Saeed Umar, Piracha Zahra Zahid
Department of Medical Research and Development, International Center of Medical Sciences Research (ICMSR), Islamabad, Pakistan.
Front Microbiol. 2023 Jan 25;14:921653. doi: 10.3389/fmicb.2023.921653. eCollection 2023.
Human parvulin peptidyl prolyl cis/trans isomerases PIN1 and PIN4 play important roles in cell cycle progression, DNA binding, protein folding and chromatin remodeling, ribosome biogenesis, and tubulin polymerization. In this article, we found that endogenous PIN1 and PIN4 were upregulated in selected hepatocellular carcinoma (HCC) cell lines.
In this study, we inhibited PIN1 and PIN4 parvulin inhibitors (Juglone, PiB, ATRA, 6,7,4'-THIF, KPT6566, and EGCG). The native agarose gel electrophoresis (NAGE) immunoblotting analysis revealed that upon PIN1 and/ or PIN4 inhibition, the HBc protein expression and core particle or capsid synthesis reduced remarkably. The effects of PIN4 inhibition on hepatitis B virus (HBV) replication were more pronounced as compared to that of PIN1. The Northern and Southern blotting revealed reduced HBV RNA and DNA levels.
During the HBV course of infection, Juglone, PiB, ATRA, 6,7,4'-THIF, KPT6566, and EGCG-mediated inhibition of PIN1 and PIN4 significantly lowered HBV transcriptional activities without affecting total levels of covalently closed circular DNA (cccDNA). Similar to the inhibitory effects of PIN1 and PIN4 on HBV replication, the knockdown of PIN1 and PIN4 in HBV infection cells revealed significantly reduced amounts of intracellular HBc, HBs, HBV pgRNA, SmRNAs, core particles, and HBV DNA synthesis. Similarly, PIN1 and PIN4 KD abrogated extracellular virion release, naked capsid levels, and HBV DNA levels. In comparison with PIN1 KD, the PIN4 KD showed reduced HBc and/or core particle stabilities, indicating that PIN4 is more critically involved in HBV replication. Chromatin immunoprecipitation (ChIP) assays revealed that in contrast to DNA binding PIN4 proteins, the PIN1 did not show binding to cccDNA. Similarly, upon PIN1 KD, the HBc recruitment to cccDNA remained unaffected. However, PIN4 KD significantly abrogated PIN4 binding to cccDNA, followed by HBc recruitment to cccDNA and restricted HBV transcriptional activities. These effects were more pronounced in PIN4 KD cells upon drug treatment in HBV-infected cells.
The comparative analysis revealed that in contrast to PIN1, PIN4 is more critically involved in enhancing HBV replication. Thus, PIN1 and PIN4 inhibition or knockdown might be novel therapeutic targets to suppress HBV infection. targets to suppress HBV infection.
人源小分子脯氨酰异构酶PIN1和PIN4在细胞周期进程、DNA结合、蛋白质折叠和染色质重塑、核糖体生物合成以及微管蛋白聚合中发挥重要作用。在本文中，我们发现内源性PIN1和PIN4在选定的肝癌（HCC）细胞系中上调。
在本研究中，我们使用小分子脯氨酰异构酶抑制剂（胡桃醌、PiB、全反式维甲酸、6,7,4'-三羟基异黄酮、KPT6566和表没食子儿茶素没食子酸酯）抑制PIN1和PIN4。原生琼脂糖凝胶电泳（NAGE）免疫印迹分析显示，在抑制PIN1和/或PIN4后，乙肝核心蛋白（HBc）的表达以及核心颗粒或衣壳的合成显著减少。与PIN1相比，抑制PIN4对乙型肝炎病毒（HBV）复制的影响更为明显。Northern印迹和Southern印迹显示HBV RNA和DNA水平降低。
在HBV感染过程中，胡桃醌、PiB、全反式维甲酸、6,7,4'-三羟基异黄酮、KPT6566和表没食子儿茶素没食子酸酯介导的对PIN1和PIN4的抑制显著降低了HBV转录活性，而不影响共价闭合环状DNA（cccDNA）的总量。与PIN1和PIN4对HBV复制的抑制作用类似，在HBV感染细胞中敲低PIN1和PIN4后，细胞内HBc、HBs、HBV pgRNA、亚基因组RNA（SmRNAs）、核心颗粒以及HBV DNA合成的量显著减少。同样，敲低PIN1和PIN4可消除细胞外病毒颗粒的释放、裸衣壳水平以及HBV DNA水平。与敲低PIN1相比，敲低PIN4显示出HBc和/或核心颗粒稳定性降低，表明PIN4在HBV复制中起更关键的作用。染色质免疫沉淀（ChIP）分析显示，与DNA结合的PIN4蛋白不同，PIN1不显示与cccDNA的结合。同样，在敲低PIN1后，HBc与cccDNA的募集不受影响。然而，敲低PIN4显著消除了PIN4与cccDNA的结合，随后HBc募集到cccDNA并限制了HBV转录活性。在HBV感染细胞中进行药物处理后，这些效应在敲低PIN4的细胞中更为明显。
对比分析显示，与PIN1相比，PIN4在增强HBV复制中起更关键的作用。因此，抑制或敲低PIN1和PIN4可能是抑制HBV感染的新治疗靶点。
