Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Chem Phys Lipids. 2020 Nov;233:105004. doi: 10.1016/j.chemphyslip.2020.105004. Epub 2020 Oct 31.
Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts.
氧化固醇是胆固醇代谢物，具有控制细胞内稳态的多种功能。特别是，27-羟胆固醇（27-OH-Chol）已被证明可以调节多种生理功能，但对于其摄取、细胞内运输和流出细胞的机制知之甚少。这主要是因为缺乏与 27-OH-Chol 紧密模拟的合适的类似物。在这里，我们提出了内源性荧光 27-羟基胆固醇（27-OH-CTL），它与 27-OH-Chol 仅在甾体环系统中多两个双键不同。基于分子动力学（MD）模拟，我们表明 27-OH-CTL 与 27-OH-Chol 相比具有几乎相同的膜性质。通过对 27-OH-CTL 和胆固醇类似物胆甾烯醇（CTL）在活细胞中的比较成像，我们评估了单个羟基对甾醇运输的影响。我们发现人成纤维细胞摄取的 CTL 比 27-OH-CTL 多，但更有效地排出该氧化固醇类似物。对于两种固醇，流出包括从质膜脱落囊泡。在细胞内，27-OH-CTL 主要积聚在脂滴（LDs）中，而 CTL 主要存在于内体和溶酶体中。通过荧光恢复后光漂白（FRAP），我们发现对于两种固醇，都有一个快速交换池，其移动速度比含有固醇的囊泡和 LDs 快几个数量级。总之，通过应用 27-OH-Chol 的新荧光衍生物，我们证明人类细胞可以根据侧链上的单个羟基区分固醇，从而导致不同的运输途径、动力学和流出动力学。内源性荧光胆固醇和氧化固醇类似物在人成纤维细胞中均显示出快速的非囊泡运输。
