1). This
unprecedented resolution will allow the PI to ask how IDPs misfold and aggregate in phase separated droplets.
The PI have begun this direction with a focus on a group of intrinsically disordered RNA binding proteins (RBPs),
which harbor RNA binding domains (RBD) and disordered prion-like domains (PLD). While PLD has been the
primary focus in literatures, preliminary data have led to a novel hypothesis that whether RBD misfolds
contributes to whether RBP misfolds during and after formation of droplets. This hypothesis will be tested both
in vitro and in live cells, using a combination of the AggTag method and biochemical assays (Project 2). Finally,
the PI will develop chemical strategies to control phase separation and membraneless organelles. Although
LLPS can be prevented and dissolved by small molecules, disruption of the liquid droplets could obstruct their
physiological functions. Till now, no small molecules have been discovered to promote formation of liquid
droplets and prevent RBP misfolding. Preliminary data indicate that sugar phosphates are a novel class of
molecules that promote droplet formation, stabilize liquid droplets, and prevent RBP misfolding in droplets. Based
on these results, the PI will use efforts from multiple disciplines to understand the mechanisms underlying the
observed effects of sugar phosphates and further develop them into a class of chemical regulators with proper
selectivity and efficacy (Project 3). In s...

## Key facts

- **NIH application ID:** 10207682
- **Project number:** 5R35GM133484-03
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** Xin Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $391,596
- **Award type:** 5
- **Project period:** 2019-09-01 → 2022-09-22

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10207682

## Citation

> US National Institutes of Health, RePORTER application 10207682, Chemically Probing and Regulating Misfolding and Aggregation of Intrinsically Disordered Proteins in Membraneless Organelles (5R35GM133484-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10207682. Licensed CC0.

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