Department of Medicine and Surgery, Physiology and Biochemistry Section, University of Perugia, 06132, Perugia, Italy.
Department of Neurosciences, Biomedicine and Movement Sciences, Biochemistry Section, University of Verona, 37134, Verona, Italy.
Biochimie. 2022 Nov;202:110-122. doi: 10.1016/j.biochi.2022.08.005. Epub 2022 Aug 11.
AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.
AGXT1 编码丙氨酸：乙醛酸氨基转移酶 1（AGT1），一种肝脏过氧化物酶体吡哆醛 5'-磷酸依赖性酶，其缺乏导致原发性高草酸尿症 1 型（PH1）。PH1 是一种罕见的疾病，其特征是草酸盐过度产生，首先导致肾结石形成，并可能发展为危及生命的系统性草酸中毒。少数 PH1 患者对吡哆醇有反应，而无反应者的选择是肝肾移植。因此，目前正在努力寻找新的治疗方法，包括最近批准的基于 RNA 沉默的有前途的方法。许多 PH1 相关突变是错义突变，导致 AGT1 的各种动力学和/或折叠缺陷。在这种情况下，能够获得可靠的体外疾病模型对于更好地理解已知或新发现的致病性变异的表型以及测试新的候选药物至关重要。在这里，我们利用 CRISPR/Cas9 技术特异性敲除 HepG2 细胞中的 AGXT1，HepG2 是一种具有保守乙醛酸代谢的肝癌衍生细胞模型。AGXT1-KO HepG2 显示出 AGT1 表达缺失和转氨基酶活性显著降低，导致甘醇酸盐挑战时草酸盐分泌增加。在 AGXT1-KO HepG2 细胞中表达的已知致病性 AGT1 变体在蛋白水平和特定转氨基酶活性方面均发生改变，并且与常见多态性相关时部分线粒体靶向错误。值得注意的是，吡哆醇治疗能够部分挽救临床反应性变体的活性和定位。总的来说，我们的数据验证了 AGXT1-KO HepG2 细胞作为研究 PH1 病理生理学的新型细胞模型，以及作为药物发现和开发的平台。
