Bhat Rajiv
School of Biotechnology, Jawaharlal Nehru University, New Delhi 100067, India.
Biochemistry. 2024 Dec 17;63(24):3194-3212. doi: 10.1021/acs.biochem.4c00362. Epub 2024 Oct 9.
The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.
生物细胞的拥挤环境会显著影响蛋白质的聚集和相互作用。了解大分子拥挤对淀粉样蛋白聚集和纤维化的影响对于治疗许多淀粉样蛋白相关疾病至关重要。大多数关于蛋白质淀粉样形成及其小分子抑制作用的研究是在稀释缓冲液中进行的，这种缓冲液无法模拟细胞环境的复杂性。在本研究中，我们使用聚乙二醇（PEGs）来模拟大分子拥挤，并研究了黄酮类化合物二氢黄酮（DHF）、黄芩素和木犀草素在pH 2条件下对人溶菌酶（HuL）聚集的抑制作用。天然存在的黄酮类化合物已被证明是某些蛋白质淀粉样形成的有效抑制剂。我们的研究结果表明，虽然黄酮类化合物在稀释缓冲溶液中可抑制HuL的聚集和纤维化，但通过硫黄素T（ThT）荧光、光散射、透射电子显微镜（TEM）和原子力显微镜（AFM）研究表明，黄酮类化合物与PEGs联合使用时可实现完全抑制。与对照样品相比，在聚乙二醇8000和黄酮类化合物存在下形成的物种疏水性更低、毒性更小且富含α-螺旋，而对照样品则疏水性强且富含β-折叠，这已通过1-苯胺基萘-8-磺酸（ANS）疏水性、MTT法和圆二色光谱（CD）得到证实。黄酮类化合物与HuL的荧光滴定研究表明，在拥挤条件下结合常数显著增加。这些发现突出了大分子拥挤在调节蛋白质聚集和淀粉样抑制中的重要性。需要进一步使用HuL的致病突变体和其他淀粉样蛋白进行研究，以探索大分子拥挤在小分子介导的蛋白质聚集和淀粉样形成的调节和抑制中的作用。
