Miki Takayuki, Hashimoto Masahiro, Shimizu Masatoshi, Takahashi Hiroki, Mihara Hisakazu
School of Life Science and Technology, Tokyo Institute of Technology 4259 Nagatsuta-cho, Midori-ku Yokohama Kanagawa 226-8501 Japan.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-0033 Japan
Chem Sci. 2025 Jul 11. doi: 10.1039/d5sc00871a.
Protein droplet formation is a crucial process involved in transient cellular responses and pathogenic protein aggregations. Conventionally, the droplet-forming capability of target proteins has been evaluated through reconstitution studies, where purified proteins are dissolved in buffer solutions. However, such droplets are highly sensitive to environmental factors, including temperature, ionic strength, and molecular crowding. Therefore, evaluation within living cells is highly desirable. Additionally, since droplet formation is typically initiated by nucleation involving dynamic protein oligomerization, simply expressing proteins in cells often fails to induce droplet formation, making intracellular evaluation challenging. In this study, we present an intracellular droplet-forming assay based on our peptide tag technique. This system employs short self-assembling YK peptide tags (7-15 residues), genetically fused to target proteins, to artificially induce oligomerization. Using this approach, we discover that the co-chaperone Hsp70/Hsp90 organizing protein possesses droplet-forming capability and identify the essential region required for its droplet formation.
蛋白质液滴形成是参与瞬时细胞反应和致病性蛋白质聚集的关键过程。传统上，目标蛋白质的液滴形成能力是通过重组研究来评估的，即把纯化的蛋白质溶解在缓冲溶液中。然而，这种液滴对环境因素高度敏感，包括温度、离子强度和分子拥挤程度。因此，在活细胞内进行评估非常必要。此外，由于液滴形成通常由涉及动态蛋白质寡聚化的成核作用引发，在细胞中简单表达蛋白质往往无法诱导液滴形成，这使得细胞内评估具有挑战性。在本研究中，我们基于肽标签技术提出了一种细胞内液滴形成测定法。该系统利用与目标蛋白质基因融合的短自组装YK肽标签（7 - 15个残基）来人工诱导寡聚化。通过这种方法，我们发现伴侣蛋白Hsp70/Hsp90组织蛋白具有液滴形成能力，并确定了其液滴形成所需的关键区域。
