Name: fractionation for resolution of soluble and insoluble huntingtin species
Uploaded: 2018-02-27T14:30:00.000+00:00
Duration: 7 min 8 s
Description: Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

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。

动物伦理学声明-实验是严格按照《国家卫生研究院的实验室动物的护理和使用指南》和机构动物保育和使用委员会批准的动物研究议定书进行的 (IACUC) 在加利福尼亚大学欧文分校, AAALAC 认证机构。尽一切努力尽量减少动物的痛苦。
1. 裂解缓冲液的制备
<ol><li>通过150µm 过滤器制备 "可溶性" 裂解缓冲液 (10 毫米三 pH 7.4, 1% 个海卫 x 100, 10% 毫米氯化钠, 0.22 甘油) 和无菌过滤器。<ol>
<li>对于一个工作的 "可溶性" 溶解缓冲, 测量 n-Ethylmaleimide (NEM) 和彻底溶解在100毫米 phenylmethysulfonyl 氟 (PMSF) 溶解在100% 乙醇。 注: 裂解缓冲抑制剂: (i.) 20 毫米 n-ethylmaleimide (NEM);(二) 0.2 毫米 PMSF;(iii) 1 毫米原钒酸钠 (Na3VO4);(iv) 1 µg/毫升 leupeptin;(v) 1 µg/毫升抑肽酶;(vi) 20 毫米氟化钠 (NaF)。 注意: 在整个协议中使用 NEM 时, 戴口罩。必须使工作裂解缓冲液保持新鲜, 以确保活性抑制剂。</li>
		<li>对于具有蛋白酶抑制剂的可溶性溶解缓冲剂, 在以下顺序添加抑制剂: 氟化钠 (NaF), 高达容积与冷裂解缓冲, 原钒酸钠 (Na3VO4), 抑肽酶和 Leupeptin。</li>
	</ol>
</li>
	<li>对于 "不溶性" 溶解缓冲, 补充 4% sds, 增加500µL 20% sds 到2毫升的 "可溶性" 溶解缓冲。在室温下离开。 注: SDS 将沉淀, 当保持在4摄氏度;把这个留在室温下。最后溶解缓冲成分:10 毫米三 (pH 7.4);1% 海卫 X-100;150毫米氯化钠;10% 甘油;4% SDS (仅不溶)。</li>
</ol>2. 可溶性/不溶性分馏协议
注意: 有关示意图, 请参见图 1 。
<ol><li>标签两套管为每个样品: "可溶性" 和 "不溶性"。</li>
	<li>添加µL 的冰冷工作可溶性溶解缓冲剂, 补充蛋白酶抑制剂 (见 1.1.1) 组织。 注: X = 体积随组织量的不同而变化;请参见表 1。</li>
	<li>对于小鼠脑组织 (即, 纹状体), dounce 缓慢30倍, 在玻璃1毫升 douncer 和吸管成 "不溶" 标记管 (保持冰)。 注: 在启动 douncing 后要小心不要打破液体表面, 因为气泡会形成, 可能产生不完全均匀化。</li>
