Name: in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease
Uploaded: 2017-12-20T14:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

作者想感谢曼迪 Loebert 和蒂娜 Czajka 的优秀技术支持。我们感谢弗洛拉罗 (哈佛医学院, 波士顿, 马萨诸塞州, 美国) 的语言编辑帮助。

1. 制备突变 pcDNA3.1/pcDNA3.1/GAA 结构
注意: 玻璃和GAA编码序列 (cd) 的克隆策略已在较早的15、18中报告。
<ol>
	<li>用定点诱变的定点诱变
	<ol>
		<li>分别使用参照序列 NM_000169.2 和 NM_000152.4 作为玻璃化和 GAA基因突变的模板。有一套高纯度无盐底漆 (25-37-市面) 由一个商业供应商合成, 与意义和反义引物运载其中一个各自序列修改中央对他们的长度单独地介绍变异。使用免费入门设计工具支持入门设计19。</li>
		<li>对于反应混合物, 使用制造商提供的反应溶液和 PCR 条件的标准条件。<ol>
			<li>混合5µL 10x 反应缓冲器, 10 ng 双模板质粒 DNA (pcDNA3.1/pcDNA3.1/GAA), 每个底漆 125 ng, 1 µL 提供 dNTP 混合物, 3 µL 的亚甲基亚砜试剂在适当量的去离子水 (最终反应量:50 µL)。最后, 添加 2.5 U 的 DNA 聚合酶和混合由移上下。作为一个负控制, 随身携带一个 no-primer 样本。</li>
		</ol>
		</li>
		<li>使用以下程序启动 PCR: 步骤 1:95 ° c 为1分钟, 步 2:95 ° c 为五十年代, 步 3:60 ° c 为五十年代, 步 4:68 ° c 为 8 min, 重复步骤 2-4 18 次, 并且步骤 5:68 ° c 为 10 min。<ol>
			<li>以下 PCR, 添加1µL 的DpnI 限制性酶 (10 U/µL) 和进一步孵化反应瓶在37° c 为 1 h。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>所需克隆的转换和筛选
	<ol>
		<li>根据制造商的建议, 转换分的 ultracompetent 细胞。使用 SOC 介质 (胰 2% (w/v), 酵母提取物 0.5% (w/v), 氯化钠10毫米, 氯化钾2.5 毫米, 在121° c 消毒, 然后添加无菌过滤解决方案的氯化镁2和葡萄糖高达最终浓度10和20毫米, 分别) 而不是制造商的中.程序后, 250 µL 的样本突变的 LB 板含有100µg/毫升氨苄西林和孵化在37° c 18 小时。</li>
		<li>确保转化的数量和 #62; 10 和反应产生至少三倍的殖民地作为 no-primer 控制反应,例如, 使用发光板, 以促进菌落计数。然后选择3殖民地和准备3毫升的隔夜文化在 LB 肉汤培养基。</li>
		<li>第二天, 用标准的试剂盒进行质粒制备, 并使用 T7 (5ʹ-TAA TAC GG-3ʹ) 和 BGHr (5ʹ-标记 AAG-TCG AGG-3ʹ) 通过标准桑格测序对整个序列进行分析。</li>
		<li>使用合适的分子生物学工具分析序列。当检测到所需的突变, 并没有进一步的序列异常相比, NM_000169.2 (α-乳糖 A) 或 NM_000152.4 (酸α-酶), 选择克隆的转染级质粒纯化。</li>
		<li>通过测量分光光度计的吸收度来确定 DNA 的纯度。 注: 只允许生产质粒纯度与 #62 的260/280 吸光度比的制剂; 1.8 用于细胞培养实验。</li>
	</ol>
	</li>
</ol>
2. HEK293H 细胞的培养
<ol>
	<li>维持 HEK293H 细胞在高葡萄糖 (4.5 克/升) Dulbecco´s 改良鹰培养基 (DMEM) 补充10% 胎牛血清 (FBS) 和1% 青霉素/链霉素。将细胞保持在37° c 以下 5% CO2大气压下的套培养箱中。</li>
	<li>培养细胞到密度为 80-90%。</li>
	<li>在没有 Ca2 +和 Mg2 +的情况下, 使用磷酸盐缓冲盐水 (PBS) 吸入培养基并进行洗涤。</li>
	<li>通过加入0.05% 胰蛋白酶-EDTA 和孵育5分钟在37° c 和 5% CO2。</li>
	<li>将新培养基和种子中的1:15 细胞分裂成一种新型的 T75 烧瓶, 以维持永久的文化。不要使用超过25通道的细胞。</li>
</ol>
3. pcDNA3.1/pcDNA3.1/GAA 质粒转染治疗 HEK293H
<ol>
	<li>在转染前24小时, 用 PBS2 +、Mg2 +将 T75 细胞培养瓶中的 HEK293H 细胞洗涤一次。收获的细胞与0.05% 胰蛋白酶-EDTA 如上所述, 种子 1.5 x 105细胞在24井培养板的洞使用500µL DMEM 培养基补充 10% FBS 没有抗生素。</li>
	<li>根据制造商手册执行转染协议。通常, 使用1µg 质粒 DNA 和2.5 µL 转染试剂的混合物在100µL 无血清 DMEM。在室温下孵育20分钟, 随后以 drop-wise 的方式添加到细胞中。</li>
	<li>在37° c/5% CO2的4小时后, 取出含有转染试剂的培养基, 并加入 500 FBS/10% µL 的新鲜 DMEM。 注: 在此步骤中, 可将 1-Deoxygalactonojirimycin 盐酸盐 (DGJ) 或 1-素盐酸盐 (DNJ) 添加到培养基中 (使用10毫米的水溶液, 以获得20µM DGJ 和 DNJ 的最终浓度。).新的 DGJ 或 DNJ 在质粒转染后增加42小时。</li>
</ol>
4. 细胞收获和α-乳糖 A 或酸α-酶活动测量
<ol>
	<li>细胞收获
	<ol>
		<li>在收割的当天, 从孵化箱中取出细胞, 并吸入培养基。使用带有 Ca2 +和 Mg2 +的 PBS 仔细清洗单元格2次。 注意: 这一步是至关重要的, 因为 DGJ 和 DNJ 分别是α-乳糖和α-酶的强力抑制剂, 任何残留物都将使测试无效。</li>
		<li>在细胞顶部直接添加200µL 的去离子水。从盘子里冲洗细胞, 然后将它们转移到1.5 毫升的反应管。</li>
	</ol>
	</li>
	<li>冻融均匀化
	<ol>
		<li>将样品放入适当的泡沫机架和涡流中5秒, 使裂解更加有效。在十年代和室温水浴中, 将样品交替放入液氮中, 直到解冻完成 (5 分钟)。</li>
		<li>重复此步骤5次, 然后旋转样本5分钟, 在 1万 x g。</li></ol></li></ol>在新的反应管中保留上清和吸管。
	
