Name: a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9
Uploaded: 2018-08-28T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 at JoVE.com

提交人感谢 NHLBI/nih (K08 HL124068 和 LRP HMOT1243)、NCATS/nih 通过 UCSF 临床和转化科学研究所催化剂方案 (UL1 TR000004)、UCSF 学术参议院、赫尔曼基金会提供的慷慨资助,基列科学研究学者奖, 辉瑞渴望心血管奖 (全部对 John s Chorba) 和霍华德休斯医学研究所 (阿德里 m. 盖文和凯万 m. Shokat)。

1. 蛋白酶载体的定点诱变
<ol><li>设计和订购自定义合成的寡核苷酸, 通过修改标准站点定向突变协议来安装感兴趣的变异12。标准淡化底漆 (没有额外的净化) 是完全可以接受的。 注: 对底漆设计的一般方法涉及创建部分重叠的底漆, 如表 1所示, 使用的熔融温度 (Tm) 计算器特定的聚合酶的兴趣。</li>
	<li>如表 2所示, 在冰上设置聚合酶链反应 (PCRs) 进行现场定向突变。</li>
	<li>按表 3所示循环 PCRs。</li>
	<li>运行 2-5 µL 的每一个 PCR 在1% 琼脂糖凝胶和可视化的产品, 以确认成功的放大。 注意: 一个成功的 PCR 将显示一个单一的, 突出的 DNA 波段在大约大小标记的模板 (~ 7.8 kB)。</li>
	<li>添加0.5 µL (每 25-µL 反应) 的 DpnI 和孵化样品37°c 1 小时。</li>
	<li>将反应的2µL 转化为具有化学 能力的大肠杆菌。 注: 快速生长的大肠杆菌菌株将减少潜伏期, 但任何克隆菌株都是可以接受的。</li>
	<li>将转换盘到含有 50-100 µg/毫升羧苄青霉素的 Luria-Bertani (磅) 琼脂上。在37摄氏度一夜之间孵化盘子。</li>
	<li>选择 2-4 个殖民地生长在一个小规模的文化 (2-5 毫升的 LB 培养基与 50-100 µg/毫升羧苄青霉素)。孵化的文化在37°c 在 220 rpm, 直到它是混浊的。</li>
	<li>根据制造商的指示, 用质粒 dna 纯化 (即"miniprep") 试剂盒将质粒 dna 从细胞中分离出来。用 microvolume 分光光度法定量测定洗脱 DNA 的浓度。</li>
	<li>用一个桑格测序服务 (如核心或商业设施) 广泛地序列质粒 DNA。根据服务规范准备 DNA 样品。在前测序反应中成功使用的底漆如表 4所述。 注: 由于整个质粒的 PCR 扩增, 建议每个突变体对整个 PCSK9 编码区域进行排序。</li>
</ol>2. 高通量荧光素酶的蛋白质水解测定方法
<ol><li>
细胞电镀 (0 天)
	<ol>
<li>使用低通道 HEK293T 细胞进行实验, 并在 Dulbecco 修饰的鹰培养基 (DMEM) 中培养10% 胎牛血清 (血清)。在组织培养罩中进行无菌条件下的所有细胞工作, 直到准备好化验荧光素酶活性。估计试验所需的水井和板材的数量, 预计 transfections 将在每一个条件下进行三个测试。</li>
		<li>将 HEK293T 细胞从母瓶中分离出来, 以最小体积的0.05% 胰蛋白酶-乙二胺-四乙酸 (EDTA) 来覆盖细胞。用2卷 DMEM 补充10% 的血清, 并将细胞转移到无菌管中, 使胰蛋白酶-EDTA 失效。使用自动单元格计数器计数单元格 (并用台盼蓝染色)。离心管在 500 x g 5 分钟恢复细胞。</li>
