A tool to dissect heterotypic determinants of homotypic protein phase behavior

https://doi.org/10.1101/2025.01.01.631016

 

Abstract

Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each protein’s sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.Lay summary Protein self-assemblies are essential for cellular function, but can also contribute to disease. We develop a new tool to study how their formation is influenced by other cellular factors. This tool allows us to control the location and number of interactions between a protein of interest and other proteins that may influence it. Our results provide new insight into mechanisms of self-assembly and will aid research toward treating diseases associated with aberrant assembly.

 

요약

 

1. 새로운 단백질 상분리 분석 도구 개발: 단백질의 상분리(phase separation) 및 자가응집(self-assembly)을 분석하기 위한 mEos3 결합 나노바디(mEosNb) 기반 도구를 개발했습니다.
2. 나노바디를 활용한 단백질 응집체 형성 기작 연구: mEosNb를 통해 특정 단백질을 mEos3에 선택적으로 결합시켜 heterotypic 상호작용을 유도하고, LLPS(액-액 상분리)와 아밀로이드 핵형성(amyloid nucleation) 메커니즘을 비교 분석했습니다.
3. 상분리 및 응집체 형성에 미치는 다가성(valency)의 영향: mEos3와 mEosNb의 다가성을 조절하여, 단백질 농도와 결합 부위 수가 상분리에 미치는 영향을 체계적으로 분석하였습니다.
4. 단백질 상분리 핵형성 기작 규명: ASC는 농도 의존적 핵형성(concentration-dependent nucleation)을 보였으나, SesA는 특정 구조적 전이(conformational transition)에 의해 핵형성이 시작됨을 발견했습니다.
5. 질환 연구 및 생명공학적 응용 가능성: 이 연구에서 개발된 mEosNb 기반 DAmFRET 도구는 알츠하이머병, 파킨슨병 등 아밀로이드 기반 질환 연구에 적용될 수 있습니다.