Schlafen (Slfn) 基因作为一类干扰素刺激因子 (ISG), 可以被Ⅰ型干扰素诱导表达。1998年, Schwarz等人首次在研究小鼠胸腺发育过程中发现了Slfn 家族基因。后续研究发现, Slfn 基因家族成员在小鼠、马和人等多个物种中均广泛分布, 且具有较高的同源性。研究表明, SLFN家族蛋白参与机体多种重要的生命活动, 它们在调节细胞增殖、细胞分化、免疫细胞生长和成熟以及抑制病毒复制方面发挥着不同的作用。

SLFN11是目前研究最多最深入的SLFN家族蛋白, 主要在肿瘤的治疗和对抗病毒的感染方面发挥重要的作用。基于DNA损伤机制的化疗和放疗是癌症最主要的治疗方式之一, 而癌症细胞的抗药性会严重影响放化疗的效果。SLFN11则可以通过多种分子机制增强肿瘤细胞对化疗药物的耐药性, 是决定癌细胞化疗敏感性的重要因素。

SLFN11另一个重要的生物学功能是限制病毒的感染, 可抑制多种病毒的复制和感染, 包括HIV, ZIKA和HCMV, 尤其是在抑制HIV感染方面研究较为透彻。病毒基因组的核苷酸使用频率和方式与人类基因组不同, 如慢病毒的基因序列和流感病毒的基因序列均存在A/T碱基偏倚。与宿主的偏好性相比, HIV-1编码的蛋白具有低的GC含量。2012年Li等人首次报道了SLFN11可根据蛋白密码子使用频率的不同, 特异性的抑制HIV-1病毒蛋白的表达, SLFN11可通过对tRNA的调节, 抑制HIV病毒蛋白的合成, 从而抑制病毒的繁殖。

但是由于 SLFN11蛋白结构信息的缺乏, 极大限制了SLFN家族蛋白功能分子机制的研究; 本研究以SLFN11为主要研究对象, 成功解析了Sus scrofa SLFN11-NTD的晶体结构, 分辨率为2.69 Å。sSLFN11-NTD的结构与已解析的其它SLFN家族蛋白的NTD结构折叠方式类似, 整体呈 “钳形” 假二聚体形式。通过一系列的生化实验证实, sSLFN11-NTD是一种能够降解核酸酶tRNA和rRNA的核酸酶, 且优先降解II 型 tRNA。此外, 体外酶活实验表明sSLFN11-NTD对同一氨基酸的不同密码子对应的tRNA降解效率也存在差异。通过系统全面的突变体酶活性实验，我们鉴定了多个决定核酸酶活性区域 (如

Connection loop) 和氨基酸位点。我们发现E42可以严重限制sSLFN11-NTD核酸酶活性, 并且E42所有非保守突变都增强了其核酸酶活性。此外, 我们还鉴定并拓展了SLFN11的酶活性中心, 即除了保守的强负电性的催化三联体 (E204-E209-D248) 外, 还包含其临近的3个正电氨基酸 (R139-K211-K251), 这6个氨基酸位点共同构成了一个较为完整的酶活性中心, 且它们在所有已知的有核酸酶活性的蛋白中都很保守, 而在无核酸酶活性的蛋白中相对不保守, 这可以作为判断SLFN蛋白有无核酸酶活性的标准之一。且细胞实验表明，SLFN11可通过蛋白翻译时密码子使用频率不一致，抑制HIV-1 Gag和p24的表达，且该过程主要依赖其NTD的核酸酶活性。

综上, 我们的研究结果首次解析了SLFN11 蛋白的晶体结构, 这不仅有助于我们对 Schlafen 家族的理解, 更为我们阐明其核酸酶活性调控的具体分子机制提供了坚实的基础，有助于我们进一步研究其抗病毒活性的机制。

Schlafen genes are a subset of interferon-stimulated response genes (ISGs) unique to mammals. SLFNs were initially found to be important for thymocyte maturation/activation and T cell quiescence in mice. Accumulating evidence demonstrates that SLFNs play vital roles in numerous biological processes, including T cell-mediated immunity, cell proliferation, cell differentiation, cancer biology, and restriction of viral infection.

SLFN11 is one of the most studied Schlafen members and attracts significant attention in the areas of cancer treatment and virus restriction. SLFN11 is considered a promising biomarker for various cancer types and can enhance chemosensitivity through different mechanisms. Another important function of SLFN11 is virus restriction. SLFN11 expression is upregulated in CD4+ cells of human immunodeficiency (HIV) elite controllers and HIV patients, suggesting its role in suppressing HIV replication. Mechanisms underlying SLFN11-mediated HIV restriction include codon usage-based inhibition of viral protein expression, which is a similar mechanism adopted by SLFN11 in translational control of host proteins ATR and ATM. In addition to HIV, SLFN11 was also found to restrict replication of other viruses, including flavivirus.

Structural characterization of SLFNs has proven challenging, which hinders elucidation of the molecular mechanism underlying this unique protein family. Herein, we determined the crystal structure of the Sus scrofa SLFN11 N-terminal domain (NTD) to 2.69 Å resolution. sSLFN11-NTD is a pincer-shaped molecule that shares an overall fold with other SLFN-NTDs but exhibits distinct biochemical characteristics. sSLFN11-NTD is a potent RNase targeting type I and II tRNAs and rRNAs, and preferentially digests type II tRNAs. Consistent with the codon usage-based translation suppression activity of SLFN11, sSLFN11-NTD digested synonymous serine tRNAs with different efficiencies in vitro. Mutational analysis revealed key determinates of sSLFN11-NTD nucleolytic activity, including the connection loop, active site, and key residues essential for substrate recognition, among which E42 constrains sSLFN11-NTD RNase activity, and all nonconservative mutations of E42 stimulated RNase activities. sSLFN11 inhibited the translation of proteins with a low codon adaptation index in cells, which mainly dependent on the RNase activity of the NTD because E42A enhanced the inhibitory effect, but E209A abolished inhibition. Our findings provide the first structural characterization of SLFN11 protein and expand our understanding of the Schlafen family.