		<li>吸入含有胰蛋白酶的培养基, 并将培养基中的细胞重组为 2 x 105细胞/毫升的浓度。使用多通道吸管, 将100µL 的细胞转移到白色 (不透明的底部) 96 井板的每个井中, 从而使最终的播种浓度为 2 x 104细胞/井。 注: 对于最初的实验, 可能还会有帮助, 另外种子一个姐妹, 清底96井板, 以监测细胞生长和遵守在协议期间, 并指导未来的诊断。</li>
		<li>孵育细胞在37°c 和 5% CO2为 24 h。</li>
		<li>用96井格式准备一盘质粒的主板。将洗脱缓冲液中的每种质粒 (三 HCl、pH 值 8.5) 稀释成96井板的单个井中的50µL。<ol>
<li>为正控制质粒8、阴性对照 (S386A) 质粒8、无质粒缓冲 (模拟 transfections) 准备4口井。如果计划采用药物或其他细胞治疗方法, 则在每口井中用正控制质粒 (S386A) 和4口井分别制备主板, 每种均为阴性对照 () 质粒和无质粒缓冲液。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
转染 (1 天)
	<ol>
<li>用深井96井板, 用96井板格式制备转染混合物。执行 transfections, 确保准备足够的试剂, 以解释吹打和转移损失。 注: 以下计算准备足够的试剂来运行一个96井板 (420 井保守估计)。<ol>
<li>将稀释后的脂质转染试剂混合, 将50.4 µL 试剂添加到2050µL 的降血清培养基中。制作一个 DNA precomplexation 试剂的总组合, 将33.6 µL 试剂添加到减少血清培养基的1730µL 中。</li>
			<li>使用多通道吸管, 整除16.8 µL 稀释的 DNA precomplexation 试剂混合物进入深井板块的每一井。</li>
			<li>使用多通道吸管, 整除3.2 µL (160 ng) 的每种质粒从主板进入深井板块的每一井。</li>
			<li>使用多通道吸管, 整除20µL 稀释脂转染试剂混合物的每一个良好的板块, 并混合的内容, 每个井使用多通道吸管。盖上盘子, 让他们坐在室温下 (RT) 10-15 分钟形成脂质: DNA 复合物。 注: 在转染后, 每井的最终成分将如下:40 的 dna, 0.12 µL 的转染试剂, 0.08 µL 的 dna 预络试剂, 每个井的含量将是10µL 在总容积 (减少血清培养基)。</li>
		</ol>
</li>
		<li>用多通道吸管轻轻地在96井板上的293T 细胞上交换培养基, 注意不要扰乱细胞。更换吸气介质与95µL DMEM 补充10% 的血清。 注意: 这将是一个适当的时间来治疗细胞与任何药物的利益。</li>
		<li>通过多通道吸管将转染混合物的10µL 添加到每个适当的井中。轻轻地旋转盘子, 混合在井中的内容。孵育板材在37°c 与 5% CO2为 24 h。 注: 油井的最终容积为105µL, 占24小时的蒸发量。</li>
	</ol>
</li>
	<li>
化验 (2 天)
	<ol>
<li>准备腔肠素试剂的库存溶液: 3 M 抗坏血酸钠 [溶于磷酸盐缓冲盐水 (PBS), 制备新鲜], 5 米氯化钠, 10 毫克/毫升牛血清白蛋白 (BSA; 在 PBS 中溶解, 准备新鲜) 和2毫米腔肠素 (溶于酸化甲醇, 含200µL 3 氮盐酸每10毫升)。 注: 2 毫米腔肠素可以储存2周, 当它保持在-80 摄氏度和没有光。</li>
