首页> 外文期刊>Journal of Molecular Biology >Full-length Rat Amylin Forms Fibrils Following Substitution of Single Residues from Human Amylin.
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Full-length Rat Amylin Forms Fibrils Following Substitution of Single Residues from Human Amylin.

机译:全长大鼠胰岛淀粉样多肽从人类胰岛淀粉样多肽替代单个残基后形成原纤维。

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摘要

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to thosegenerated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.
机译:由37个氨基酸残基的肽胰岛淀粉样多肽组成的胰淀粉样蛋白沉积物代表2型糖尿病病理的组成部分。人胰岛淀粉样多肽(hA)在体外形成原纤维,对培养的胰岛β细胞有毒性。相反,大鼠胰岛淀粉样多肽(rA)与hA的区别仅在于肽中心区域的六个氨基酸残基,即残基18-29,不形成原纤维并且没有细胞毒性。为了阐明单个残基在原纤维形成中的作用,我们产生了一系列全长rA变体,并检查了它们在体外形成原纤维的能力。来自hA序列相应位置的氨基酸(即R18H,L23F或V26I)的单残基取代足以使rA具有原纤维形成的能力,尽管产率很低。这些取代中的两个或三个的组合通常增加了产生原纤维的能力。通过负染色电子显微镜检查了变异的rA原纤维形态,发现与hA本身产生的形态相似。大量测定,即涉及硫代黄素-T荧光和沉降,表明与相同条件下的hA相比,这些rA变体的原纤维形成量相对较小。延时原子力显微镜显示了原纤维的生长,并使用MALDI-TOF质谱法验证了原纤维由全长肽组成。我们的观察结果证实了先前的报道,即三个脯氨酸残基在原纤维形成中起主要的负作用。但是,它们的存在不足以完全消除rA形成原纤维的能力,因为其他三个相关残基(即R18,L23和V26)中的每一个也具有主要的调节作用。

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