	
	<li>用二辛可宁酸测定蛋白质浓度
	<ol>
		<li>为每个样品准备一个新的管, 其中包含40µL 的去离子 H2O, 并添加10µL 的样品。混合溶液由涡流简要地和转移10µL 入一个96井板的洞 (每个样品三份)。在去离子 H2O 中稀释2毫克/毫升牛血清白蛋白 (bsa) 的储存液, 如下所示获取标准曲线:50 µL H2O/50 µL BSA;60µL H2O/40 µL BSA;70µL H2O/30 µL BSA;80µL H2O/20 µL BSA;90µL H2O/10 µL BSA;100µL H2O。</li>
		<li>开始反应, 加入200µL 的综合分析试剂 (试剂 a 和试剂 B 混合在一个50:1 的比率) 和孵化1小时在黑暗中, 在37° c 下轻微搅拌在一个轨道振动筛 (300 rpm)。在平板阅读器中测量 560 nm 的吸光度。 注: 样品通常包含每µL 1 至1.5 µg 蛋白。</li>
	</ol>
	</li>
	<li>人工 4-甲基基质 (4 杯) 的酶活性测定
	<ol>
		<li>稀释每个样品的计算量和吸管到新鲜的1.5 毫升反应管获得 0.05 (α-乳糖 A) 或 0.5 (对于酸性α-酶) µg 蛋白/µL 溶液。漩涡样品为 5 s 再和吸管10µL 这稀释入96井板 (每个样品在一重复)。</li>
		<li>开始反应, 加入20µL 各自的基板解决方案: α-乳糖甲: 2 毫米 4-甲基-α-d-乳糖 (4-亩-加仑) 在0.06 米磷酸盐柠檬酸盐缓冲, pH 4.7。 酸性α-酶: 2 毫米 4-甲基α-d-喃 (4-亩-谷氨酸) 在0.025 米醋酸钠, pH 4.0。</li>
		<li>在37° c 下, 在轨道振动筛 (300 rpm) 的轻微搅动下, 在黑暗中孵育 1 h 的酶反应。终止反应由增加200µL 1.0 M, pH 10.5 被调整的甘氨酸-氢氧化钠缓冲。</li>
		<li>从0.01 毫克/毫升的库存中制备 4-伞形 (4-亩) 的标准曲线, 如下所示: 100µL H2O/4-亩;80µL H2O/20 µL 4-亩;60µL H2O/40 µL 4-亩;40µL H2O/60 µL 4-亩;20µL H2O/80 µL 4-亩;没有 H2O/100 µL 4-亩, 吸管10µL 每稀释入96井板 (在重复) 和增加200米甘氨酸氢氧化钠缓冲液每井为了调整容量和 pH 值。</li>
		<li>在装有适当过滤器设置的荧光阅读器中测量酶活性, 并使用合适的软件对荧光读写器进行数据分析。 注: 在接触α-乳糖 A 或酸性α-酶的过程中, 两个4杯基片的衬底都减少到4μ。释放4亩是一个荧光, 可以分别测量为360和 465 nm 的激发和发射波长, 使用微荧光阅读器。</li>
	</ol>
	</li>