		<li>根据表 5, 为荧光素酶的读数准备2x 腔肠素试剂, 每个细胞和培养基都有一个单独的试剂。混合所有试剂保存腔肠素首先, 过滤混合物通过一个0.22 µm 注射器过滤器, 然后添加腔肠素。保护试剂不受光, 直到它们准备好添加到盘子里。 注: 由于过滤器失去了溶液, 所以要做足够的试剂来说明过滤器的损耗以及转移的损失。表 5显示了2x 试剂的最终浓度 (以及添加到一个试剂的 96-井板中的库存溶液的数量)。</li>
		<li>在转染后从孵化箱中取出细胞24小时。使用多通道吸管, 把50µL 的条件介质从每个井转移到一个新鲜的, 白色底 (不透明) 的96井板。<ol>
<li>将车牌标记为它们是否包含介质或单元格。如果有超过一个96井板被转染, 标签的板块, 以确保每一个中载板与其母板的细胞。</li>
		</ol>
</li>
		<li>使用多通道吸管, 添加50µL 2x 非裂解腔肠素试剂的板只含有条件的培养基。在 5-10 分钟的 RT 中, 轻轻摇动或摇动盘子。</li>
		<li>使用多通道吸管, 添加50µL 的2x 裂解腔肠素试剂的板块包含细胞。在 5-10 分钟的 RT 中, 轻轻摇动或摇动盘子。</li>
		<li>孵化后, 读出平板阅读器中只中板的发光。然后, 阅读同一版阅读器中的含细胞板的发光。</li>
		<li>丢弃车牌并保存文件进行数据分析。</li>
	</ol>
</li>
</ol>3. 数据分析
<ol><li>使用电子表格软件执行初始数据分析。创建包含单元格和中板结果的电子表格。</li>
	<li>手动检查单元格中的数据以识别转染不良的油井。显示 < 5%-10% 的读数的负控制 (S386A) 质粒应该被认为是不良的转染, 使这些数据的解释可疑的水井。</li>
	<li>计算模拟转染井各板块的平均背景发光。从该板块的值中减去每个板块的背景。 注意: 这个值可能是微不足道的, 这取决于使用的板阅读器。</li>
	<li>通过计算荧光素酶活性在培养基中的比例, 与各井的总荧光素酶活性相比, 对数据进行处理。由于单元格板包含除细胞外的条件介质, 而中只板含有与细胞板相同数量的条件介质, 因此使用以下方程式是适当的: <img alt="Equation 1" src="/files/ftp_upload/58265/58265eq1.jpg"> 在这里, RLU = 相对发光单位, 背景减去读数从板读取器。</li>
	<li>计算阳性对照 (S386A) 和负控 (i.) 井的平均分泌荧光素酶。</li>
	<li>通过计算 Z 因子13来评估实验的整体质量: <img alt="Equation 2" src="/files/ftp_upload/58265/58265eq2.jpg"> 注意: 活动值来自正控制 (小波), 非活动值来自负控制 (S386A) 井。接近1的值表明实验质量较高。如果值低于 0, 请考虑重复该实验或优化工作流。</li>
	<li>将数据从电子表格程序传输到科学数据分析软件中。</li>
	<li>将分泌的荧光素酶活性规范化为阳性对照值和阴性对照 (S386A), 将阳性对照设置为 1, 负控制为0。</li>
	<li>使用回归和离群删除 (溃败) 方法清除异常值的数据, 将最大错误发现率设置为1%。</li>
	<li>通过将每个突变条件 (或突变体) 的数据与 "重量" 活动的平均值进行比较 (规范化为 1) 来评估统计学意义上的差异。使用 Sidak 法和α = 0.05 进行多个配对t检验, 修正多种比较。</li>
</ol>

