Ribosome-mediated folding of partially unfolded ricin A-chain
Ribosome-mediated folding of partially unfolded ricin A-chain
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DOI：
10.1074/jbc.275.13.9263
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发表时间：
2000-03-31
影响因子：
4.8
通讯作者：
Radford, SE
中科院分区：
文献类型：
--
作者：
Argent, RH;Parrott, AM;Radford, SE
在被哺乳动物细胞内吞摄取后，细胞毒性蛋白蓖麻毒素被转运到内质网，于是A链必须穿过内腔膜到达其核糖体底物。据推测，膜遍历之前的解折叠蓖麻毒素A链，然后在胞质溶胶中重折叠，以产生天然的，生物活性的毒素。在这里，我们描述了蓖麻毒素A-链的展开及其体外重折叠的生物化学和生物物理分析。我们发现，天然蓖麻毒素A链在pH 7.0时令人惊讶地不稳定，在37 ℃以上非协同地展开以产生部分展开状态，该物种具有典型的熔融球的构象性质，并且不能通过操纵缓冲液条件或通过添加茎环十二核糖核苷酸或脱蛋白质的大肠杆菌核糖体RNA而重折叠至天然状态，两者都是蓖麻毒素A链的底物。相比之下，在盐洗核糖体的存在下，部分未折叠的蓖麻毒素A链恢复全部催化活性。这些数据表明，蓖麻毒素A链的构象稳定性是理想的准备易位从内质网，在胞质溶胶中，蓖麻毒素A链分子，然后在核糖体的存在下重新折叠，导致核糖体脱嘌呤和细胞死亡。
After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state, This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manip ulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum, Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.