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A., 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=Oral+pharmacological+chaperone+migalastat+compared+with+enzyme+replacement+therapy+in+Fabry+disease:+18-month+results+from+the+randomised+phase+III+ATTRACT+study.">Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.</a> J Med Genet. 54, 288-296 (2017).</li><li>Parenti, G., Andria, G., Valenzano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Chaperone+Therapy:+Preclinical+Development,+Clinical+Translation,+and+Prospects+for+the+Treatment+of+Lysosomal+Storage+Disorders.">Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.</a> Mol Ther. 23, 1138-1148 (2015).</li><li>Parenti, G., 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=A+Chaperone+Enhances+Blood+&#945;-Glucosidase+Activity+in+Pompe+Disease+Patients+Treated+With+Enzyme+Replacement+Therapy.">A Chaperone Enhances Blood &#945;-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy.</a> Mol Ther. 22, 2004-2012 (2014).</li></ol>

作者声明他们没有竞争的金融利益。

本文所述的协议为遗传性溶酶体代谢疾病的酵素损伤评估提供了健壮的结果。本手稿是对先前发布的15协议的修正。最关键的修改涉及严格 (即, 在突变体的载体构造准备过程中), 细胞培养协议 (即, HEK293H 细胞维护和转染条件) 的标准化, 以及高实验重复次数 (至少 5), 对结果的重现性有很高的贡献。总之, 两个目标基因都很容易获得诱变和多样性的突变可以并行组装。...

