Division of Interdisciplinary Program in Precision Public Health (BK21 FOUR Program), Department of Biomedical Engineering, Korea University 145 Anam-ro, Seongbuk-gu, Seoul 02842, Korea.
Int J Mol Sci. 2021 Sep 16;22(18):9998. doi: 10.3390/ijms22189998.
The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat-TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 μM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment.
天然蛋白质折叠对细胞内拥挤环境中无规卷曲蛋白质 (IDPs) 的稳定性和折叠速率的各种影响在生物医学中很重要。虽然大多数蛋白质折叠研究都是在体外进行的，提供了有价值的见解，但细胞内拥挤环境中蛋白质折叠的研究却很少。本研究旨在基于细胞内相互作用，包括 TAR RNA，探索细胞内分子拥挤对突变型 HIV-1 Tat 折叠的影响，以此证明之前报道的 chaperna-RNA 概念。考虑到 Tat-TAR RNA 基序与 RNA 结合，我们评估了 TAR RNA 作为 chaperna 的潜在功能，用于折叠突变型 R52Tat，该突变体通过定点突变将精氨酸 (R) 残基替换为丙氨酸 (A)。我们通过荧光显微镜和生物层干涉法监测 HeLa 细胞中 Tat-EGFP 和 Tat 的折叠，使用 EGFP 融合作为折叠状态的指示剂。这些结果表明，在 0.3 μM TAR RNA 浓度下，R52A Tat 的折叠得到了很好的刺激；野生型 Tat 的折叠基本上被废除了，因为在该浓度下与 TAR RNA 的亲和力降低。TAR RNA 的刺激主要促进了 R52Tat 的折叠和重折叠。我们的发现通过研究尚未探索的 RNA 介导的蛋白质折叠以及调节细胞内拥挤环境中病毒遗传变异体病毒进化联系的病毒遗传变异体，为 chaperna 介导的折叠的治疗潜力提供了新的见解。
