# The Role and Mechanisms of UBQLN2-mediated Phase Transitions in the Assembly and Disassembly of Biomolecular Condensates

> **NIH NIH R01** · SYRACUSE UNIVERSITY · 2022 · $300,000

## Abstract

PROJECT SUMMARY/ABSTRACT:
Biomolecular condensates are dynamic, membraneless compartments that spatiotemporally regulate a
myriad of cellular functions from gene transcription to cellular stress response. Liquid-liquid phase
separation (LLPS) is increasingly appreciated as the biophysical mechanism for how these condensates
assemble. Key to proper condensate function is the maintenance of their dynamics and
assembly/disassembly processes, but little is known about these mechanisms. Hints are provided from
disease states whereby condensates may undergo liquid-to-solid transitions into cytoplasmic inclusions
that contain protein quality control components and are characteristic of proteinopathies such as
amyotrophic lateral sclerosis. We have identified UBQLN2, a member of ubiquitin-mediated protein
quality control systems, as a contributor to condensate function. We recently showed that UBQLN2 forms
condensates in vitro, and is recruited to stress granules, cytoplasmic condensates that form in response
to stress. The multitude of UBQLN2 functions are driven through interactions with proteasomal subunits,
polyubiquitin chains, and client proteins. Ubiquitin and polyubiquitin, biological signals for the
maintenance of protein homeostasis through degradation and autophagy, drive disassembly of UBQLN2
condensates in vitro. These observations have broad implications for how phase separation mechanisms
regulate the function of protein quality control systems. In this project, we aim to identify the molecular
and cellular mechanisms that drive how UBQLN2 condensates assemble and disassemble. Aim 1
determines how domain-domain interactions promote or inhibit phase separation of UBQLN2 via
construction of phase diagrams for constructs from a combination of UBQLN2 domain deletion and
disease-linked mutations. These domain deletions will be used to mimic the different “states” of UBQLN2
when specific domains are engaged with binding partners and unable to contribute to LLPS. We will use
UBQLN2 disease-linked mutations as a nature-provided library to elucidate how intra- and intermolecular
UBQLN2 interactions promote or inhibit condensate assembly and alter condensate morphology and
material properties both in vitro and in mammalian cell culture models. Aim 2 quantifies how UBQLN2
condensates are affected by UBQLN2 engagement with protein quality control components, including
proteasomal receptors, client proteins, and different types of polyubiquitin chains. We monitor these
effects in vitro and with designed mutants in vivo. Importantly, we develop a reconstituted UBQLN2
condensate model to quantify the parameters of how polyubiquitin and polyubiquitinated substrates
engage with UBQLN2 to disassemble condensates. These studies will lay the foundation for determining
the physiological roles of phase separation as it pertains to protein homeostasis through ubiquitin-
mediated pathways.

## Key facts

- **NIH application ID:** 10334494
- **Project number:** 5R01GM136946-03
- **Recipient organization:** SYRACUSE UNIVERSITY
- **Principal Investigator:** Carlos Antonio Castaneda
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $300,000
- **Award type:** 5
- **Project period:** 2020-04-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10334494

## Citation

> US National Institutes of Health, RePORTER application 10334494, The Role and Mechanisms of UBQLN2-mediated Phase Transitions in the Assembly and Disassembly of Biomolecular Condensates (5R01GM136946-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10334494. Licensed CC0.

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