Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex
Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex
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DOI：
10.1074/jbc.m001786200
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Greeve, J
中科院分区：
文献类型：
--
作者：
Lellek, H;Kirsten, R;Greeve, J
载脂蛋白 B (apoB) mRNA 的编辑需要催化成分 APOBEC-1 以及迄今为止尚未确定特征的“辅助”蛋白。在这里，我们报告了从大鼠肝脏中纯化 apoB mRNA 编辑酶复合物的这些附加成分，以及新型 APOBEC-1 刺激蛋白 (ASP) 的 cDNA 克隆，两种蛋白质共纯化为最终活性组分，并通过肽测序和质谱分析进行表征：KSRP，一种最初被描述为剪接调节因子的 75 kDa 蛋白质，和 ASP，一种迄今为止未知的 65 kDa 蛋白质。这两种蛋白质的分离导致 APOBEC-1 刺激活性降低。 ASP 代表一种新型的 RNA 结合蛋白，在氨基末端含有三个单链 RNA 结合结构域，在羧基末端含有一个推定的双链 RNA 结合结构域。纯化的重组谷胱甘肽 S-转移酶 (GST)-ASP（而非重组 GST-KSRP）在体外刺激重组 GST-APOBEC-1 编辑 apoB RNA。这些数据表明ASP是apoB mRNA编辑酶复合物的第二个基本成分。在大鼠肝脏中，ASP显然与KSRP相关，这可能赋予编辑酶复合物稳定性，其底物apoB RNA作为额外的辅助成分。
Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP), Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex, In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.