An investigation into the formation of annular aggregates of human islet amyloid polypeptide on tantalum oxide surfaces.-猫眼课题宝

DOI：

10.1002/chem.201102215

发表时间：

2012-02

期刊：

Chemistry

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Mei-sha Chen;Shuai Zhang;Qian-Ying Liu;Peng Liu;K. Busuttil;Chen Wang;F. Besenbacher;Yan‐Mei Li;M. Dong

蛋白质错误折叠和积聚是蛋白质淀粉样变性相关疾病的共同特征。到目前为止，蛋白质淀粉样变性的发生被广泛认为与四十多种人类疾病有关，如阿尔茨海默氏症、朊病毒和II型糖尿病（T2 DM）。在T2 DM中，人胰岛淀粉样多肽（hIAPP），一种由b细胞分泌的37个氨基酸的肽激素，在胰岛中形成淀粉样蛋白聚集体，并参与b细胞功能障碍。参与hIAPP诱导的细胞毒性的普遍接受的机制包括可溶性hIAPP寡聚体形成、膜去稳定化、膜透化和氧化应激。hIAPP被认为附着在细胞膜上，在此过程中破坏细胞膜并导致细胞死亡。实验证据表明，脂质膜的表面可以催化hIAPP的错误折叠和积累，作为在正常生理条件下带负电荷的膜和带正电荷的hIAPP之间的静电相互作用的结果。膜介导的hIAPP聚集表明，在细胞膜存在的情况下，可能促进潜在毒性hIAPP物质的形成，这可能是T2 DM病理学中蛋白-膜相互作用和hIAPP诱导的b细胞功能障碍之间的联系。因此，深入研究hIAPP在带负电荷的表面上的聚集对于充分理解表面介导的淀粉样肽组装的机制至关重要。原子力显微镜（AFM）是一种强大的表面表征技术，可以有效地检测淀粉样蛋白的不同形态。13]除了记录表面形态，AFM还可以检测分子间/分子内的相互作用，为蛋白质的纳米力学性质提供独特的见解。然而，传统的基于AFM的力谱是耗时的，远远慢于标准成像。最近发展起来的微秒力谱（mFS）可以克服这一限制。mFS同时记录高分辨率地形图像和它们的力学图，从而区分具有不同力学性质的物种成为可能。在此，我们结合联合收割机标准液体原子力显微镜和mFS研究有趣的环状结构的hIAPP聚集体。聚集过程由带负电荷的氧化钽（Ta 2 O 5）介导，其具有高度生物相容性，并被选择用于模拟细胞膜的电荷和表面性质。Ta 2 O 5在中性缓冲液中像细胞膜表面一样带负电荷，并且能够在其表面积累带正电荷的hIAPP。此外，表面粗糙度可以为聚集提供更多的接触点。将hIAPP溶解在盐酸（HCl，pH 2）中以形成初始结构，其被认为是无规卷曲结构。通过以VHCl/VPBS = 1：9的比率添加磷酸盐缓冲盐水缓冲液（PBS; 10 mm，pH 7.4），实现80 mm hIAPP溶液的最终浓度。将新鲜的hIAPP溶液立即以10 mL/min的流速注入到含有预安装的Ta 2 O 5表面的40 mL液体室中。温和的流动使聚集期间的物理扰动最小化，使得Ta 2 O 5表面成为结构形成的主要贡献因素。样品在37 8 ℃下孵育48 h，然后用液体AFM和mFS进行原位表征。图1a中的AFM图像清楚地显示，hIAPP在给定的实验环境下形成各种尺寸和高度的环形结构，而不是通常在别处观察到的b-折叠原纤维。为了全面了解环状结构，提取一组环状圆（n=66）并进行统计分析。图1 B和1 c描述了瓣环直径[a] M的直方图和概率-概率图（P-P图）。陈角，澳-地Liu，P. Liu，Prof. Dr. Y.- M.清华大学化学系生物有机磷化学与化学生物学教育部重点实验室，北京100084中国）传真：（+86）10-62781695 E-mail：liym@mail.tsinghua.edu.cn [B] S. Zhang，Dr. K. Busuttil，F.贝森巴赫博士Dong跨学科纳米科学中心（iNANO）和奥胡斯大学物理与天文学系，8000奥胡斯码头区（丹麦）传真：（+45）8942-3690电子邮件：fbe@inano.au.dk dong@inano.au.dk [c] Prof. C.国家纳米科学技术中心北京100190中国）这篇文章的支持信息可以在http://dx.doi.org/10.1002/chem.201102215下获得。

