Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil; Laboratory of Molecular Research in Cardiology, Institute Dante Pazzanese of Cardiology, Sao Paulo 04012-909, Brazil; Department of Internal Medicine VIII, University Hospitals Tübingen, Tübingen DE72076, Germany.
Gene. 2023 Jan 30;851:146979. doi: 10.1016/j.gene.2022.146979. Epub 2022 Oct 17.
PCSK9 gain-of-function (GOF) variants increase degradation of low-density lipoprotein receptor (LDLR) and are potentially associated with Familial Hypercholesterolemia (FH). This study aimed to explore the effects of PCSK9 missense variants on protein structure and interactions with LDLR using molecular modeling analyses and in vitro functional studies. Variants in FH-related genes were identified in a Brazilian FH cohort using an exon-target gene sequencing strategy. Eight PCSK9 missense variants in pro- [p.(E32K) and p.(E57K)], catalytic [p.(R237W), p.(P279T) and p.(A443T)], and C-terminal histidine-cysteine rich (CHR) [p.(R469W), p.(Q619P) and p.(R680Q)] domains were identified. Molecular dynamics analyses revealed that GOF variants p.(E32K) and p.(R469W) increased extreme motions in PCSK9 amino acid backbone fluctuations and affected Hbond and water bridge interactions between the pro-domain and CM1 region of the CHR domain. HEK293FT cells transfected with plasmids carrying p.(E32K) and p.(R469W) variants reduced LDLR expression (8.7 % and 14.8 %, respectively) compared to wild type (p < 0.05) but these GOF variants did not affect PCSK9 expression and secretion. The missense variants p.(P279T) and p.(Q619P) also reduced protein stability and altered Hbond interactions. In conclusion, PCSK9 p.(E32K), p.(R469W), p.(P279T) and p.(Q619P) variants disrupt intramolecular interactions that are essential for PCSK9 structural conformation and biological activity and may have a potential role in FH pathogenesis.
PCSK9 功能获得性（GOF）变异可增加低密度脂蛋白受体（LDLR）的降解，与家族性高胆固醇血症（FH）有关。本研究旨在使用分子建模分析和体外功能研究探索 PCSK9 错义变异对蛋白质结构和与 LDLR 相互作用的影响。采用外显子靶向基因测序策略，在巴西 FH 队列中鉴定了与 FH 相关基因的变异。在 PCKS9 的前区 [p.(E32K)和 p.(E57K)]、催化区 [p.(R237W)、p.(P279T)和 p.(A443T)]和 C 末端组氨酸-半胱氨酸富含区（CHR） [p.(R469W)、p.(Q619P)和 p.(R680Q)]中发现了 8 种 PCSK9 错义变异。分子动力学分析表明，GOF 变异 p.(E32K)和 p.(R469W)增加了 PCSK9 氨基酸骨架波动的极端运动，并影响了前区和 CHR 区 CM1 区之间的氢键和水桥相互作用。与野生型相比，转染携带 p.(E32K)和 p.(R469W)变异体的质粒的 HEK293FT 细胞 LDLR 表达降低（分别为 8.7%和 14.8%，p<0.05），但这些 GOF 变异不影响 PCSK9 的表达和分泌。错义变异 p.(P279T)和 p.(Q619P)也降低了蛋白质稳定性并改变了氢键相互作用。总之，PCSK9 p.(E32K)、p.(R469W)、p.(P279T)和 p.(Q619P)变异破坏了 PCSK9 结构构象和生物学活性所必需的分子内相互作用，可能在 FH 发病机制中起潜在作用。