有十几个溶酶体存储紊乱 (LSDs) 与糖苷功能障碍相关的主要基因突变的结果。在法布里 (OMIM #301500) 和庞贝氏病 (OMIM #232300) 疾病, 超过500和200义突变1,2,3分别报告, 这相当于大约60% 的总变异计数。许多新的基因变种仍在被识别, 其中很多是未知的意义。广泛的生物化学研究表明, 某些基因型不会导致一个完整的功能的玻璃基因 (OMIM * 300644) 在法布里病, 但导致相应的酶未能达到一个热力青睐的折叠状态 4.这导致 ER 保留和过早退化, 否则功能酶。类似的结论也在其他 LSDs 包括庞贝氏病5中得出。此外, 分子特性的酶变体可以促进临床解释的突变在诊断时的6, 这表明 LSD 进展是一个个体的过程中, 基于突变的性质。因此, 为了简化临床咨询和治疗决策, 应重新评估传统的2到3种不同临床类型的分类。
两种疾病都有酶替代疗法。然而, 在受影响的组织/器官, 如大脑和骨骼肌肉的功效有限。此外, 专家审评可以引起免疫的反应, 危及其治疗效益。药理陪护 (PCs) 是一种有吸引力的治疗方案, so-called 反应性突变患者。pc 作为正确的蛋白质折叠和稳定的分子支架, 从而防止内质网 (er) 保留和 er 相关的酶降解。此外, 个人电脑可以口服和潜在的能够跨越血脑屏障。因此, PCT 可能是一个更可行的选择, 以治疗患者的某些基因型。有关 LSDs 中 PC 应用程序的广泛审查, 请参阅 Parenti7的优秀评审。
发现数以百计的疾病, 导致突变等位基因挑战前临床药物测试, 并要求一个简单, 快速, 和高度标准化的评估, 以适合病人的个性化药物的方法。为了评估 LSD 基因突变的不利影响, 并测试候选突变, 以预测适合的患者 PCT, 一个高度标准化的表达系统的 HEK293H 细胞, 允许快速和可靠的酶活性测量开发.类似的表达系统曾被描述为法布里和庞贝氏病, 使用 COS-78,9,10,11, HeLa 细胞12, 或 HEK29313 ,14,15,16糖苷基因的单元格。
一个非常类似的方法甚至被授予专利作为 "预测反应的方法, 药理陪护治疗疾病"17表明的相关性, 细胞培养系统能够融入临床实践。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>QuikChange II XL Site-Directed Mutagenesis Kit&amp;nbsp;</td><td>Stratagene, La Jolla, CA, USA</td><td>#200522</td><td></td></tr><tr><td>Primer GLA[R301Q]-frw: 5&amp;acute;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GLA[R301Q]-rev: 5&amp;acute;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GLA[A156V]-frw: 5&amp;acute;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GLA[A156V]-rev: 5&amp;acute;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GLA[A156V]-frw: 5&amp;acute;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GLA[A143T]-rev: 5&amp;acute;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[F455Y]-frw: 5&amp;acute;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[F455Y]-rev: 5&amp;acute;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[P545L]-frw: 5&amp;acute;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[P545L]-rev: 5&amp;acute;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[L552P]-frw: 5&amp;acute;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer GAA[P545L]-rev: 5&amp;acute;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer T7: 5&amp;acute;-TAA TAC GAC TCA CTA TAG GG-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Primer BGHrev: 5&amp;acute;-TAG AAG GCA CAG TCG AGG-3&amp;acute;</td><td>MWG Eurofins Operon, Ebersberg, Germany</td><td>n.a.</td><td></td></tr><tr><td>Tryptone</td><td>Carl Roth, Karlsruhe, Germany</td><td>8952.1</td><td></td></tr><tr><td>Yeast Extract</td><td>Merck, Darmstadt, Germany</td><td>103,753</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck, Darmstadt, Germany</td><td>1,064,041,000</td><td></td></tr><tr><td>Potasium chloride</td><td>Merck, Darmstadt, Germany</td><td>1,049,360,500</td><td></td></tr><tr><td>MgCl2 x 6H2O</td><td>Merck, Darmstadt, Germany</td><td>1,058,330,250</td><td></td></tr><tr><td>D (+)-Glucose monohydrate</td><td>Merck, Darmstadt, Germany</td><td>1,083,422,500</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Merck, Darmstadt, Germany</td><td>1,064,980,500</td><td></td></tr><tr><td>Glycine</td><td>Carl Roth, Karlsruhe, Germany</td><td>3908.2</td><td></td></tr><tr><td>Citric acid</td><td>Carl Roth, Karlsruhe, Germany</td><td>X863.3</td><td></td></tr><tr><td>disodium hydrogen phosphate</td><td>Merck, Darmstadt, Germany</td><td>1,065,860,500</td><td></td></tr><tr><td>Sodium acetate</td><td>Merck, Darmstadt, Germany</td><td>1,062,640,050</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma Aldrich, Munich, Germany</td><td>A6283</td><td></td></tr><tr><td>LB Agar</td><td>Carl Roth, Karlsruhe, Germany</td><td>X969.2</td><td></td></tr><tr><td>LB Medium</td><td>Carl Roth, Karlsruhe, Germany</td><td>X968.2</td><td></td></tr><tr><td>Zyppy Plasmid Miniprep Kit</td><td>ZymoResearch, Freiburg, Germany</td><td>D4020</td><td></td></tr><tr><td>QIAfilter Plasmid Midi Kit</td><td>Qiagen, Hilden, Germany</td><td>12245</td><td></td></tr><tr><td>HEK293H cell line</td><td>Invitrogen, Karlsruhe, Germany</td><td>11631-017&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&amp;acute;s Modified Eagle Medium&amp;nbsp;</td><td>Invitrogen, Karlsruhe, Germany</td><td>31966-047</td><td></td></tr><tr><td>HyClone fetal bovine serum&amp;nbsp;</td><td>GE Healthcare, South