Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.
Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.
J Mol Biol. 2020 Mar 27;432(7):2319-2348. doi: 10.1016/j.jmb.2020.02.030. Epub 2020 Mar 3.
Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.
脑表达 X 连锁（BEX）蛋白家族在人类中由五个成员组成，在神经元发育过程中高度表达。它们已知参与细胞周期和参与神经变性和癌症的信号通路。BEX3 具有保守的富含亮氨酸的核输出信号，实验数据证实了 BEX3 的核质穿梭。先前的数据表明，小鼠 BEX3 在富含固有无序的寡聚体中自动缔合。在这项工作中，我们表明，在 tRNA（tRFs）的小片段存在下，人 BEX3（hBEX3）具有明确的三维结构。相反，核酸游离的纯化 hBEX3 呈现无序结构。小角度 X 射线散射数据显示，在 tRFs 的存在下，hBEX3 采用紧凑的球状折叠，与由纯蛋白形成的伸长的高阶寡聚物非常不同。此外，显微镜显示 hBEX3 在体外经历富含蛋白质的微米级液滴的浓缩。在 tRFs 的存在下，生物分子凝聚体更小且数量更多，显示吖啶橙绿色荧光发射，这与碱基配对核酸的存在相符。此外，我们发现随着时间的推移，hBEX3 从液体凝聚物转变为聚集体，这些聚集体在温度升高时是可逆的，并被 1,6-己二醇溶解。hBEX3 组装在 tRFs 的存在下显示出不同的形态，这似乎可以防止淀粉样形成。总的来说，我们的发现支持 tRFs 在 hBEX3 无序到有序转变和相转变调节中的作用。此外，hBEX3 易于聚集的特征和与 tRNA 片段的特异性相互作用表明，对于理解 BEX 家族在神经发育和细胞死亡中的参与具有至关重要的意义。