<ol>
	<li>Park, S. W., Moon, Y. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+low+density+lipoprotein+receptor+protein+by+proprotein+convertase+subtilisin/kexin+type+9a+in+mouse+liver.">Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.</a> Journal of Biological Chemistry. 279 (48), 50630-50638 (2004).</li><li>Cohen, J. C., Boerwinkle, E., Mosley, T. H., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variations+in+PCSK9,+low+LDL,+and+protection+against+coronary+heart+disease.">Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.</a> New England Journal of Medicine. 354 (12), 1264-1272 (2006).</li><li>Ridker, P. M., 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=Cardiovascular+Efficacy+and+Safety+of+Bococizumab+in+High-Risk+Patients.">Cardiovascular Efficacy and Safety of Bococizumab in High-Risk Patients.</a> New England Journal of Medicine. 376 (16), 1527-1539 (2017).</li><li>Sabatine, M. 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=Evolocumab+and+Clinical+Outcomes+in+Patients+with+Cardiovascular+Disease.">Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease.</a> New England Journal of Medicine. 376 (18), 1713-1722 (2017).</li><li>Kazi, D. 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=Cost-effectiveness+of+PCSK9+Inhibitor+Therapy+in+Patients+With+Heterozygous+Familial+Hypercholesterolemia+or+Atherosclerotic+Cardiovascular+Disease.">Cost-effectiveness of PCSK9 Inhibitor Therapy in Patients With Heterozygous Familial Hypercholesterolemia or Atherosclerotic Cardiovascular Disease.</a> Journal of the American Medical Association. 316 (7), 743-753 (2016).</li><li>Kazi, D. 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=Updated+Cost-effectiveness+Analysis+of+PCSK9+Inhibitors+Based+on+the+Results+of+the+FOURIER+Trial.">Updated Cost-effectiveness Analysis of PCSK9 Inhibitors Based on the Results of the FOURIER Trial.</a> Journal of the American Medical Association. 318 (8), (2017).</li><li>Pettersen, D., Fjellstr&#246;m, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulators+of+PCSK9+-+A+literature+and+patent+overview.">Small molecule modulators of PCSK9 - A literature and patent overview.</a> Bioorganic &amp; Medicinal Chemistry Letters. 28 (7), 1155-1160 (2018).</li><li>Chorba, J. S., Galvan, A. M., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+processing+analyses+of+the+single-turnover+PCSK9+protease+reveal+its+substrate+sequence+specificity+and+link+clinical+genotype+to+lipid+phenotype.">Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype.</a> Journal of Biological Chemistry. 293 (6), 1875-1886 (2018).</li><li>Maxwell, K. N., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+expression+of+Pcsk9+in+mice+results+in+a+low-density+lipoprotein+receptor+knockout+phenotype.">Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (18), 7100-7105 (2004).</li><li>Benjannet, 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=NARC-1/PCSK9+and+its+natural+mutants:+zymogen+cleavage+and+effects+on+the+low+density+lipoprotein+(LDL)+receptor+and+LDL+cholesterol.">NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.</a> Journal of Biological Chemistry. 279 (47), 48865-48875 (2004).</li><li>Cunningham, D., 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=Structural+and+biophysical+studies+of+PCSK9+and+its+mutants+linked+to+familial+hypercholesterolemia.">Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.</a> Nature Structural &amp; Molecular Biology. 14 (5), 413-419 (2007).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8, 91 (2008).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Hall, M. P., 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=Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate.">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS Chemical Biology. 7 (11), 1848-1857 (2012).</li><li>McNutt, M. C., Lagace, T. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+activity+is+not+required+for+secreted+PCSK9+to+reduce+low+density+lipoprotein+receptors+in+HepG2+cells.">Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.</a> Journal of Biological Chemistry. 282 (29), 20799-20803 (2007).</li><li>Chorba, J. S., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprotein+convertase+subtilisin/kexin+type+9+(PCSK9)+active+site+and+cleavage+sequence+differentially+regulate+protein+secretion+from+proteolysis.">The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.</a> Journal of Biological Chemistry. 289 (42), 29030-29043 (2014).</li><li>Benjannet, S., Rhainds, D., Hamelin, J., Nassoury, N., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprotein+convertase+(PC)+PCSK9+is+inactivated+by+furin+and/or+PC5/6A:+functional+consequences+of+natural+mutations+and+post-translational+modifications.">The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications.</a> Journal of Biological Chemistry. 281 (41), 30561-30572 (2006).</li><li>Zhao, Z., 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=Molecular+characterization+of+loss-of-function+mutations+in+PCSK9+and+identification+of+a+compound+heterozygote.">Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.</a> American Journal of Human Genetics. 79 (3), 514-523 (2006).</li></ol>