Logan, Utah, USA</td><td>SV30160.03</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen, Karlsruhe, Germany</td><td>15140-122</td><td></td></tr><tr><td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td><td>VWR International GmbH, Hannover, Germany</td><td>82050-854</td><td></td></tr><tr><td>Cell culture plates (24 well)</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>831,836</td><td></td></tr><tr><td>Cellstar 96 well plate</td><td>Greiner bio one, Frickenhausen, Germany</td><td>655185</td><td></td></tr><tr><td>SafeSeal reaction tubes, 1,5mL&amp;nbsp;</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>72,706</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen, Karlsruhe, Germany</td><td>11668-019</td><td></td></tr><tr><td>1-Deoxygalactonojirimycin Hydrochloride&amp;nbsp;</td><td>Sigma Aldrich, Munich, Germany</td><td>D9641</td><td></td></tr><tr><td>1-Deoxygalactonojirimycin Hydrochloride&amp;nbsp;</td><td>Toronto Research Chemicals, Toronto, Canada</td><td>D236500</td><td></td></tr><tr><td>1-Deoxynojirimycin&amp;nbsp;</td><td>Sigma Aldrich, Munich, Germany</td><td>D9305</td><td></td></tr><tr><td>PBS Dulbecco w/o Calcium, w/o Magnesium</td><td>Biochrom, Berlin, Germany</td><td>L 1825</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Scientific, Braunschweig, Germany</td><td>25300054</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific, Braunschweig, Germany</td><td>23225</td><td></td></tr><tr><td>4-Methylumbelliferone</td><td>Sigma Aldrich, Munich, Germany</td><td>M1381</td><td></td></tr><tr><td>4-Methylumbelliferyl &amp;alpha;-D-galactopyranoside</td><td>Sigma Aldrich, Munich, Germany</td><td>M7633</td><td></td></tr><tr><td>4-Methylumbelliferyl &amp;alpha;-D-glucopyranoside</td><td>Sigma Aldrich, Munich, Germany</td><td>M9766</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&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>incubator type T6</td><td>Heraeus Instruments, Hanau, Germany</td><td>n.a.</td><td></td></tr><tr><td>Luminous Plate Gr. 2 E</td><td>Carl Roth, Karlsruhe, Germany</td><td>P265.1</td><td></td></tr><tr><td>3130 xl Genetic Analyzer&amp;nbsp;</td><td>Applied Biosystems, Darmstadt, Germany</td><td>n.a.</td><td></td></tr><tr><td>GFL-3032 bacterial shaker</td><td>GFL, Burgwedel, Germany&amp;nbsp;</td><td>n.a.</td><td></td></tr><tr><td>Avanti J-25 centrifuge</td><td>Beckman/Coulter, Krefeld, Germany</td><td>n.a.</td><td></td></tr><tr><td>Ultraspec 3100 &lt;em&gt;pro&lt;/em&gt; Spectrophotometer</td><td>Amersham Biosciences, Buckinghamshire, United Kingdom</td><td>n.a.</td><td></td></tr><tr><td>water-jacket incubator&amp;nbsp;</td><td>Binder, Tuttlingen, Germany</td><td>n.a.</td><td></td></tr><tr><td>Vortex Genie 1 touch mixer</td><td>Scientific Industries, Bohemia, NY, USA</td><td>n.a.</td><td></td></tr><tr><td>Z 233 MK-2 refrigerated microtube centrifuge</td><td>Hermle, Tuttlingen, Germany&amp;nbsp;</td><td>n.a.</td><td></td></tr><tr><td>LaboStar ultrapure water device</td><td>Siemens, Berlin, Germany</td><td>n.a.</td><td></td></tr><tr><td>Thermo Shaker PST-60HL-4 orbital shaker</td><td>Biosan, Riga, Latvia</td><td>n.a.</td><td></td></tr><tr><td>Tecan GENios Reader</td><td>Tecan, M&amp;auml;nnedorf, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>HI 223 Microprocessor pH Meter</td><td>HANNA Instruments, Arvore, Portugal</td><td>n.a.</td><td></td></tr><tr><td>CASY&lt;br/&gt; Cell Counter + Analyser System&lt;br/&gt; Model TT</td><td>Innovatis AG, Cham/Zug, Switzerland</td><td></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;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Magellan data analysis software v6.6</td><td>Tecan, M&amp;auml;nnedorf, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>GraphPad Prism 5.04</td><td>GraphPad Software, Inc., La Jolla, CA; USA</td><td>n.a.</td><td></td></tr><tr><td>Microsoft Office 2010</td><td>Microsoft Corporation, Redmond, WA, USA</td><td>n.a.</td><td></td></tr><tr><td>BioEdit Alignment Editor, V7.0.1</td><td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td><td>n.a.</td><td></td></tr><tr><td>&lt;strong&gt;Abbreviations&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>frw = forward; rev = reverse</td><td></td><td></td><td></td></tr><tr><td>n.a. = not applicable</td><td></td><td></td><td></td></tr></tbody></table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

