SAMHD1肿瘤相关突变在逆转座子LINE-1异常激活中的作用机制研究

Long interspersed nuclear elements (LINEs) are transposable elements in the genome of some organisms，and has played a major role in disrupting the integrity of human genome. Movement of these LINE-1 sequences to new loci in the genome has the potential of causing sporadic cases of disease. SAM domain and HD domain with protein 1 contained (SAMHD1) is a dual-function enzyme containing both nuclease and deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) activity. It has been recently reported that SAMHD1 mutations are present in several human cancers. SAMHD1 has another significant function, the capacity to block long interspersed element 1 (LINE-1, L1) retrotransposition activity. The previous general conclusion was that the dNTPase of SAMHD1 is essential for suppressing LINE-1. Mutations of SAMHD1 are found in a variety of tumor tissues and autoimmune diseases. however, the mechanism by which tumor-related SAMHD1 mutants inhibit LINE-1 remains unknown. In previous work, we found two special mutants of SAMHD1 from the carcinoma of intestine, the SAMHD1 V133I mutation functioned as a normal dNTPase while lost the ability of inhibition LINE-1 retro-transposition, and the SAMHD1 K288T mutant was defective for dNTPase activity but showed potent activity against LINE-1 retrotransposition, suggesting that the nucleotide metabolism function of SAMHD1 is not required for its role in regulating endogenous retroelements. It may show a novel mechanism in the modulation of LINE-1 retrotransposition by SAMHD1. The project intends to study the relationship between the functional changes of SAMHD1 and its inhibition of LINE-1 retrotransposition. The association between the SAMHD1 mutation and the line-1 reverser was determined using animal models and clinical tumor samples. The completion of this project will expand the knowledge of mechanism on the modulation of LINE-1 retrotransposition, and reveal the molecular mechanism of cancer and autoimmune disease, which may provide a new idea on looking for disease diagnosis and new drug development.

LINE-1是人体内具有自主转座活性的逆转座子，几乎在所有自身免疫疾病和肿瘤细胞中LINE-1均为激活状态。SAMHD1突变导致LINE-1异常活化可能是多种疾病及癌变的重要诱因。SAMHD1 dNTP水解酶活性是否是其抗 LINE-1 的重要分子机制，仍是悬而未决的难题。前期工作中，我们从临床SAMHD1突变中发现了两个特殊突变体V133I和K288T，提示SAMHD1对LINE-1的抑制可能并不依赖其dNTPs代谢能力。本项目从临床来源的SAMHD1突变信息出发，构建突变体；明确突变对SAMHD1的功能性改变和抑制LINE-1能力的影响；以K288T和V133I为突破口探讨SAMHD1抑制LINE-1的未知机制；借助小鼠荷瘤模型和收集的临床肿瘤样本，验证SAMHD1突变与LINE-1异常激活的直接关联。项目的成果将拓展人们对于逆转座子调控机制的认识，为相关疾病的诊断和治疗提供科学参考。

LINE-1 是人体内具有自主转座活性的逆转座子，几乎在所有自身免疫疾病和肿瘤细胞中LINE-1均为激活状态。了解机体对于LINE-1的调控机制，对于理解这些疾病的发生至关重要。SAMHD1的突变导致LINE-1异常活化可能是多种免疫性疾病及癌变的重要诱因。普遍认为SAMHD1的dNTPase活性与其对LINE-1的抑制密切相关。为了研究SAMHD1抑制LINE1的机制。本项目成功的构建了SAMHD1肿瘤相关突变体的真核、原核表达重组体。SAMHD1肿瘤相关突变体形成四聚体的能力变差、并伴随着dNTPase活性降低乃至丧失，SAMHD1肿瘤相关突变普遍影响了SAMHD1功能；多数SAMHD1肿瘤相关突变体失去了抑制LINE-1的能力，而突变体K288T，几乎丧失了dNTPase活性，但对于逆转座子LINE-1的抑制能力几乎不影响，这表明SAMHD1抑制LINE-1并不完全依赖其dNTPase活性；肿瘤相关突变影响了SAMHD1的核定位情况，但是这种亚细胞定位的改变与其抗LINE-1能力的差异没有直接的联系；突变体抑制LINE-1能力的缺失，不能通过592位的磷酸缺失实现恢复；SAMHD1肿瘤相关突变体对LINE-1的抑制情况与其对LINE-1 RNPs 的ORF2p的逆转录能力的调节程度一致。对于临床肿瘤样本的检测表明，逆转座子LINE-1在肝癌组织中活性显著增高；在LINE-1异常活化的肝癌组织中存在着更多的SAMHD1突变；在同一份肿瘤样本中检测到LINE-1的活性增加并伴随着SAMHD1的突变，证明肝肿瘤组织中SAMHD1的突变与LINE-1的活性增加很可能存在着直接关联。本项目成果拓展了人们对于哺乳动物逆转座子调控机制的认识，为相关疾病的诊断和治疗提供新的分子机制和靶点。