Name: recombinant α- β- and γ-synucleins stimulate protein phosphatase 2a catalytic subunit activity in cell free assays
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Description: Watch this Scientific Journal Video about Recombinant Î±- Î²- and Î³-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays at JoVE.com

作者赞赏以前的实验室成员 Mackett, 桑德拉 Slusher 和杰陈谁工作的优化方法的努力。作者还感谢国立卫生研究院, 迈克尔 j ·福克斯基金会, 德克萨斯理工大学健康科学中心埃尔帕索学术活动研究项目 (SARP), Lizanell 和科尔伯特科德韦尔基金会, 多系统萎缩联盟,霍伊的家庭研究, 和安娜。

1. 缓冲器和试剂的制备
<ol><li>
对硝基苯基磷酸酯 (pNPP) 缓冲液的制备
	<ol>
<li>准备50毫升的 pNPP 缓冲器, 如下所示。</li>
		<li>称量出0.30285 克的三基色 (见材料表), 并将其溶解在25毫升的超纯去离子水中。<ol>
<li>添加41.6 µL 120 mM CaCl2。调整到 pH 值7.0 与 1 M HCl 和带来最后的解决方案体积到50毫升与超纯去离子水。</li>
			<li>确保最终浓度是50毫米的三盐酸, 100 毫米 CaCl2, pH 7.0。</li>
			<li>将 pNPP 缓冲区存储在 4 oC 处。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
对于孔雀石绿解决方案, 请准备以下内容。
	<ol>
<li>准备解决方案 A , 将32.8 毫升浓缩 hcl 添加到67.2 毫升的超纯去离子水中, 以创建4米盐酸溶液。 注意: 孔雀石溶液是在通风柜中使用一次性手套、面罩和安全护目镜制备的。</li>
		<li>通过称量4.2 克钼酸铵并将其添加到95.8 毫升溶液 A 中, 准备溶液 B 。</li>
		<li>通过称量0.045 克孔雀石绿草酸盐和添加超纯去离子水来制备解决方案 C , 使最终的总容量为100毫升。</li>
		<li>通过将解决方案 B 和 C 在 1:3 (v/v) 比混合, 在室温下搅拌1小时, 准备解决方案 D 。</li>
		<li>通过使用50毫升注射器的0.22 µm 孔径硝化纤维膜过滤装置过滤解决方案 D 。</li>
		<li>存储准备的孔雀石溶液在 4 oC 保护免受光照。</li>
	</ol>
</li>
	<li>
活化剂的制备
	<ol>
<li>要准备一个1% 吐温20溶液, 吸管10µL 的吐温20成990µL 的超纯去离子水 (v/v) 然后漩涡, 直到补间20完全溶解。</li>
	</ol>
</li>
	<li>
孔雀石绿吐温 20 () 工作液的制备。
	<ol>
<li>将激活器 (步骤 1.3) 与孔雀石绿解决方案 (解决方案 D, 步骤 1.2.5) 的比例为 1:100 (例如1 µL 活化剂到99µL 孔雀石绿溶液)。</li>
	</ol>
</li>
	<li>
苏氨酸酪 (KRpTIRR) (pT) 基质的制备。
	<ol>
<li>准备一个2毫米的股票, 在冰上放置一个1毫克酪瓶, 添加550µL 的超纯去离子水, 和漩涡轻轻地溶解。<ol>
<li>使〜10等分和存储 pT 在-20oC。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
核制剂
	<ol>
<li>使用一次性手套、面罩和安全护目镜准备 synucleins 在通风罩中。</li>
		<li>重1毫克的重组核在1毫升超纯去离子水中, 最终浓度为1毫克/毫升。</li>
		<li>轻轻的涡旋以溶解和放置在冰上。</li>
		<li>准备50µL 等分并存储在-80 oC 上。</li>
	</ol>
</li>
	<li>
磷酸酯标准的制备
	<ol>
<li>准备0.1 毫米磷酸盐库存溶液如下。<ol>
<li>称0.1361 克2PO4 , 并在烧杯中放置80毫升超纯去离子水。加入搅拌棒和混合溶解。把所有的解决方案转移到一个毕业的气缸, 并带来最终的总体积为100毫升, 超纯去离子水。</li>
			<li>通过使用50毫升注射器的0.22 µm 孔径硝化纤维膜过滤器来过滤整个溶液;最后的浓度是10毫米。</li>
			<li>稀释溶液 (10 mM) 1:100;最终浓度将是0.1 毫米磷酸盐 (PO4) 用于化验标准。</li>
			<li>将磷酸盐库存解决方案存储在 4 oC 上。</li>
		</ol>
</li>
		<li>有关限制稀释的表 1, 用于制定 PO4标准, 最终总容量为1250年µL。按表1添加适量的磷酸盐和超纯去离子水到1.5 毫升管。<ol>
<li>在-20 oC 处存储准备好的 PO4标准。</li>
		</ol>
</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="7">磷酸盐标准</td>
		</tr>
<tr>
<td>0.1 mM PO4解决方案 (mL)</td>
			<td>0</td>
