可溶性和不溶性杭丁顿蛋白的分离分解
Summary
本文介绍了一种从小鼠脑和细胞培养中分离不溶性和可溶性突变体杭丁顿蛋白的方法。该方法对杭丁顿蛋白蛋白通量和艾滋病在分析疾病发病机制中蛋白质稳态和扰动存在时的定性和定量具有重要意义。
Abstract
错误折叠蛋白的积累是亨廷顿病 (HD) 和许多其他神经退行性疾病的病理中枢。具体来说, HD 的一个关键病理特征是突变 HTT (mHTT) 蛋白在高分子量复合体中的异常积累, 以及由片段和其他蛋白质组成的细胞内包裹体内。传统的测量和理解各种形式的 mHTT 的贡献的方法包括荧光显微术, 西方印迹分析, 过滤器陷阱化验。
然而, 这些方法大多是构象特异性的, 因此由于聚合溶解度和分辨率的复杂性, 无法解决 mHTT 蛋白通量的全态。
为确定聚合 mHTT 和各种改良的形式和配合物, 细胞团聚体和碎片的分离和增溶是强制性的。在这里, 我们描述了一种分离和可视化可溶性 mHTT、单体、寡聚物、片段和不溶性高分子量 (mHTT) 积累的方法。高分子量蛋白 mHTT 跟踪与疾病进展, 对应于鼠标行为读数, 并已受益于某些治疗干预措施1。这种方法可用于小鼠大脑, 周边组织和细胞培养, 但可以适应其他模型系统或疾病的上下文。
Introduction
蛋白质质量控制网络的中断, 确保适当折叠和降解细胞蛋白, 可能是阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和其他 “蛋白质错误折叠” 病理学的核心。异常2,3。因此, 对 proteostasis 网络组件及其对病理学的贡献的详细了解, 对于发展改进的治疗干预至关重要。hd 是由 hd 基因内的 CAG 重复异常扩展引起的, 导致杭丁顿蛋白 (HTT) 蛋白4中 polyglutamines (polyQ) 的扩张.HD 发病机制的一个一致的病理特征是产生的错误折叠, 积累和聚集的 HTT 在大脑区域和周围组织, 这已经表明干预的几种方式, 正常细胞稳态和功能5,6. 虽然 HTT 无所不在表达, 纹状体中的中刺神经元有选择性的脆弱性, 最明显的影响与显着皮质萎缩也与 HD 发病机制。
HD 患者和动物模型脑中 HTT 聚集的形成, 通常作为 mHTT7的疾病进展和显性负增益功能的代理标记。虽然蛋白质错误折叠和聚集可能导致 mHTT 毒性的精确机制仍不清楚, 但在 HD 患者和各种动物模型脑中形成这些包裹体是一个不变的、不可避免的特征。夹杂物似乎主要由累积的 HTT 片段组成, 其中包含 N 终端扩展 polyQ, 表明对全长 HTT 的分解和处理以及可选拼接8,9可能会起到在 HD 发病机制中的重要作用. n-端 HTT 片段可能构成一种病理形式的 HTT, 可以快速聚合, 并加速或传播聚合过程10。
然而, 这些包裹体的存在不一定与 HTT 诱导的毒性或细胞死亡有关11。HTT 已被建议从可溶性单体通过可溶性寡聚物种和β薄片纤维到不溶性骨料和夹杂物的聚合过程中, 12.通过标准生化检测方法解决这些不同的蛋白质物种在低洗涤剂裂解缓冲液中的稳定性和使用标准生化检测的难以可视化方面一直是一个挑战。因此, 方法学考虑对于检测累积和聚集 mHTT 的程度和模式至关重要。
这里提出的协议提供了一种方法, 以可视化几个中间体的 HTT, 特别是形成一个不溶性的高分子量 HTT 物种, 似乎密切跟踪 HD 发病机制和疾病进展1,13 ,14。能够解决和跟踪多种 mHTT 为研究人员提供了一种生化工具来研究疾病的发病机制和评估潜在的治疗干预, 通过其调节和对疾病发病机制的影响。
Protocol
Representative Results
Discussion
为了确保一致性和定量的结果, 上述协议需要一些预防措施。首先, mHTT 在两个组分将自发地形成聚集体随着时间的推移在多个冻融循环, 特别是当在高浓度。因此, 冻结蛋白质眼影的整除数是至关重要的, 在按照上文议定书所述的方法运行化验之前, 只解冻所需的体积。此外, 如果不溶性分数产生白色沉淀后解冻, 重建可能是必要的额外的 5 s 超声波脉冲, 5 分钟煮沸, 并重新定量之前通过生化化验分?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NIH (RO1-NS090390) 的支持。我们还要感谢琼 Steffanfor 博士在这项试验的开发过程中提供的技术援助和讨论。
Materials
| Sterile Filter | Millipore | SCGP505RE | Screw cap, sterile vaccum filter |
| 1 mL Tissue Grinder, Dounce | Wheaton | 357538 | |
| Sonicator | Qsonica | Model Q125 | |
| DC Protein Assay | Biorad | 5000111 | Comparable to Lowry assay |
| Tris 1M buffer solution | Alfa Aesar | J60636 | |
| Triton X-100 | Fisher | BP151-100 | |
| NaCl 5M solution | Teknova | S0251 | |
| Glycerol | Fisher | BP229-1 | |
| 20% SDS solution | Teknova | S0295 | |
| N-ethylmaleimide | Sigma | E1271 | |
| Phenylmethylsulfony flouride | Sigma | P7626 | create 100mM stock solution in 100%EtOH, store at 4°C |
| Sodium orthovanadate | Sigma | S6508 | Create 0.5M stock solution in water |
| Leupeptin | Sigma | L2884 | Create 10mg/ml stock solution in water |
| Aprotinin | Sigma | A1153 | Create 10mg/ml stock solution in water |
| Sodium Fluoride | Sigma | S4504 | Create 500mM stock solution in water |
| Anti-HTT | Millipore | MAB5492 | Use 1:1000 for western blot, 1:500 for filter retardation assay |
| Anti-GAPDH | Novus Biologicals | NB100-56875 | Use 1:1000 for soluble western blot |
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