使用个性化药物治疗罕见的基因疾病, 如溶酶体存储紊乱 (LSDs) 是挑战的复杂的临床试验设计, 高成本, 和低的病人人数。在大多数的 LSDs 中, 有数百个变种等位基因被牵连。根据病情轻重, 这些疾病通常分为2到3种不同的临床类型。此外, 基因型的分子特性可以帮助预测临床结果和告知病人护理。因此, 我们开发了一个简单的细胞培养方法的基础上 HEK293H 细胞 heterologously 发酵的突变发现的法布里和庞贝氏病。最近还引入了类似的检测方法作为临床前检查, 以确定在法布里病中药物陪护治疗 (PCT) 的易变异。这份手稿描述了一个修订的细胞培养法, 使位变异的快速表型评估, 以确定符合标准的患者, 并可能有助于发展新的 pharmacochaperones。

诱变过程 为了评估玻璃化基因突变的效率, 突变被归类为以下类别之一. 这种产生突变的方法显示, 在第一次尝试中, 大约有66.5% 的玻璃化突变得到了. 另有25% 可在稍加修饰的第二 PCR 后获得。
1类: 突变 PCR 在第一次尝试中是有效的。
类别 2 : 第一诱变 PCR...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

有一个要求, 使临床前测试的一个新的类 "孤儿" 药物称为药理伴侣可再生, 快速, 高效。我们开发了一个简单, 高度标准化, 多功能细胞文化检测, 以筛选合格的病人以及新的药理陪护药物。

体外酶法检测药物陪护与庞贝氏症的反应

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

in-vitro-enzyme-measurement-to-test-pharmacological-chaperone

Research

JoVE Journal

Medicine

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

8.0K 次观看.  University Rostock Medical Center. 有一个要求, 使临床前测试的一个新的类 "孤儿" 药物称为药理伴侣可再生, 快速, 高效。我们开发了一个简单, 高度标准化, 多功能细胞文化检测, 以筛选合格的病人以及新的药理陪护药物。

视频: In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

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 ...

科研

生物化学

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

<meta charset="utf8"></head><body>使用切割测定评估神经发育障碍中的 DUB 变异

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

<meta charset="utf8"></head><body>通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

医学

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

细胞内复性检测

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

生物学

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

生物样品中赤子酶活性的测定

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

免疫与感染

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外酶法检测药物陪护与庞贝氏症的反应

摘要

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此视频中的章节

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

磷31磁共振波谱分析:一种测量工具

细胞内复性检测

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

生物样品中赤子酶活性的测定

体外试验来测量磷脂酰乙醇胺甲基转移酶活

体外酶法检测药物陪护与庞贝氏症的反应

摘要

探索更多视频

此视频中的章节

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

通过体外泛素链切割测定法测量神经发育障碍相关去泛素化酶的酶活性

磷31磁共振波谱分析:一种测量工具

细胞内复性检测

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

生物样品中赤子酶活性的测定

体外试验来测量磷脂酰乙醇胺甲基转移酶活

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的