			<td>75</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1200</td>
		</tr>
<tr>
<td>超纯水去离子水 (毫升)</td>
			<td>1250</td>
			<td>1175</td>
			<td>1100</td>
			<td>950</td>
			<td>650</td>
			<td>50</td>
		</tr>
<tr>
<td>PO4的最终浓度 [pmol]</td>
			<td>0</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1200</td>
			<td>2400</td>
		</tr>
</tbody></table>表 1: 磷酸盐标准。
2. PP2A 测定对重组 Synucleins 的反应
注: 每个反应的最终总体积为60µL, 因此实验者必须适当调整 pNPP 缓冲器的初始容积, 以容纳每个样品的 synucleins 量。在大多数情况下, pNPP 缓冲区的容量将介于40和44µL 之间。
<ol><li>
准备每 PP2Ac 反应如下。
	<ol>
<li>在1.5 毫升的管, 增加39µL 的 pNPP 缓冲和地方在冰上。</li>
		<li>添加1µL (105 ng/µL 或 0.0007 U/µL) 的人类重组 PP2Ac (rPP2Ac)。</li>
		<li>增加4µL 0, 1.5, 7.5, 15 或30µM synucleins 到 rPP2Ac 在 pNPP 缓冲器为最后的浓度 0, 0.1, 0.5, 1.0 和2.0 µM 和孵育在4° c 为30分钟的旋转单位;最后一卷将是60µL。</li>
		<li>30分钟后, 将样品移动到冰上, 加入16µL 2 mM 的铂基板。</li>
		<li>在间歇晃动的水浴中孵育每 PP2A 化验混合物10分钟, 30 ° c。</li>
		<li>对平底96井板, 在从低到高的重复井中添加25µL 的每个 PO4标准 (0、150、300、600、1200和 2400 pmol)。</li>
		<li>接下来添加25µL 的每一个实验样品 (PP2A 化验±核), 准备在步骤 2.1.1 2.1.6, 以重复井在同一 96-井板。</li>
		<li>接下来, 在每个包含标准和样品的井中添加75µL 的工作解决方案。</li>
		<li>室温下留板10分钟。</li>
		<li>观察在磷酸盐水平上有可测量的差异的样品的颜色变化。</li>
	</ol>
</li>
</ol>3. 光度分析
<ol><li>用波长设置为 630 nm29的分光光度板阅读器分析包含标准和样品的96井板。</li>
	<li>绘制产生的吸收曲线 (在 630 nm 上读取的样本), 并注意它保持线性 2400 pmol 浓度的 PO4。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine/threonine+protein+phosphatase+assays.">Serine/threonine protein phosphatase assays.</a> Curr Protoc Mol Biol. Chapter 18, (2010).</li><li>Anwar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+alterations+to+the+nigrostriatal+system+in+mice+lacking+all+three+members+of+the+synuclein+family.">Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.</a> J Neurosci. 31 (20), 7264-7274 (2011).</li><li>Vargas-Medrano, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+FTY720-based+compounds+stimulate+neurotrophin+expression+and+phosphatase+activity+in+dopaminergic+cells.">Novel FTY720-based compounds stimulate neurotrophin expression and phosphatase activity in dopaminergic cells.</a> ACS Med Chem Lett. 5 (7), 782-786 (2014).</li><li>Swingle, M., Ni, L., Honkanen, R. E., Moorhead, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Small-molecule+inhibitors+of+Ser/Thr+protein+phosphatases.">Chapter 3, Small-molecule inhibitors of Ser/Thr protein phosphatases.</a> Methods in Molecular Biology. 365, Protein Phosphatase Protocols, 23-38 (2007).</li></ol>