作者没有什么可透露的。

上述实验程序提出了一种克服单周转蛋白酶 PCSK9 内在低活性的方法, 并以稳健的方式评价其蛋白水解功能。该检测的关键概念依赖于将单个周转事件转换为酶放大读数。该检测的优点包括相对较短的时间框架和易用性荧光素酶的记者, 以及它的可伸缩性高吞吐量的方法。此外, 该化验结果评估蛋白质水解在其本机, 细胞的上下文。此外, 通过这种检测, 临床鉴定的 snp 可以评估其对 PCSK9 蛋白水解的影...

PCSK9 靶向 ldl 受体 (ldl R) 降解, 提高 ldl 胆固醇 (ldl C) 和驱动动脉粥样硬化性心脏病1,2。治疗靶向 PCSK9 稳健低密度脂蛋白 C 和改善心血管结局的患者, 即使添加到积极的降脂治疗与他汀类药物3,4。然而, 目前批准的疗法仅限于基于抗体的方法, 而且缺乏成本效益5,6。为了解决这个问题, 不那么昂贵的治疗方案, 一种方法来识别患者可能获得更大的利益, 或两者, 是需要的。
小分子方法可以针对细胞内 PCSK9, 提供一个改进的管理路线, 并降低成本, 使它们成为这个领域的 "圣杯"7。然而, PCSK9 已经被小分子所证明是难以用药的。作为蛋白酶, 靶向 PCSK9's 蛋白水解功能是一个很有吸引力的策略, 因为自我分解是 PCSK9 成熟8的速率限制步骤, 它对 LDL-R9的最大影响是必需的。然而, 到目前为止, 这项战略还没有成功, 可能是由于 PCSK9's 独特的生物化学: PCSK9 仅10, 执行单翻转反应, 自解裂后, PCSK9 prodomain 仍然绑定在活动站点作为自动抑制剂11, 防止任何进一步的蛋白酶活动的读数。
本文提出了一种评价高通量时尚8PCSK9 蛋白水解功能的方法。通过现场定向诱变, 研究者可以利用这一方法来探讨在临床中发现的编码 snp 的影响, 以评估它们对蛋白质水解的影响, PCSK9 成熟的速率限制步骤。此外, 这种方法将有助于设计高通量的屏幕, 以确定 PCSK9 蛋白降解的调制器, 这将最终扰乱 PCSK9 的表现为 LDL R (和调制 PCSK9's 胆固醇效应).最后, 该协议可以适应其他具有本质上低活性的蛋白酶, 只要 i) 一个特定的基质蛋白酶对可以找到, 和 ii) 一个合适的细胞内锚可以建立的基质。

