Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
Sci Rep. 2022 Jul 13;12(1):11909. doi: 10.1038/s41598-022-16233-z.
Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of the antiviral ribonucleotide, 3'-deoxy-3',4'-didehydroCTP. The enzyme is conserved across all kingdoms of life, and in higher animals viperin is localized to the ER-membrane and lipid droplets through an N-terminal extension that forms an amphipathic helix. Evidence suggests that the N-terminal extension plays an important role in viperin's interactions with other membrane proteins. These interactions serve to modulate the activity of various other enzymes that are important for viral replication and constitute another facet of viperin's antiviral properties, distinct from its catalytic activity. However, the full-length form of the enzyme, which has proved refractory to expression in E. coli, has not been previously purified. Here we report the purification of the full-length form of viperin from HEK293T cells transfected with viperin. The purification method utilizes nanodiscs to maintain the protein in its membrane-bound state. Unexpectedly, the enzyme exhibits significantly lower catalytic activity once purified, suggesting that interactions with other ER-membrane components may be important to maintain viperin's activity.
Viperin 是一种激进的 S-腺苷甲硫氨酸酶，可催化抗病毒核苷酸 3'-脱氧-3'，4'-二脱氢 CTP 的形成。该酶在所有生命领域中都得到了保守，在高等动物中，viperin 通过形成两性螺旋的 N 端延伸定位于内质网膜和脂滴。有证据表明，N 端延伸在 viperin 与其他膜蛋白的相互作用中起着重要作用。这些相互作用有助于调节对病毒复制很重要的各种其他酶的活性，这是 viperin 抗病毒特性的另一个方面，与它的催化活性不同。然而，之前一直难以在大肠杆菌中表达的全长形式的酶尚未被纯化。在这里，我们报告了从转染了 viperin 的 HEK293T 细胞中纯化全长形式的 viperin 的方法。该纯化方法利用纳米盘将蛋白质保持在其膜结合状态。出乎意料的是，一旦纯化，该酶的催化活性显著降低，表明与其他内质网膜成分的相互作用对于维持 viperin 的活性可能很重要。