Dr. 佩雷斯是1999年至2011在匹兹堡大学医学院神经学系的成员。

采用 pT 的孔雀石绿法测定 PP2Ac 活性, 因为它比使用32P 标记基板的经典方法更容易执行。这种分析的另一个优点是, 放射性同位素会带来一些风险, 而且成本高昂, 特别是32P 标记的分子, 它们的半衰期很短, 这就限制了在试剂到期前可以执行的化验数量。使用孔雀石绿而不是放射性, 也消除了处理32P 废料的需要。在测定丝氨酸/苏氨酸磷酸的活性时, 孔雀石绿测定法与使用基体 p-硝基苯 (pNPP) 的另一种色度分析方法相比, 也相当敏感, 这是非选择性的, 因为它可以化由广泛的酵素32。
商业孔雀石绿包测量 PP2A 的活动已经有一段时间, 并在实验室以前使用。然而, 当做许多孔雀石绿的化验, 这种套件变得令人望而却步的昂贵。此外, 用于 PP2A 活动的商用套件只稳定一年, 同时具有粉状和适当贮存的试剂, 可提供在结构上较长时间内稳定的现成的化验组件。
在测试 bSyn 和 gSyn 之前, 异步电动机被用作一种积极的控制来刺激 PP2Ac 的活动, 并确定化验所需的 PP2Ac 量, 还要确认所产生的数据与标准曲线保持比例 (150-2400 pmol 的 PO4).值得注意的是, 如果异步电动机刺激 PP2Ac 太强烈, 虽然反应通常是线性的, 数据将脱落的规模和化学品在该方案的沉淀, 使它无法测量准确数量的发布自由 PO4与一个板块读者.
鉴于异步电动机有助于 PP2Ac 活动在体内和在体外12,19,21,23,25, 所有 synucleins 有相当的同源性, 它已经假设所有核家族成员都可以在细胞自由测定中刺激 PP2Ac。使用这里描述的比色法, 发现事实上, 所有三重组 synucleins (异步电动机, bSyn, gSyn) 刺激 PP2Ac 活动, 提高的可能性, 所有三 synucleins 可能执行类似的功能在神经系统。然而, 大脑从帕金森氏症或痴呆与路易体体病例有较少的 PP2Ac 活动的组织中含有高度聚集的异步电动机23。鼠组织窝藏路易体体样病变也表现为减少 PP2Ac 活动25。这一点, 以及图 3中的新数据以及来自异步电动机击倒鼠标12的先前数据, 强烈建议 bSyn 和 gSyn 都不能补偿大脑中可溶性异步电动机的丢失, 或者说那些核同系物可能不与 PP2Ac 相关联, 如异步电动机所示 (图 3)。艾伦脑图谱显示定位的所有三核基因在许多脑区, 虽然三核淘汰赛小鼠已生成9,33, PP2Ac 活动尚未评估在该模型。有趣的是, 异步电动机磷酸化在 Ser129 上的马球样激酶 2, 是不太能够激活 PP2Ac12, 但这里显示的证据 bSyn 表明 bSyn 磷酸化不太可能影响 PP2Ac 的活动, 因为很少 bSyn 下来PP2Ac 在鼠标的大脑中 (图 3)。同时, 数据表明, 内源性 PP2Ac 活性调节剂, 如异步电动机, 可能有助于健康和疾病。
这里概述的协议是一个简单而强大的技术, 以确定 PP2Ac dephosphorylate 的能力, 以响应各种处理的酪基板。例如, 同样的方法可以用来测试其他 PP2Ac 激活剂, 如神经和潜在的新疗法34, 或评估 PP2Ac 抑制剂的影响,体外。同样重要的是要指出, 类似的化验可以测量 PP2Ac 的活动, 已被1D6 沉淀从细胞提取物或身体组织, 如先前所描述的作者12,19, 25. 孔雀石绿检测的未来应用存在于测量 PP2Ac 活性之外, 因为 ATP 活性也可以通过这种检测来确定。
注意, 每一次独立的 PP2Ac 化验都必须生成标准曲线。这是强制性的, 以确保所有使用的试剂是无磷酸盐, 由于自由磷酸盐人工增加背景信号和产生错误的结果。在化验的当天, 必须准备足够的新鲜的解决方案, 所有样品都要化验。此外, 公司是唯一的好, 它是做的一天, 往往在几个小时后沉淀。从供应商购买的 PP2Ac 必须 aliquotted, 并在收到后立即保存, 以防止冻结解冻循环减少其活动。由于其他磷酸可以 dephosphorylate pT 衬底 (例如PP1 和 PP535), 因此在分析 PP2Ac 未被分离的细胞或组织提取物时, 必须通过柱上的样品来除去游离磷酸盐。和特定的磷酸抑制剂必须用于确认磷酸酶的特异性, 如前所述18。

