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首页> 外文期刊>The FEBS journal >Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris
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Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris

机译：人类溶菌酶变体的天然状态稳定性对巴斯德毕赤酵母蛋白质分泌的影响

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摘要

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.
机译：我们报告了毕赤酵母的两种人类溶菌酶变异F57I和W64R，与系统性淀粉样变性病相关的分泌表达，并描述了它们通过生物物理方法的表征。与野生型人溶菌酶相比，这两种变异体的热稳定性都大大降低，这一发现为它们增加形成纤维状聚集体和产生疾病的倾向提供了解释。巴斯德毕赤酵母的F57I和W64R变体的分泌产量分别比野生型人溶菌酶低200倍和30倍。对10种溶菌酶变体的分泌水平进行更全面的分析表明，在受控条件下，这些分泌蛋白的低产量可能直接与其天然状态的热稳定性降低相关。在这种选择的变体中对mRNA水平的分析表明，较低的分泌水平是由于转录后过程造成的，并且分泌的蛋白质减少是通过酵母质量控制系统降解了部分折叠或错误折叠的蛋白质造成的。重要的是，我们的结果表明，与人类疾病相关的突变所表达的水平与其他位点的不稳定突变不相符。这些发现表明，降低的天然状态稳定性，较低的分泌水平和蛋白质聚集倾向之间的复杂相互作用影响了引起家族性淀粉样疾病的突变类型。

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