	<li>对于细胞 (即, HeLa, HEK293T), 用冷的 1x PBS 冲洗一次细胞。移除 PBS 并将最小的溶解缓冲量应用到细胞中, 用细胞刮刀提升。 注: 对于上述细胞线, 细胞被镀在 5 x 105细胞每6个井板和生长到接近融合。一个井相当于小鼠纹状体的体积。</li>
	<li>将匀浆或细胞裂解物转化为标签, "不溶性" 管和溶解在冰 1 h (开始时钟后, 最终管处理)。 注: 对于细胞裂解物, triturate 多次在开始孵化前分解细胞团簇。避免气泡的形成。简要地漩涡脉冲每个样品为 1 s 中途通过株溶藻。</li>
	<li>离心机样品在4°c 在 1.5万 x g 为20分钟。</li>
	<li>恢复上清为 "可溶性" 分数。<ol>
<li>删除与吸管, 小心不要扰乱颗粒/混浊层, 因为这将污染的分数。吸管可溶性分数为 "可溶性" 标记管。保持冰块。</li>
	</ol>
</li>
	<li>洗涤颗粒与500µL 溶解缓冲 (x2) 和离心机在4°c 在 1.5万 x g 在每洗涤以后5分钟;如果有些多云的层被 pipetted 了, 这是可以的。</li>
	<li>最后清洗后, 取出所有剩余的洗涤缓冲器只留下组织颗粒。 注意: 从这一点看, 不溶性样品现在应该保存在室温下。</li>
	<li>并用重悬颗粒与 X µL 裂解缓冲剂辅以 4% SDS (体积可根据颗粒大小调整; 请参见表 1)。</li>
	<li>油脂实验每个样品三十年代在室温与探针 sonicator (125 瓦特, 20 赫, 脉冲, 振幅 40%, 看见材料目录)。</li>
	<li>煮沸样品 (轧煮) 30 分钟;离心机简要 (十五年代) 在 6000 x g 为不丢失任何样品。</li>
	<li>执行 DC (洗涤剂兼容) 蛋白质化验 (参见材料表) 或劳瑞蛋白质测定以测量蛋白质浓度 (建议开始稀释1/5 样品在各自的溶解缓冲细胞培养和1/10 为组织裂解物)。 注: 由于高洗涤剂浓度, 蛋白质浓度将必须使用 DC 或劳瑞化验。</li>
	<li>整除样品为50µL 批次, 以避免冻融问题。 注意: 通过将两个分数存储在-20 摄氏度以上, 可以在进一步的生化分析之前暂停协议。</li>
</ol>3. 西方污点和量化
<ol><li>按照其他地方报告的西方印迹1,13通过稀释30µg 样本1:1 在加载缓冲区 (1.6x 加载染料, 1x 减少)。 注:30 µg 的蛋白质每分数是足够的, 但可以调整, 以最佳分辨率。3-8% 三乙酸酯聚酰胺凝胶应用于不溶性分数。推荐用于可溶性分数的 4-12% 双三凝胶。</li>
	<li>煮沸样品 (辗煮) 为5分钟离心机简要地 (十五年代) 在 6000 x g 收集样品。</li>
	<li>根据制造商关于减少样品的协议, 通过 SDS 页和西方印迹分析分数。 注: 当转移到0.45 µM 硝化棉膜时, 不溶性分数最佳。0.45 或0.2 µM 硝化纤维素可用于可溶性分数, 取决于蛋白质的大小的兴趣。对高分子量谷蛋白材料进行最佳转移可能需要进行一些优化。</li>
	<li>染色膜与全蛋白染色 (例如, MEMcode, 见材料表), 以可视化的蛋白质加载效率。 注: 西方印迹可以用平均像素强度进行分析。可溶性馏分可归一化为最适合于实验系统的保家蛋白。不溶性分数作为相对蛋白质丰度通过可逆蛋白染色或组蛋白3在分离印迹上的规范化验证。</li>
</ol>