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>
One of the authors&#39; names was corrected from:
Brian Marcus
to:
Brian S. Marcus

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>

Erratum: Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

synucleins 在脑和脊髓中的分布的研究表明, 所有三 synucleins 是丰富的神经组织, 虽然不同的本地化模式已经报告了1。例如, 异步电动机是最丰富的在前部位的大脑皮层和基底节2,3, bSyn 在大脑和脊髓中有更均匀的分布4,5, 而 gSyn 更丰富的脊髓和周边神经节6。虽然所有 synucleins 的一些胚胎表达可能发生, 三同系物在新生儿期间变得更加丰富4,5,6,7,8。值得注意的是, 来自核三联击倒小鼠的数据表明, 所有三 synucleins 的丢失导致年龄开始的神经元功能障碍9, 支持重要的核功能在中枢神经系统 (CNS)10,11. 目前尚不清楚核三联击的小鼠是否显示 PP2Ac 分布或活动的变化。来自单个核击倒小鼠的数据表明, 单异步电动机的丢失能够显著减少脑部12的 PP2Ac 活动。除了中枢神经系统, 所有的 synucleins 本地化到其他组织的整个身体13,14,15。
由于其与神经16,17的成熟的遗传链接, 异步电动机是最好的研究三 synucleins。佩雷斯实验室长期以来一直致力于定义异步电动机的正常功能活动, 特别是在 CNS11、12、18、19、20、21中, 22,23和最近的外围组织24,25。其中, 发现异步电动机在刺激 PP2Ac 活动中起着关键的调节作用19。PP2A 活动对正常的脑功能有广泛的贡献, 包括对多巴胺生产的调节26。PP2A 全包括三独立亚基, 催化 C 亚基, PP2Ac, 脚手架 a 亚基, 和几个不同的管理/目标 B 亚基, 帮助本地化 PP2A 特定亚细胞微, 从而提供 PP2A功能多样性27。证据显示, 异步电动机与 PP2Ac 的相互作用对其磷酸酶活性有显著的贡献在体外和在体内12,19,21,23, 25。当异步电动机积累在蛋白质聚集物, 因为它做当路易体身体形成在帕金森氏症的神经元和老年痴呆与路易体身体 (DLB), 组织与聚集的异步电动机减少了 PP2Ac 活动23,25, 当可溶性异步电动机水平降低。鉴于所有三 synucleins 具有显著的同源性28 (异步电动机与 bSyn 和56% 与 gSyn 共享66% 身份), 并且异步电动机有助于激活 PP2Ac, 因此假设核蛋白家族的所有成员都可能能够增加 PP2Ac 活动, 至少是体外。为了评估这一点, PP2Ac 的活动是为了响应重组异步电动机, bSyn 和 gSyn 使用一个简单的色度测定, 仿照 Fathi 的方法et al.29. 本文详细介绍了该方法和新的研究结果。

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M Trizma Base</td><td>Sigma-Aldrich</td><td>T15003</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;(calcium chloride)</td><td>Fisher Scientific</td><td>C70-500</td><td></td></tr><tr><td>Concentrated HCl&amp;nbsp;&amp;nbsp;</td><td>JT Baker</td><td>9535-33</td><td></td></tr><tr><td>Ammonium molybdate</td><td>Sigma-Aldrich</td><td>A1343</td><td></td></tr><tr><td>Malachite green oxalate salt</td><td>Sigma-Aldrich</td><td>M6880</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>Threonine phosphopeptide (KRpTIRR)</td><td>New England peptide LLC</td><td>BP12-011</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate)</td><td>Sigma-Aldrich</td><td>795488</td><td></td></tr><tr><td>Recombinant human PP2A C subunit</td><td>Cayman Chemical&amp;nbsp;</td><td>10011237</td><td></td></tr><tr><td>Recombinant human &amp;alpha;-synuclein</td><td>rPeptide</td><td>S-1001-2</td><td></td></tr><tr><td>Recombinant human &amp;beta;-synuclein</td><td>rPeptide</td><td>S-1003-2</td><td></td></tr><tr><td>Recombinant human &amp;gamma;-synuclein</td><td>rPeptide</td><td>S-1007-2</td><td></td></tr><tr><td>PP2Ac 1D6 antibody</td><td>Millipore</td><td>05-421</td><td></td></tr><tr><td>PP2Ac antibody</td><td>Novus Biologicals</td><td>NB100-92217</td><td></td></tr><tr><td>aSyn antibody C-20</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>bSyn antibody EP1537Y</td><td>Novus Biologicals</td><td>NB100-79903</td><td></td></tr><tr><td>gSyn antibody E-20</td><td>Santa Cruz Biotechnology</td><td>sc-10698</td><td></td></tr><tr><td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td><td>Thermo-Fisher Scientific</td><td>89882</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multiskan Spectrum Plate Reader</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>51118650</td><td></td></tr><tr><td>pH Meter&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>13-636-AB15P</td><td></td></tr></tbody></table>

recombinant &#945;- &#946;- and &#947;-synucleins stimulate protein phosphatase 2a catalytic subunit activity in cell free assays

