Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)
Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)
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DOI：
10.1016/s1074-5521(00)00034-x
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发表时间：
2000-11-01
影响因子：
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通讯作者：
Kapurniotu, A
中科院分区：
文献类型：
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作者：
Krampert, M;Bernhagen, J;Kapurniotu, A
背景：胰腺淀粉样蛋白与II型糖尿病有关。胰腺淀粉样蛋白的主要成分是37个残基的肽胰岛淀粉样多肽（IAPP）。IAPP表达为称为ProIAPP的67个残基的前肽，其在刺激后加工成IAPP。虽然已经研究了体外IAPP淀粉样蛋白形成的分子事件，但对ProIAPP在胰腺淀粉样蛋白形成中的作用知之甚少。这已部分是由于有限的可用性纯化ProIAPP的构象和生化studies.Results：我们提出了一种有效的重组表达和纯化的ProIAPP和加工位点突变体，mutProIAPP，硫氧还蛋白（Trx）融合蛋白的方法。通过圆二色谱、电子显微镜和刚果红染色对切割的ProIAPP和mutProIAPP以及融合蛋白的构象和淀粉样变性进行评估。我们发现，ProIAPP和mutProIAPP表现出很强的自缔合潜力，并能够形成淀粉样蛋白。然而，在老化和淀粉样蛋白生成过程中ProIAPP和mutProIAPP的构象转变与IAPP的随机卷曲到P-折叠转变不同。发现这两种蛋白质的淀粉样蛋白生成比IAPP少，并且除了原纤维之外，在ProIAPP的老化期间形成许多非原纤维但有序的聚集体。ProIAPP聚集体对胰腺细胞具有细胞毒性，但细胞毒性低于IAPP，而mutProIAPP聚集体基本上缺乏细胞毒性。Trx融合蛋白既不是淀粉样蛋白也不是细胞毒性的。结论：我们的研究表明，ProIAPP具有典型的属性的淀粉样蛋白多肽，但也表明，前区域抑制淀粉样蛋白和细胞毒性潜力的IAPP。
Background: Pancreatic amyloid has been associated with type II diabetes. The major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide called ProIAPP which is processed to IAPP following stimulation. While the molecular events underlying IAPP amyloid formation in vitro have been studied, little is known about the role of ProIAPP in the formation of pancreatic amyloid. This has been due in part to the limited availability of purified ProIAPP for conformational and biochemical studies.Results: We present a method for efficient recombinant expression and purification of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAPP and mutProIAPP and the fusion proteins were assessed by circular dichroism, electron microscopy and Congo red staining. We find that ProIAPP and mutProlAPP exhibit strong self-association potentials and are capable of forming amyloid. However, the conformational transitions of ProIAPP and mutProIAPP during aging and amyloidogenesis are distinct from the random coil-to-P-sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggregates form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancreatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essentially lack cytotoxicity. The Trx fusion proteins are neither amyloidogenic nor cytotoxic.Conclusions: Our studies suggest that ProIAPP has typical properties of an amyloidogenic polypeptide but also indicate that the pro-region suppresses the amyloidogenic and cytotoxic potentials of IAPP.