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作者没有什么可透露的。

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析。
通过将可溶性分数的平均像素强度与负荷控制或保存在每个样本中的保留基因进行规范化, 可实现此检测的定量分辨率。如下所示, GAPDH 被用作可溶分数的一家保留基因, 因为它主要本地化细胞质 (图 1)。其他常用的保家蛋白有肌动蛋白、α蛋白和 Erk 1/2。然而, 为了保证稳态表达的正确性和自主规范化, 应优化合适的规范化蛋白质。虽然不溶性分数作为一个粗略的核分数, 如出现的组蛋白3和缺乏 GAPDH1, 分辨率 3-8% 页凝胶限制可探测性的常用核标记物。因此, 用原始信号强度值作为蛋白质丰度的相对表示, 不溶性分数适合于定量化。然而, 整个蛋白质标记, 如可逆蛋白污渍, 是用来控制蛋白质负载的变化, 并可作为规范化的因素, 不溶性分数。
此外, 虽然分馏提供了一种解决不溶性和可溶性 mHTT 物种的方法, mHTT 在整个疾病发病机制中经历了许多构象变化, 其中一些在不溶性分数中是不可区分的。此外, 一些构象可能有利于疾病的结果, 并作为稳定的清除中间体。因此, 重要的是要确定其他 mHTT 构象与发病机制的结果相关。一些优化可能是必要的可视化其他 mHTT 中间体。为解决这一问题, 如果聚合体抗低浓度, SDS 百分比可以增加到20%。
该协议表明蛋白质分馏条件和结果检测, 以发现各种构象和片段的 mHTT 积累和聚集的细胞培养和转基因小鼠模型的 HD。这种类型的生化蛋白分离被设想用于评估其他 HD 模型的疾病进展, 并可能有助于加深对细胞内 mHTT 动力学的理解及其对疾病病理学的影响。
分馏后, 样品可以用多种方式处理。SDS 页其次是西方印迹分析将解决可溶性和不溶性 mHTT 物种, 以提供一个相对评估的水平 mHTT 物种1。此外, 还可以评估额外生化化验中的分数, 以进一步评估 mHTT 物种。例如, 琼脂糖凝胶电泳 (年龄) 可以解决和检测可溶性, 寡聚 mHTT, 而过滤器迟缓 (FR) 检测是一个强大的工具, 以检测不溶性, 纤维 mHTT 物种 16, 17.
这项试验的另一个优点是对与发病机制相关的蛋白质 mislocalization 的评估。正如我们在18中演示的那样, 此分馏协议允许可视化和跟踪诸如 Rangap1 这样的蛋白质, 这些蛋白似乎被大的不溶性骨料所隔离, 并且可能无法在标准页凝胶上解析。此外, 由于这一化验也作为一个粗略的核/细胞质分离, 你也可以测量蛋白质的 mislocalization,例如, p65, 在 HD 鼠标模型1。
分析 mHTT 聚集和积累的一个显著动机是新的 HD 诊断和治疗方法的发展。抑制或反转 mHTT 聚合的化合物已从高通量屏幕和目标测试中识别出来1,12,19,20,21。然而, 考虑到解决 mHTT 的许多形式的困难, 聚合调制的评价并不总是可行的。传统的裂解方法可以处理可能含有不溶性骨料蛋白的颗粒状细胞碎片, 或者裂解缓冲剂可能不含足够的洗涤剂来增溶。此处描述的方法提供了一个平台, 用于隔离和检测 mHTT 的聚合形式, 可用作基于细胞或临床前体内评估的生化标记。
这里描述的化验可以应用于其他错误折叠蛋白。由于错误折叠蛋白的动态特性, 这些测定可以有效地与其他分析相结合, 揭示蛋白质稳态和聚集的深层机制。这些分析包括测量不同细胞分数 (图 1) 中聚合体及其分区的稳态水平, 或使用纯化的重组蛋白分析蛋白质的折叠/聚合。这些化验将有助于区分遗传或药理调控对蛋白质降解或聚集的直接或间接影响。在以前的体外synuclein 分级聚合评估22中, 我们已经将此工作应用于帕金森病小鼠模型, 以解决 synuclein23的各种构象。因此, 这项工作中描述的协议可应用于其他蛋白质错误折叠和聚合模型, 并可能有助于发现和发展针对聚合蛋白的新疗法。

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。

<table><tbody><tr><td>Sterile Filter</td><td>Millipore</td><td>SCGP505RE</td><td>Screw cap, sterile vaccum filter</td></tr><tr><td>1 mL Tissue Grinder, Dounce</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sonicator</td><td>Qsonica</td><td>Model Q125</td><td></td></tr><tr><td>DC Protein Assay</td><td>Biorad</td><td>5000111</td><td>Comparable to Lowry assay</td></tr><tr><td>Tris 1M buffer solution</td><td>Alfa Aesar</td><td>J60636</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>NaCl 5M solution</td><td>Teknova</td><td>S0251</td><td></td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td></td></tr><tr><td>20% SDS solution</td><td>Teknova</td><td>S0295</td><td></td></tr><tr><td>N-ethylmaleimide</td><td>Sigma</td><td>E1271</td><td></td></tr><tr><td>Phenylmethylsulfony flouride</td><td>Sigma</td><td>P7626</td><td>create 100mM stock solution in 100%EtOH, store at 4&amp;deg;C</td></tr><tr><td>Sodium orthovanadate</td><td>Sigma</td><td>S6508</td><td>Create 0.5M stock solution in water</td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td>Create 10mg/ml stock solution in water</td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td>Create 10mg/ml stock solution in water</td></tr><tr><td>Sodium Fluoride</td><td>Sigma</td><td>S4504</td><td>Create 500mM stock solution in water</td></tr><tr><td>Anti-HTT</td><td>Millipore</td><td>MAB5492</td><td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td></tr><tr><td>Anti-GAPDH</td><td>Novus Biologicals</td><td>NB100-56875</td><td>Use 1:1000 for soluble western blot</td></tr></tbody></table>