α-核 (异步电动机), β-核 (bSyn), 和γ-核 (gSyn) 是一个保守的家庭的成员类似的伴侣的蛋白质, 是高度表达的脊椎动物神经组织。在三 synucleins, 只有异步电动机有强烈的牵连神经退行性疾病, 如帕金森氏病, 痴呆与路易体体, 和多系统萎缩。在研究正常异步电动机功能时, 数据表明异步电动机刺激了大量表达的 dephosphorylating 酶的催化亚单位的活性, PP2Ac体外和体内。先前的数据表明, 异步电动机聚集在人脑减少 PP2Ac 活动在路易体体病理, 其中可溶性异步电动机已成为不溶性。然而, 由于所有三 synucleins 在氨基酸序列中有相当的同源性, 实验被设计来测试是否都能调节 PP2Ac 活性。采用重组 synucleins 和重组 PP2Ac 蛋白, 采用孔雀石绿比色法测定活性。数据显示, 所有三重组 synucleins 刺激 PP2Ac 活性在无细胞的检测, 提高的可能性, 保守的同源性之间的 synucleins 可能赋予所有三同系物的能力绑定和激活 PP2Ac。然而, 沉淀数据表明, PP2Ac 调制可能发生在异步电动机和 PP2Ac 之间的内生相互作用的在体内。

