Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

中文翻译：

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LINE-1 ORF2p 表达在人类癌症中几乎难以察觉

长散布元件 1 (LINE-1, L1) 是现代人类移动 DNA 活动的主要驱动因素。表达时，LINE-1 基因座产生双顺反子转录本，编码两种逆转录转座必需的蛋白质：ORF1p 和 ORF2p。许多类型的人类癌症的特征是 L1 启动子低甲基化、L1 转录、L1 ORF1p 蛋白表达和体细胞 L1 逆转录转座。ORF2p 编码 L1 逆转录转座所需的核酸内切酶和逆转录酶活性。它的表达在人体组织和细胞系中的特征很少。我们报告了基于质谱的肿瘤蛋白质组分析研究，其中 ORF2p 逃避检测。为了测试是否可以用特定试剂检测 ORF2p，我们开发并验证了五种对蛋白质上特定表位具有免疫反应性的兔单克隆抗体。这些试剂可轻松检测双顺反子 L1 构建体表达的异位 ORF2p。然而，尽管 ORF1p 表达水平很高，但通过蛋白质印迹、免疫沉淀或免疫组织化学在人类肿瘤样本或细胞系中并未检测到内源性 ORF2p。此外，我们报告了内源性 ORF1p 相关的相互作用组，从结直肠癌中分离出亲和力，但我们同样未能检测到 ORF2p。这些样本包括含有数百个体细胞获得性 L1 插入的原发性肿瘤。新数据可通过 ProteomeXchange 获取，标识符为 PXD013743。尽管体细胞逆转录转座为人类癌症中 ORF2p 的表达提供了明确的遗传证据，但我们无法使用几种标准方法直接测量其存在。实验系统先前已表明 ORF1p 和 ORF2p 之间的化学计量不相等，但在体内，这两种蛋白质的表达可能更加惊人地脱钩。这些发现与 ORF2p 不能耐受细胞生长的观察结果一致。