Kretschmer Stefanie, Wolf Christine, König Nadja, Staroske Wolfgang, Guck Jochen, Häusler Martin, Luksch Hella, Nguyen Laura A, Kim Baek, Alexopoulou Dimitra, Dahl Andreas, Rapp Alexander, Cardoso M Cristina, Shevchenko Anna, Lee-Kirsch Min Ae
Department of Pediatrics, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.
Biotechnology Center, Technische Universität Dresden, Dresden, Germany.
Ann Rheum Dis. 2015 Mar;74(3):e17. doi: 10.1136/annrheumdis-2013-204845. Epub 2014 Jan 20.
The HIV restriction factor, SAMHD1 (SAM domain and HD domain-containing protein 1), is a triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs). Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory disorder that shares phenotypic similarity with systemic lupus erythematosus, including activation of antiviral type 1 interferon (IFN). To further define the pathomechanisms underlying autoimmunity in AGS due to SAMHD1 mutations, we investigated the physiological properties of SAMHD1.
Primary patient fibroblasts were examined for dNTP levels, proliferation, senescence, cell cycle progression and DNA damage. Genome-wide transcriptional profiles were generated by RNA sequencing. Interaction of SAMHD1 with cyclin A was assessed by coimmunoprecipitation and fluorescence cross-correlation spectroscopy. Cell cycle-dependent phosphorylation of SAMHD1 was examined in synchronised HeLa cells and using recombinant SAMHD1. SAMHD1 was knocked down by RNA interference.
We show that increased dNTP pools due to SAMHD1 deficiency cause genome instability in fibroblasts of patients with AGS. Constitutive DNA damage signalling is associated with cell cycle delay, cellular senescence, and upregulation of IFN-stimulated genes. SAMHD1 is phosphorylated by cyclin A/cyclin-dependent kinase 1 in a cell cycle-dependent manner, and its level fluctuates during the cell cycle, with the lowest levels observed in G1/S phase. Knockdown of SAMHD1 by RNA interference recapitulates activation of DNA damage signalling and type 1 IFN activation.
SAMHD1 is required for genome integrity by maintaining balanced dNTP pools. dNTP imbalances due to SAMHD1 deficiency cause DNA damage, leading to intrinsic activation of IFN signalling. These findings establish a novel link between DNA damage signalling and innate immune activation in the pathogenesis of autoimmunity.
HIV限制因子SAMHD1（含SAM结构域和HD结构域蛋白1）是一种降解脱氧核糖核苷三磷酸（dNTP）的三磷酸水解酶。SAMHD1突变会导致Aicardi-Goutières综合征（AGS），这是一种炎症性疾病，与系统性红斑狼疮存在表型相似性，包括抗病毒1型干扰素（IFN）的激活。为了进一步明确SAMHD1突变导致的AGS自身免疫的发病机制，我们研究了SAMHD1的生理特性。
检测原发性患者成纤维细胞的dNTP水平、增殖、衰老、细胞周期进程和DNA损伤。通过RNA测序生成全基因组转录谱。通过免疫共沉淀和荧光交叉相关光谱法评估SAMHD1与细胞周期蛋白A的相互作用。在同步化的HeLa细胞中并使用重组SAMHD1检测SAMHD1的细胞周期依赖性磷酸化。通过RNA干扰敲低SAMHD1。
我们发现，由于SAMHD1缺乏导致的dNTP池增加会导致AGS患者成纤维细胞中的基因组不稳定。持续性DNA损伤信号与细胞周期延迟、细胞衰老以及IFN刺激基因的上调相关。SAMHD1在细胞周期依赖性方式下被细胞周期蛋白A/细胞周期依赖性激酶1磷酸化，其水平在细胞周期中波动，在G1/S期观察到最低水平。通过RNA干扰敲低SAMHD1可重现DNA损伤信号的激活和1型IFN激活。
SAMHD1通过维持平衡的dNTP池对基因组完整性至关重要。由于SAMHD1缺乏导致的dNTP失衡会引起DNA损伤，导致IFN信号的内在激活。这些发现确立了DNA损伤信号与自身免疫发病机制中固有免疫激活之间的新联系。
