PDF(Pubmed)
Abstract：
Splice modulating antisense oligomers (AOs) are increasingly used to modulate RNA processing. While most are investigated for their use as therapeutics, AOs can also be used for basic research. This study examined their use to investigate internally and terminally truncated proprotein convertase subtilisin/kexin type 9 (PCSK9) protein isoforms. Previous studies have used plasmid or viral-vector-mediated protein overexpression to study different PCSK9 protein isoforms, creating an artificial environment within the cell. Here we designed and tested AOs to remove specific exons that encode for PCSK9 protein domains and produced protein isoforms at more physiologically relevant levels. We evaluated the isoforms\' expression, secretion, and subsequent impact on the low-density lipoprotein (LDL) receptor and its activity in Huh-7 cells. We found that modifying the Cis-His-rich domain by targeting exons 10 or 11 negatively affected LDL receptor activity and hence did not enhance LDL uptake although the levels of LDL receptor were increased. On the other hand, removing the hinge region encoded by exon 8, or a portion of the prodomain encoded by exon 2, have the potential as therapeutics for hypercholesterolemia. Our findings expand the understanding of PCSK9 isoforms and their impact on the LDL receptor and its activity at physiologically relevant concentrations.
摘要：
剪接调节反义寡聚体(AOs)越来越多地用于调节RNA加工。虽然大多数人被研究作为治疗药物，AOs也可用于基础研究。这项研究检查了它们在研究内部和末端截短的前蛋白转化酶枯草杆菌蛋白酶/kexin9型（PCSK9）蛋白质亚型中的用途。以前的研究已经使用质粒或病毒载体介导的蛋白过表达来研究不同的PCSK9蛋白亚型，在细胞内创造一个人造环境。在这里，我们设计并测试了AOs以去除编码PCSK9蛋白质结构域的特定外显子，并在更生理相关的水平上产生蛋白质同工型。我们评估了同工型的表达，分泌，以及随后对低密度脂蛋白（LDL）受体及其在Huh-7细胞中的活性的影响。我们发现,通过靶向外显子10或11修饰富含Cis-His的结构域对LDL受体活性产生负面影响,因此尽管LDL受体的水平增加,但并未增强LDL摄取。另一方面,去除由外显子8编码的铰链区,或由外显子2编码的前结构域的一部分,具有治疗高胆固醇血症的潜力.我们的发现扩展了对PCSK9亚型及其在生理相关浓度下对LDL受体及其活性的影响的理解。
