Le Quang D, Lewis Amanda, Dix-Matthews Alice, Ringler Philippe, Duff Anthony, Whitten Andrew E, Atkin Rob, Brunner Manuel, Ho Diwei, Iyer K Swaminathan, Marshall Andrew C, Fox Archa H, Bond Charles S
School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia.
Faculty of Biology, VNU University of Science, 334-Nguyen Trai Street, Ha Noi 100000, Vietnam.
Int J Mol Sci. 2025 Feb 5;26(3):1356. doi: 10.3390/ijms26031356.
Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein containing low-complexity domains (LCDs), which are known to regulate protein behavior under stress conditions. This study demonstrates the ability to control hnRNPK's transitions into four distinct material states-monomer, soluble aggregate, liquid droplet, and fibrillar hydrogel-by modulating environmental factors such as temperature and protein concentration. Importantly, the phase-separated and hydrogel states are newly identified for eGFP-hnRNPK, marking a significant advancement in understanding its material properties. A combination of biophysical techniques, including DLS and SEC-LS, were used to further characterize hnRNPK in monomeric and soluble aggregate states. Structural methods, such as SANS, SAXS, and TEM, revealed the elongated morphology of the hnRNPK monomer. Environmental perturbations, such as decreased temperature or crowding agents, drove hnRNPK into phase-separated or gel-like states, each with distinct biophysical characteristics. These novel states were further analyzed using SEM, X-ray diffraction, and fluorescence microscopy. Collectively, these results demonstrate the complex behaviors of hnRNPK under different conditions and illustrate the properties of the protein in each material state. Transitions of hnRNPK upon condition changes could potentially affect functions of hnRNPK, playing a significant role in regulation of hnRNPK-involved processes in the cell.
异质性核核糖核蛋白K（hnRNPK）是一种含有低复杂性结构域（LCDs）的RNA结合蛋白，已知这些结构域在应激条件下调节蛋白质行为。本研究表明，通过调节温度和蛋白质浓度等环境因素，能够控制hnRNPK转变为四种不同的物质状态——单体、可溶性聚集体、液滴和纤维状水凝胶。重要的是，对于eGFP-hnRNPK，新发现了相分离状态和水凝胶状态，这标志着在理解其物质特性方面取得了重大进展。使用包括动态光散射（DLS）和尺寸排阻色谱-激光散射（SEC-LS）在内的多种生物物理技术，进一步表征了单体和可溶性聚集体状态下的hnRNPK。诸如小角中子散射（SANS）、小角X射线散射（SAXS）和透射电子显微镜（TEM）等结构方法，揭示了hnRNPK单体的细长形态。诸如温度降低或拥挤剂等环境扰动，促使hnRNPK进入相分离或凝胶状状态，每种状态都具有独特的生物物理特征。使用扫描电子显微镜（SEM）、X射线衍射和荧光显微镜对这些新状态进行了进一步分析。总体而言，这些结果证明了hnRNPK在不同条件下的复杂行为，并阐明了该蛋白质在每种物质状态下的特性。条件变化时hnRNPK的转变可能会潜在地影响hnRNPK的功能，在细胞中hnRNPK参与的过程的调节中发挥重要作用。