fractionation for resolution of soluble and insoluble huntingtin species

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。

可利用西方分析和过滤器延迟检测方法 (图 2) 测定可溶性和不溶性细胞裂解物后的分解。例如, HEK293T 细胞使用转染试剂 (例如, 脂质体 2000), 与 HTT 外显子1编码 cDNA 含有97谷氨酰胺重复15 其次是聚脯氨酸丰富的区域, 这些细胞被允许用5µM MG132 对44小时的细胞进行了治疗, 以阻止蛋白酶体或亚砜在收获和裂解前的18小时的控制。根据分馏协议, 突变体 HTT 外显子1解析为约 45 kDa 4-12% 双页凝胶上的可溶性单体, 并作为一个在 3-8% 三醋酸盐页凝胶 (图 2A) 的高分子量不溶积累的物种。可溶性单体可以量化和规范化的一家保持蛋白 (这里显示为 GAPDH)。谷蛋白、不溶性 mHTT 以平均像素强度量化为相对蛋白质丰度。当用抑制剂 MG132 处理时, mHTT 可溶性单体明显减少, 而高分子量蛋白、不溶性 mHTT 的相应增加。
不溶性分数可以通过其他技术进行分析, 如过滤器延迟检测, 以解决不溶性, 纤维 mHTT16 (图 2B)。HeLa 细胞与上述 97 q HTT 外显子 1, 或 His-SUMO-1 或 His-SUMO-2 同样转染。在分馏前, 用亚砜或 MG132 对细胞进行了18小时的治疗, 并用西方印迹和过滤器延缓试验进行分析。数据显示, 抗原相扑异构体的存在增加了高分子量和纤维的不溶性 mHTT。添加 MG132 加剧了不溶性 mHTT 物种的增加。HWM mHTT 物种也可以通过过度表达或减少 HD 疾病目标, PIAS1 在 HeLa 细胞13和鼠标白芨1 (图 2C-D) 来调制。PIAS1 在快速发展的 HD 小鼠模型中被击倒, R6/2 使用病毒传递 siRNA 对抗 PIAS1, 减少了高分子量不溶性 mHTT 的积累。R6/2 小鼠 PIAS1 的病毒过度表达导致高分子量不溶性 mHTT (图 2D)1显著增加。分数之间溶解度的变化是可量化的, 表明该协议可以跟踪病原突变蛋白种类的调制, 使之成为临床疾病干预前生化评估的适宜技术。mHTT 的不溶性、纤维种的变化也捕获蛋白质种类之间的通量, 这些蛋白种通过 MG132 治疗或调节潜在的治疗目标 (如 PIAS11) 进行调制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57082/57082fig1v2.jpg"> 图 1: 可溶性/不溶分馏协议和蛋白质分辨率.(A) 用于分馏协议的可视化管线。这里描述的协议可以应用于小鼠组织或细胞培养样本, 但适合于其他样本类型的适应。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57082/57082fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57082/57082fig2.jpg"> 图 2: 可溶和不溶分馏的代表性结果和分析.(A) HEK293T 细胞的可溶性和不溶性馏分, 裂解物 4-12% 双, 3-8% 三乙酸酯页凝胶, 恭敬地解决。可溶性分数归一化, GAPDH 负荷控制显示 mHTT 外显子1可溶性单体基于接触 MG132 的波动。不溶性分数表明, mHTT 的高分子量谷蛋白积累的种类也被 MG132 处理。(B) 使用西方印迹和过滤器延迟分析裂解物细胞的可溶性和不溶性成分: hela 细胞转染 His-SUMO-1 或 SUMO-2 和/或 97 q HTT 外显子 1, 5 µM MG132 处理18小时. * (C)从 HeLa 细胞 overexpressing 97 q HTT 外显子1和抗原 PIAS1 或 siRNA 治疗 PIAS1 的可溶性和不溶性分馏显示了调节一个潜在的治疗靶后的高分子量蛋白 mHTT 积累的调控. * (D)microRNA 对 PIAS1 治疗的小鼠白芨不溶性分数及对 3-8% 三醋酸盐页凝胶的处理, 显示 HWM 不溶性 mHTT 的调制。面板 (B-c) 已使用权限13进行了修改。面板 (D) 已使用权限1进行了修改。* 从 O ' 洛克et转载。13具有权限. ** 转载自 Ochaba et 。1 , 并具有 Elsevier 的权限。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57082/57082fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组织 (每半球)</td>
			<td>近似重量 (毫克)</td>
			<td>容积可溶性 (µL)</td>
			<td>体积不溶 (µL)</td>
		</tr>
<tr>
<td>小鼠纹状体</td>
			<td>30</td>
			<td>250</td>
			<td>150</td>
		</tr>
<tr>
<td>小鼠皮质</td>
			<td>100</td>
			<td>500</td>
			<td>250</td>
		</tr>
<tr>
<td>小鼠海马</td>
			<td>30</td>
			<td>250</td>
			<td>150</td>
		</tr>
<tr>
<td>小鼠小脑</td>
			<td>40</td>
			<td>250</td>
			<td>150</td>
		</tr>
<tr>
<td>鼠标骨骼肌肉 *</td>
			<td>100</td>
			<td>300</td>
			<td>150</td>
		</tr>
<tr>
<td>小鼠肝脏 (裂片)</td>
			<td>100</td>
			<td>400</td>
			<td>200</td>
		</tr>
<tr>
<td colspan="3">* 结缔组织形成乳白色界面;移除以避免污染</td>
			<td></td>
		</tr>
</tbody></table>表 1: 建议的卷 (X) 用于可溶和不溶性裂解缓冲器的组织特异性分馏.基于物料起始量的不溶性溶解缓冲容积, 可根据不溶性粒径进行相应调整。

