Optimization of apolipoprotein B mRNA editing by APOBEC1 apoenzyme and the role of its auxiliary factor, ACF
Optimization of apolipoprotein B mRNA editing by APOBEC1 apoenzyme and the role of its auxiliary factor, ACF
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DOI：
10.1261/rna.7490704
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发表时间：
2004-09-01
期刊：
影响因子：
4.5
通讯作者：
Navaratnam, N
中科院分区：
文献类型：
--
作者：
Chester, A;Weinreb, V;Navaratnam, N
表达和纯化载脂蛋白B mRNA编辑亚基APOBEC1，证明了即使在没有任何辅助因素的情况下，该脱辅酶在最小的apoB mRNA底物上也具有相当大的残留酶活性。通过使用不完全析因和响应面实验，这种活性作为各种实验条件的函数的分析已经导致了分析条件的实质性优化。令人惊讶的是，酶是热稳定性的，其最适温度接近45℃。我们已经使用这些优化的条件，评估了APOBIEC1mRNA编辑活动在有和没有辅助因子ACF的情况下的稳态动力学参数。辅助因子的一个重要作用是拓宽APOBEC1活性的温度范围，降低最适温度，使其在较低温度下发挥最佳作用。与这一观察相一致的一个模型是，在较低的温度下，ACF促进了RNA底物中的构象转变，这种转变在较高的温度下自发发生。值得注意的是，APOBEC1的大量RNA编辑活动本身可能是在转基因小鼠中观察到APOBEC1过表达时观察到的“过度编辑”的原因。
Expression and purification to homogeneity of the apolipoprotein B mRNA editing subunit, APOBEC1, has allowed the demonstration that this apoenzyme has considerable residual enzymatic activity on a minimal apoB mRNA substrate, even in the absence of any auxiliary factors. Assay of this activity as a function of various experimental conditions has led to substantial optimization of assay conditions through the use of incomplete factorial and response surface experiments. Surprisingly, the apoenzyme is thermostable, and has a temperature optimum near 45degreesC. We have used these optimized conditions, to assess steady-state kinetic parameters for APOBIEC1 mRNA editing activity with and without the auxiliary factor, ACF. An important effect of the auxiliary factor is to broaden the temperature range of APOBEC1 activity, lowering the optimal temperature and enabling it to function optimally at lower temperatures. A model consistent with this observation is that at lower temperatures ACF promotes a conformational transition in the RNA substrate that occurs spontaneously at higher temperature. Notably, the substantial RNA editing activity of APOBEC1 alone may be responsible for the "hyperediting" observed upon overexpression of APOBEC1 in transgenic mice.