<table><tbody><tr><td>PCR Tubes</td><td>USA Scientific</td><td>1402-2900</td><td>For PCR</td></tr><tr><td>Q5 Hot Start</td><td>New England Biolabs</td><td>M0493L</td><td>High-fidelity DNA Polymerase</td></tr><tr><td>Deoxynucleotide Solution Mix</td><td>New England Biolabs</td><td>N0447L</td><td>dNTPs (for PCR)</td></tr><tr><td>pPCSK9-NLucProteaseAssay-WT</td><td>Authors</td><td>n/a</td><td>Available from authors</td></tr><tr><td>pPCSK9-NLucProteaseAssay-S386A</td><td>Authors</td><td>n/a</td><td>Available from authors</td></tr><tr><td>Agarose LE</td><td>Gold Biotechnology</td><td>A-201-100</td><td>For DNA gels</td></tr><tr><td>E-Gel Imager System with Blue Light Base</td><td>ThermoFisher Scientific</td><td>4466612</td><td>For imaging DNA gels</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>ThermoFisher Scientific</td><td>S33102</td><td>For DNA gels</td></tr><tr><td>Tris Base</td><td>ThermoFisher Scientific</td><td>BP152-1</td><td>For DNA gel running buffer</td></tr><tr><td>Glacial acetic acid</td><td>ThermoFisher Scientific</td><td>A38-500</td><td>For DNA gel running buffer</td></tr><tr><td>Ethylenediaminetetraacetic acid solution</td><td>Millipore Sigma</td><td>3690</td><td>EDTA, for DNA gel running buffer</td></tr><tr><td>1 kb DNA ladder</td><td>Gold Biotechnology</td><td>D010</td><td>DNA ladder</td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176S</td><td>Restriction enzyme</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/mL carbenicillin</td><td>Teknova</td><td>L1010</td><td>LB-Carb plates</td></tr><tr><td>One Shot Mach1 T Phage-Resistent Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>ThermoFisher Scientific</td><td>C862003</td><td>Chemically competent cells</td></tr><tr><td>LB Broth, Miller</td><td>ThermoFisher Scientific</td><td>BP1426-2</td><td>LB</td></tr><tr><td>Carbenicillin</td><td>Gold Biotechnology</td><td>C-103-5</td><td>Selective antibiotic</td></tr><tr><td>E.Z.N.A. Plasmid Mini Kit I</td><td>Omega BioTek</td><td>D6942-02</td><td>DNA Purification Miniprep kit</td></tr><tr><td>NanoDrop 2000 Spectrophotomer</td><td>ThermoFisher Scientific</td><td>ND-2000C</td><td>Spectrophotometer</td></tr><tr><td>293T Cells</td><td>American Tissue Culture Collection (ATCC)</td><td>CRL-3216</td><td>HEK 293T cells</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>ThermoFisher Scientific</td><td>11995065</td><td>DMEM, mammalian cell media</td></tr><tr><td>Fetal Bovine Sera</td><td>Axenia Biologix</td><td>F001</td><td>FBS</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>ThermoFisher Scientific</td><td>25300062</td><td>Trypsin, for cell dissociation</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>ThermoFisher Scientific</td><td>10010023</td><td>PBS</td></tr><tr><td>Countess automated cell counter</td><td>ThermoFisher Scientific</td><td>C10227</td><td>Automated cell counting</td></tr><tr><td>Countess cell counting chamber slides</td><td>ThermoFisher Scientific</td><td>C10228</td><td>Slides for cell counting</td></tr><tr><td>CELLSTAR Tissue Culture Plates, White, White-Bottom, with Lid</td><td>Grenier Bio-One</td><td>655083</td><td>White, white-bottom 96 well plate</td></tr><tr><td>TempPlate non-skirted 96-well PCR plate, natural</td><td>USA Scientific</td><td>1402-9596</td><td>96 well plate for master plasmid plate</td></tr><tr><td>Nunc 2.0mL DeepWell Plates</td><td>ThermoFisher Scientific</td><td>278743</td><td>96 well deep well plate</td></tr><tr><td>Lipofectamine 3000</td><td>ThermoFisher Scientific</td><td>L3000008</td><td>Lipid transfection reagent, Lf3K</td></tr><tr><td>P3000 Reagent</td><td>ThermoFisher Scientific</td><td>L3000008</td><td>DNA pre-complexation reagent, provided with Lf3K</td></tr><tr><td>OptiMEM I Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>31985062</td><td>Reduced serum medium for transfection</td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Millipore Sigma</td><td>A4034</td><td>Sodium ascorbate</td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>S9888</td><td>NaCl</td></tr><tr><td>Albumin, Bovine Serum, Fraction V, Low Heavy Metals</td><td>Millipore Sigma</td><td>12659</td><td>BSA</td></tr><tr><td>Methanol (HPLC)</td><td>ThermoFisher Scientific</td><td>A4524</td><td>MeOH</td></tr><tr><td>Hydrochloric acid</td><td>VWR</td><td>JT9535-2</td><td>Concentrated HCl</td></tr><tr><td>Coelenterazine</td><td>Gold Biotechnology</td><td>CZ2.5</td><td>Luciferase substrate</td></tr><tr><td>Syringe Filter, Sterile</td><td>ThermoFisher Scientific</td><td>09-720-3</td><td>Sterile filter, PVDF, 0.22 &amp;micro;m pore</td></tr><tr><td>Pipet-Lite Multi Pipette L12-200XLS+</td><td>Rainin</td><td>17013810</td><td>Multichannel pipette</td></tr><tr><td>Pipet-Lite Multi Pipette L12-20XLS+</td><td>Rainin</td><td>17013808</td><td>Multichannel pipette</td></tr><tr><td>Pipet-Lite Multi Pipette L12-10XLS+</td><td>Rainin</td><td>17013807</td><td>Multichannel pipette</td></tr><tr><td>Reagent reservoir</td><td>Corning</td><td>4870</td><td>Trough for reagents</td></tr><tr><td>Centrifuge tubes, 15 mL</td><td>ThermoFisher Scientific</td><td>05-539-12</td><td>15 mL tubes</td></tr><tr><td>Centrifuge tubes, 50 mL</td><td>Corning</td><td>430829</td><td>50 mL tubes</td></tr><tr><td>Spark Microplate Reader</td><td>Tecan</td><td>N/a</td><td>Plate Reader</td></tr><tr><td>Excel</td><td>Microsoft</td><td>2016 for Mac</td><td>Spreadsheet software</td></tr><tr><td>Prism</td><td>GraphPad Software</td><td>v7</td><td>Scientific data analysis software</td></tr></tbody></table>