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

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。

可溶性和不溶性杭丁顿蛋白的分离分解

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

fractionation-for-resolution-soluble-insoluble-huntingtin

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.5K 次观看.  University of California Irvine. 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。

视频: Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

科研

生物化学

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

利用相扑融合策略生成国产、未加标签的杭丁顿蛋白 Exon1 单体和纤维

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

使用瞬时表达系统高效、可扩展地生产哺乳动物细胞中的全长人类亨廷顿蛋白变异

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

生物学

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

可溶性和不溶性杭丁顿蛋白的分离分解

摘要

探索更多视频

此视频中的章节

脑膜分离法

人死后脑中洗涤剂不溶性蛋白的富集

化学感受器配体结合域的高效复用与纯化方法

利用相扑融合策略生成国产、未加标签的杭丁顿蛋白 Exon1 单体和纤维

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

使用瞬时表达系统高效、可扩展地生产哺乳动物细胞中的全长人类亨廷顿蛋白变异

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

Hsp104纯化，蛋白质Disaggregase

从真核细胞中快速体内固定和分离转化复合物

可溶性和不溶性杭丁顿蛋白的分离分解

摘要

探索更多视频

此视频中的章节

脑膜分离法

人死后脑中洗涤剂不溶性蛋白的富集

化学感受器配体结合域的高效复用与纯化方法

利用相扑融合策略生成国产、未加标签的杭丁顿蛋白 Exon1 单体和纤维

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

使用瞬时表达系统高效、可扩展地生产哺乳动物细胞中的全长人类亨廷顿蛋白变异

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

Hsp104纯化，蛋白质Disaggregase

从真核细胞 中快速体内 固定和分离转化复合物

从真核细胞中快速体内固定和分离转化复合物