Probing Neuroserpin Polymerization and Interaction with Amyloid-β Peptides Using Single Molecule Fluorescence
Probing Neuroserpin Polymerization and Interaction with Amyloid-β Peptides Using Single Molecule Fluorescence
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DOI：
10.1016/j.bpj.2009.07.057
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发表时间：
2009-10-21
影响因子：
3.4
通讯作者：
Klenerman, David
中科院分区：
文献类型：
--
作者：
Chiou, Albert;Haeggloef, Peter;Klenerman, David
Neuroserpin是丝氨酸蛋白酶抑制剂超家族的成员。它可以经历构象转变以形成与具有神经丝氨酸蛋白酶抑制剂包涵体的痴呆家族性脑病相关的聚合物，并且野生型蛋白质可以抑制阿尔茨海默病中淀粉样蛋白-β肽的毒性。我们已经使用了单分子荧光方法，双色符合检测，以确定荧光团标记的neuroserpin的聚合的早期阶段的限速步骤，并评估了这个过程是如何在A β（1-40）的存在下改变。我们的数据表明，neuroserpin聚合首先进行的活性单体的单分子形成，然后由两个聚合和形成的潜在单体从活化物种的竞争过程。这些数据是不符合最近提出的域交换模型的聚合物的形成，其中潜在的物种和活化的单体很可能是直接从未活化的单体丝氨酸蛋白酶抑制剂的竞争途径形成。此外，A β（1-40）肽与神经丝氨酸蛋白酶抑制剂形成弱复合物（解离常数为10 +/-5 nM），其增加活性单体的量，从而增加聚合速率。A β（1-40）从复合物中被置换，从而其充当催化剂并且不被掺入神经丝氨酸蛋白酶抑制剂聚合物中。
Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wildtype protein can inhibit the toxicity of amyloid-beta peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of A beta(1-40). Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the A beta(1-40) peptide forms a weak complex with neuroserpin (dissociation constant of 10 +/- 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The A beta(1-40) is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.