a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9

Proprotein convertase 枯草杆菌9型 (PCSK9) 是一种单周转蛋白酶, 它调节血清低密度脂蛋白 (LDL) 水平, 从而导致心血管疾病。尽管 PCSK9 蛋白水解是其完全胆固醇效果所必需的, 但对其蛋白水解功能的评价是有挑战性的: PCSK9 只知道它自己, 只经历一个单一的营业额, 并在蛋白质水解后, 保留其基底在其活性部位为自动抑制剂。这里提出的方法描述了一种克服这些挑战的试验。该方法的研究重点是细胞间蛋白水解, 并将成功的裂解与分泌的荧光素酶活性联系在一起, 可以很容易地在条件培养基中读出。通过突变、瞬变转染和荧光素酶读数的顺序步骤, 该方法可以在基因或分子摄动条件下, 在高通量的情况下 PCSK9 蛋白质水解。该系统非常适合于临床发现的无义单核苷酸多态性 (SNPs) 的生物化学评价, 以及筛选 PCSK9 蛋白水解物的小分子抑制剂。

高通量的蛋白质分解测定方法依赖于克服三大挑战。首先, 为了克服单周转 PCSK9 蛋白酶固有的低产量, PCSK9 蛋白酶缺乏抑制 prodomain, 与分裂序列的 prodomain 链接到一个荧光素酶, 可以分泌14。第二, 为了满足蛋白酶在复合体中的折叠与抑制 prodomain 的需要, 这两种多肽在细胞 coexpressed15,16,通过一个 bicistronic ?...

Watch this Scientific Journal Video about A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 at JoVE.com

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

本协议提出了一种评估细胞内一种内在的低活性、单周转蛋白酶的蛋白水解活性的方法。具体来说, 该方法用于评价 PCSK9 的蛋白水解活性, 这是脂代谢的关键驱动力, 其蛋白水解活性是其最终胆固醇功能所必需的。

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

a-high-throughput-luciferase-assay-to-evaluate-proteolysis-single

Research

JoVE Journal

Biochemistry

8.1K 次观看.  Zuckerberg San Francisco General and University of California San Francisco. 本协议提出了一种评估细胞内一种内在的低活性、单周转蛋白酶的蛋白水解活性的方法。具体来说, 该方法用于评价 PCSK9 的蛋白水解活性, 这是脂代谢的关键驱动力, 其蛋白水解活性是其最终胆固醇功能所必需的。

视频: A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

科研

癌症研究

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

生物化学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

<meta charset="utf8"></head><body>一种用于高通量筛选的测量 ER-线粒体接触的简单检测

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>作者聚焦：ER-线粒体接触的调节和失调 — 对神经退行性疾病发病机制的影响

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

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 ...

神经科学

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

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://app.jove.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

摘要

探索更多视频

此视频中的章节

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

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

使用自制双辉光萤光素酶检测的高通量筛选功能

测定法错误折叠的蛋白质在细胞中的降解

一种用于筛选蛋白酶蛋白水解活性的荧光肽切割测定法