# The role of weak multivalent interactions and phase separation in SPOP tumor suppressor function

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2021 · $359,000

## Abstract

SUMMARY
Liquid-liquid phase separation (LLPS), i.e. the ability of molecules to condense into liquid-like assemblies,
compartmentalizes cells extensively and impacts many fundamental biological processes. Whether LLPS is
required for function in cells remains largely unclear. One challenge in answering this question arises from the
difficulty in modulating the ability to form condensates without affecting the proteins' function, because
assembly and function are often mediated by the same interactions. It is possible that smaller, discrete
complexes are able to facilitate the function. We will address this question in enzymatically active condensates
of the tumor suppressor speckle-type POZ protein (SPOP). SPOP recruits substrates to a ubiquitin ligase for
ubiquitination. We have recently shown that SPOP and substrates undergo LLPS via weak, multivalent
interactions, which result in their colocalization in active, membraneless organelles. Prostate cancer mutations
blunt the ability of SPOP to phase separate with substrates, leading to their separate localization in cells, to
increased substrate levels, and transformation of susceptible cells.
We have experience in characterizing multivalent, disordered and phase-separating systems, and have built
the necessary in vitro biophysical, biochemical and cell biological approaches and reagents to tackle the above
question. In the proposed work, we will first modulate the material properties of condensates to test the
requirement of fluidity for effective enzymatic activity. Second, we will test whether designed monovalent
substrates, which bind at similar affinities as their multivalent counterparts, can be ubiquitinated effectively in
the absence of phase separation. Third, we will make use of cancer mutations that modulate the formation of
condensates and discrete complexes in opposite directions to test which of the two are the major players in
SPOP function. Forth, we will address the critical question whether the weak interactions that typically mediate
LLPS are able to compartmentalize cells specifically. We will use SPOP endometrial cancer mutations, which
alter substrate specificity, to identify the strongest motifs responsible for the specificity alteration. The results
will provide a conservative measure of specificity-mediating affinities in phase-separating systems.
Our rigorous, multidisciplinary studies will significantly advance the knowledge of the structural determinants of
specificity in weak SPOP/substrate interactions that drive phase separation, of the necessity of phase
separation for SPOP-mediated substrate ubiquitination, and of the biophysical basis for the dysfunction of
several SPOP cancer mutations that are distinct from the well-characterized prostate cancer mutations. The
expected results will therefore provide conceptual insights into the role of phase separation in biological
function. While we use rare cancer-associated mutations mainly as guides towards understanding of...

## Key facts

- **NIH application ID:** 10070106
- **Project number:** 5R01GM112846-07
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** Tanja Mittag
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $359,000
- **Award type:** 5
- **Project period:** 2015-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10070106

## Citation

> US National Institutes of Health, RePORTER application 10070106, The role of weak multivalent interactions and phase separation in SPOP tumor suppressor function (5R01GM112846-07). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10070106. Licensed CC0.

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