Refolding and polymerization pathways of neuroserpin.
Refolding and polymerization pathways of neuroserpin.
复制标题
神经丝氨酸蛋白酶抑制剂的重折叠和聚合途径。
DOI：
10.1016/j.jmb.2010.07.047
复制
发表时间：
2010
影响因子：
5.6
通讯作者：
M. Onda
中科院分区：
文献类型：
--
作者：
S. Takehara;Juan Zhang;Xiaoyan Yang;N. Takahashi;B. Mikami;M. Onda
神经丝氨酸蛋白酶抑制剂是丝氨酸蛋白酶抑制剂超家族的成员，其突变体作为有序聚合物保留在神经元的内质网内，与痴呆相关。已经提出神经丝氨酸蛋白酶抑制剂聚合物是通过折叠蛋白质中的构象变化形成的。然而，最近提出了一种替代模型，即在蛋白质折叠过程中形成聚合物，而不是从折叠的蛋白质。我们研究了野生型neuroserpin（WT）和致病突变体S49 P和H338 R的重折叠和聚合途径。复性后，变性WT立即形成初始复性中间体IIN，然后通过后期复性中间体IR进一步复性为天然形式。晚发型突变体S49 P也能够通过IIN和IR复性为天然形式，但最终复性步骤进行的速度较慢，复性产率较低，与WT相比。早发突变体H338R通过与S49P相同的途径形成IR，但该蛋白不能达到天然状态而保持IR。突变体的IR在4 °C下具有较长的寿命，因此对其进行纯化和表征。引人注目的是，当在生理条件下孵育时，IR形成了有序的聚合物，其性质与天然蛋白质形成的聚合物基本相同。结果表明，突变体在蛋白质折叠过程中形成聚合物的倾向大于从折叠的蛋白质形成聚合物的倾向。我们的发现提供了深入了解生化方法治疗丝氨酸蛋白酶蛋白病的目标是聚合物折叠中间体。
Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate IINand then underwent further refolding to the native form through a late refolding intermediate, IR. The late-onset mutant S49P was also able to refold to the native form through IINand IR, but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed IRthrough the same pathway as S49P, but the protein could not attain the native state and remained as IR. The IRs of the mutants had a long lifespan at 4 °C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, IRformed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.