蛋白质的功能和活性可以改变后的翻译修改, 如磷酸化。许多蛋白质可以磷酸化的激酶, 或化的磷酸酶。在蛋白质磷酸酶 2A (PP2A) 的情况下, 这种 multisubunit 磷酸酶促进磷酸的丝氨酸和苏氨酸残留在广泛的磷酸。异常的 PP2A 活动可能导致失调的蛋白质功能发生在许多疾病, 包括帕金森氏症的异步电动机可以成为 hyperphosphorylated, 并在阿尔茨海默氏症的地方 tau 蛋白可以成为 hyperphosphorylated。这为优化 in-house 程序以快速评估 PP2Ac 活动提供了依据, 并确定异常 PP2Ac 活动是否可能导致细胞功能障碍。
使用这种无细胞比色法测定, PP2Ac 活性是通过量化的自由 po4从 pT 衬底 (图 1A) 切割的数量, 并与标准曲线中的 po4的已知 picomolar 量进行比较 (图1B).PP2Ac 活动也在响应0.0、0.1、0.5、1.0 和2.0 µM synucleins (图 1A) 时进行测量, 与在没有添加核 (位于图 1A中的大箭头) 中的基线 PP2Ac 活动相比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55361/55361fig1.jpg"> 图 1: PP2A 活化的重组 synucleins, 是衡量相对于已知水平的磷酸盐在标准.在 (a) 中, 异步电动机、bSyn 和 gSyn 在 0-2 µM 蛋白质浓度范围内进行了评估, 以便与 0-2400 pmol PO4标准进行比较。(B) 生成孔雀石绿 PP2Ac 试验的标准曲线, 以提供已知数量的磷酸盐, 以计算在实验条件下获得的值。在所有情况下, 当 PP2Ac 活动增加时, 从 pT 衬底中切割出来的自由 PO4的数量会增加并产生较深的绿色。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
结果表明, 在初始孵化步骤中加入 synucleins PP2Ac 可以增加自由 PO4的量, 从而显示出增加的 PP2Ac 活动。经过多次重复的统计分析表明, 所有三重组 synucleins (异步电动机、bSyn、gSyn) 显著增加了 PP2Ac 活动 (图 2)。这些结果表明, 可溶性细胞 synucleins 量的变化可以改变 PP2Ac 活性;因此可能影响细胞稳态。然而, 不知道, 如果所有三 synucleins 贡献激活 PP2Ac在体内, 虽然数据在图 3建议异步电动机可能有助于 PP2Ac 活动在体内。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55361/55361fig2.jpg"> 图 2: PP2Ac 活动由所有三 synucleins 激发.图示的 PP2Ac 活化反应不同浓度的重组异步电动机, bSyn 或 gSyn 显示。所有三重组 synucleins 刺激 PP2Ac 活动。异步电动机在大多数实验中似乎是稍微更有力, 虽然 ANOVAs 执行使用统计软件是非常相似的整体。数据代表4-9 独立实验的平均± SEM。ns, 不显着;* p < 0.05;** p < 0.01;p < 0.0001。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了评估哪些核 (s) 可以与大脑中的 PP2Ac 相互作用, co-immunoprecipitation (co-IP) 进行了使用野生型鼠脑和 PP2Ac-specific-antibody, 1D6。然后用 SDS 页分离蛋白质, 用已建立的方法19探测 PP2Ac、异步电动机、bSyn 和 gSyn。重组核控制显示异步电动机, bSyn 和 gSyn 抗体特异性 (图 3A)。联合 ip 数据显示, 与 PP2Ac 在脑中的主要核是异步电动机的, 更少的 bSyn 在 co ip (图 3B) 中下来, 并且没有 gSyn 在 co ip 中出现 (数据没有显示)。这表明内源性异步电动机在调节脑 PP2Ac 中具有独特的作用。它也提高了一种可能性, PP2Ac 调制与 bSyn-based 疗法可能有可能恢复 PP2A 活动在那些与 synucleinopathy, bSyn 是已知的 nonamyloidogenic, 但在这里显示可以刺激 PP2Ac30, 31。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55361/55361fig3v2.jpg"> 图 3:PP2Ac 与内源性 synucleins 在小鼠脑中的相互作用.(A) 重组异步电动机、bSyn 和 gSyn 的核特异抗体进行了探讨, 如下所述。(B) 使用小鼠脑匀浆联合 IP 进行抗体 1D6, 然后对 immunoblots 进行样品分析。数据显示为 PP2Ac, 异步电动机和 bSyn, 但不是为 gSyn 与 PP2Ac 没有共同知识产权。开始样品显示相对水平 PP2Ac (车道 1), 异步电动机 (车道 4) 和 bSyn (车道 7) 在最初的匀。结束样品显示剩余的水平 PP2Ac (车道 2), 异步电动机 (车道 5) 和 bSyn (车道 8) 在匀以后 1D6 PP2Ac Co IP。重要水平的 PP2Ac 是 immunoprecipitated 使用1D6 抗体 (车道 3), 这也带来了大量的异步电动机 (车道 6, 在箭头), 但很少 bSyn (车道 9, 强烈曝光显示微弱的 bSyn 信号, 在箭头)。* 星号和括号标记在墨迹上的非特定信号。有关所用抗体的详细信息, 请参阅材料目录。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Recombinant Î±- Î²- and Î³-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays at JoVE.com

Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

本出版物提供了一项协议, 显示如何测量重组蛋白磷酸酶2A 催化亚基 (PP2Ac) 的活性, 以响应重组α-核, β-核, 或γ核蛋白使用简单的比色法。我们展示了关于α-核特异性向 PP2Ac 在小鼠脑中的发现。

重组αβ-γ-Synucleins 刺激蛋白磷酸酶2A 催化亚基活性在细胞自由测定中的作用

&#945;-Synuclein (aSyn), &#946;-Synuclein (bSyn), and &#947;-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly ...

recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic

Research

JoVE Journal

Neuroscience

6.7K 次观看.  Texas Tech University Health Sciences Center El Paso. 本出版物提供了一项协议, 显示如何测量重组蛋白磷酸酶2A 催化亚基 (PP2Ac) 的活性, 以响应重组α-核, β-核, 或γ核蛋白使用简单的比色法。我们展示了关于α-核特异性向 PP2Ac 在小鼠脑中的发现。

视频: Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

科研

生物化学

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

发现疾病相关的α突触核蛋白通过增强ELISA在转基因小鼠表达人A53T的突变脑α突触核蛋白

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

医学

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

神经科学

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

生物学

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

<meta charset="utf8"></head><body>化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

重组αβ-γ-Synucleins 刺激蛋白磷酸酶2A 催化亚基活性在细胞自由测定中的作用

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淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

发现疾病相关的α突触核蛋白通过增强ELISA在转基因小鼠表达人A53T的突变脑α突触核蛋白

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

使用自制双辉光萤光素酶检测的高通量筛选功能

从单体和体内使用的Α-syr核 in 预成型纤维的产生

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病