Protein misfolding and accumulation are common features of protein amyloidosis-related diseases. Until now, the occurrence of protein amyloidosis was widely considered to be associated with more than forty types of human diseases, such as Alzheimer s, Prion, and Type II Diabetes mellitus (T2DM). In T2DM, human Islet amyloid polypeptide (hIAPP), a 37-amino acid peptide hormone secreted from bcells, forms amyloid aggregates in pancreatic islets and is involved in b-cell dysfunction. The generally accepted mechanisms involved in hIAPP-induced cytotoxicity include soluble hIAPP oligomer formation, membrane destabilization, membrane permeabilization, and oxidative stress. hIAPP is thought to attach itself to cell membranes, damaging it in the process and resulting in cell death. Experimental evidence suggests that the surface of lipid membranes can catalyze misfolding and accumulation of hIAPP, as a result of electrostatic interactions between the negatively charged membranes and the positively charged hIAPP under normal physiological conditions. Membrane mediated hIAPP aggregation indicates that the formation of the potentially toxic hIAPP species may be promoted in the presence of cellular membranes, which could be the link between protein–membrane interaction and hIAPP-induced b-cell dysfunction in the pathology of T2DM. Therefore, a thorough investigation of hIAPP aggregation on negatively charged surfaces is essential for a full understanding of the mechanism involved in surface-mediated amyloid peptide assembly. Atomic force microscopy (AFM), a powerful surface characterization technique, can detect different morphologies of amyloid proteins efficiently and successfully. 13] Besides recording surface morphology, AFM can detect inter/intramolecular interactions, providing a unique insight into the nanomechanical properties of proteins. However, conventional AFM-based force spectroscopy is time consuming and far slower than standard imaging. The recently developed microsecond force spectroscopy (mFS) can overcome this limitation. mFS records high resolution topographical images and their mechanical maps simultaneously, thereby distinguishing species with different mechanical properties becomes possible. Herein, we combine standard liquid AFM and mFS to investigate the intriguing annular structures of hIAPP aggregates. The aggregation process is mediated by negatively charged tantalum oxide (Ta2O5), which is highly biocompatible and is chosen to mimic the charges and surface properties of cell membrane. Ta2O5 is negatively charged in neutral buffer like the surface of cell membranes, and able to accumulate positively charged hIAPP at its surface. In addition, the surface roughness can provide more contact points for aggregation. hIAPP was dissolved in hydrochloric acid (HCl, pH 2) to form the initial structure, which is thought to be a random coil structure. By adding a phosphate-buffered saline buffer (PBS; 10 mm, pH 7.4) at a ratio of VHCl/VPBS = 1:9, a final concentration of 80 mm hIAPP solution was achieved. The fresh hIAPP solution was immediately injected into a 40 mL liquid chamber containing a pre-mounted Ta2O5 surface, at a flow rate of 10 mLmin . The gentle flow minimizes physical disturbance during aggregation, such that the Ta2O5 surface becomes the major contributing factor to structure formation. The samples were incubated for 48 h at 37 8C, then characterized in situ by liquid AFM and mFS. The AFM image in Figure 1 a clearly shows that hIAPP forms annular structures of various sizes and heights under the given experimental environment, rather than the b-sheet fibrils commonly observed elsewhere. To get a comprehensive understanding of the annular structures, a set of ring-like circles (n=66) were extracted and analyzed statistically. Figure 1 b and 1 c depict the histogram plot and probability–probability plot (P–P plot) of the annular diameters, [a] M. Chen, Q. Liu, P. Liu, Prof. Dr. Y.-M. Li Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education) Department of Chemistry, Tsinghua University Beijing 100084 (P.R. China) Fax: (+86) 10-62781695 E-mail : liym@mail.tsinghua.edu.cn [b] S. Zhang, Dr. K. Busuttil, Prof. F. Besenbacher, Dr. M. Dong Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy Aarhus University, 8000 Aarhus C (Denmark) Fax: (+45) 8942-3690 E-mail : fbe@inano.au.dk dong@inano.au.dk [c] Prof. C. Wang National Center for Nanoscience and Technology Beijing 100190 (P.R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201102215.

An investigation into the formation of annular aggregates of human islet amyloid polypeptide on tantalum oxide surfaces.

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