酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征
1Chemistry Department,Brooklyn College, 2Ph.D. Program in Biochemistry,Graduate Center of the City University of New York, 3Ph.D. Program in Chemistry,Graduate Center of the City University of New York, 4Ph.D. Programs in Chemistry, Biochemistry, and Biology,Graduate Center of the City University of New York
Summary
该协议概述了实验程序, 以描述全基因组的变化, 在与 ALS 和帕金森病相关的蛋白质过度表达的情况下, 组蛋白翻译后修饰 (PTM) 的水平发生。酿酒酵母模型。SDS-PAGE 分离后, 通过西方印迹, 通过修正特异性抗体检测单个组蛋白 PTM 水平。
Abstract
神经退行性疾病, 如肌萎缩侧索硬化症 (ALS) 和帕金森病 (PD), 每年造成数十万人丧生。缺乏能够阻止疾病进展的有效治疗方案。尽管在大量患者中进行了广泛的测序工作, 但大多数 ALS 和 PD 病例仅由于基因突变就无法解释。表观遗传学机制, 如组蛋白的翻译后修饰, 可能参与神经退行性疾病的病因和进展, 并导致药物干预的新目标。哺乳动物在体内和体外模型 als 和 PD 是昂贵的, 往往需要长期和费力的实验方案。在这里, 我们概述了一种实用、快速和具有成本效益的方法, 利用酿酒酵母作为模型系统, 确定组蛋白修饰水平的全基因组变化。该协议允许对与神经退行性蛋白病有关的表观遗传变化进行全面调查, 证实了以前在不同模型系统中的发现, 同时显著扩大了我们对神经退行性疾病表观基因组。
Introduction
神经退行性疾病是毁灭性的疾病, 几乎没有或根本没有治疗选择。其中, 肌萎缩侧索硬化症 (ALS) 和帕金森病 (PD) 尤其可怕。大约90% 的 als 和 pd 病例被认为是零星的, 没有家族病史, 而其余病例在家庭中运行, 一般与特定的基因突变1,2有关。有趣的是, 这两种疾病都与蛋白质错位和聚集3,4,5,6有关。例如, 融合肉瘤 (fus) 和 tar dna 结合蛋白 43 (tdp-43) 是 rna 结合蛋白, 在 als7、8、9、10、 11,12, 而α-合核蛋白是蛋白质聚集物的主要组成部分, 称为 le七烯体在pd5,13,14,15。
尽管在大量患者中进行了广泛的全基因组关联工作, 但绝大多数 ALS 和 PD 病例在基因上仍无法解释。表观遗传学能在神经退行性疾病中发挥作用吗？表观遗传学包括基因表达的变化, 在不改变基础 DNA 序列16的情况下发生。一个主要的表观遗传机制涉及组蛋白16的翻译后修饰 (Ptm).在真核细胞中, 遗传物质紧紧地包裹在染色质中。染色质的基本单位是核糖体, 由146个基对 dna 组成, 包裹在一个组蛋白八分体上, 由四对组蛋白组成 (分别为两个组蛋白 H2A、H2A、H3 和 H4)17。每个组蛋白都有一个 n 端的尾巴, 从核糖体中伸出来, 可以通过添加各种化学分子来修饰, 通常是在赖氨酸和精氨酸残留物18上。这些 Ptm 是动态的, 这意味着它们可以很容易地添加和删除, 并包括乙酰化、甲基化和磷酸化等群体。Ptm 控制 DNA 对转录机制的可访问性, 从而有助于控制基因表达18。例如, 组蛋白乙酰化降低了高度基本的组蛋白蛋白和负电荷 DNA 主干之间的静电相互作用的强度, 使乙酰化组蛋白包装的基因更容易获得, 从而更高表示19。最近, 特定组蛋白 ptm 及其组合的显著生物学特异性导致组蛋白编码假说20, 21, 其中写、擦除和读取组蛋白 ptm 的蛋白质都协同作用调节基因表达。
酵母是研究神经退行性变的一个非常有用的模型。重要的是, 许多神经元细胞通路从酵母保存到人类22,23,24。酵母重述 fus、tdp-43 或α-共核蛋白 22、23、24、25、26的过度表达后的细胞毒性表型和蛋白质夹杂物.事实上,酿酒酵母的 als 模型已被用来识别遗传危险因素在人类27。此外, 酵母过度表达人α-共核蛋白允许将 rsp5 网络定性为可吸毒的靶点, 以改善 28,29神经元中α-共核蛋白的毒性。
在这里, 我们描述了一个利用酿酒酵母检测全基因组组蛋白 PTM 变化与神经退行性蛋白病变相关的协议 (图 1)。与其他体外和动物神经退行性模型相比, 酿酒酵母的使用具有很强的吸引力, 因为它使用方便、成本低、速度快。利用以前开发的 als 和 pd 模型22,23,25, 26, 我们在酵母中过度表达了人类 fus、tdp-43 和α-合核蛋白, 并发现了明显的组蛋白 ptm 变化发生在与每个蛋白病30的连接。我们在这里描述的协议可以在从转换到数据分析的不到两周的时间内完成。
Protocol
1. 用神经退行性蛋白质与病理相关的蛋白质结构转化酿酒酵母 在30°c 晃动 (200 转/分) 的酵母提取物肽葡萄糖 (YPD) 肉汤中生长野生型 (WT) 303 酵母。 经过12-16小时的生长, 用 YPD 将酵母稀释到600纳米 (OD600) 的光学密度。由于每次转化都需要10毫升的酵母液体培养, 因此为5个转化制备50毫升的酵母液体培养, 这些转化与 FUS、TDP-43、a-synuclein 和向量仅 (ccdB) 结构相对应, 以及负…
Representative Results
为了说明这个方法, 我们将利用最近发布的结果30。WT 人 FUS 和 TDP-43 被过度表达 5小时, 而 WT α-合核蛋白被过度表达8小时。将 ccdB 构造用作矢量负控制。图 2显示了固体和液体培养物中的生长抑制。酵母被收获了, 并且西部印迹与修改特异性抗体被执行。采用抗总 H3 作为加载控制。在 FUS 过度表达模型中, H3S10ph 和 H3S10ph 的水平明显下…
Discussion
这里描述的协议提供了一种简单、方便和具有成本效益的方法, 可对与神经退行性蛋白病变相关的全基因组蛋白组蛋白 PTM 变化进行分类。虽然 als 和 PD 还有其他模型, 如体外人类细胞系和小鼠模型32, 但由于其易用性,酿酒酵母仍然很有吸引力。例如, 酵母模型不需要使用无菌罩, 也不需要在细胞培养工作的同时进行强化培训。此外, 酵母的培养试剂也比哺乳动物细胞?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢罗耶娜·塔纳兹、胡达·优素夫和萨迪卡·塔森的技术帮助。我们非常感谢詹姆斯·肖特教授在蔗糖调谐实验的设计方面慷慨提供试剂和智力援助。酵母浆浆体是 Aaron Gitler 教授的慷慨礼物 (包括 303Gal-FUS;添加基因质粒 # 29614)。布鲁克林学院和高级科学研究中心 (纽约市立大学) 以及国家卫生研究院 NINSS 高级博士后过渡奖 (K22NS09131401) 支持 m. p. t。
Materials
| -His DO Supplement | Clontech | 630415 | |
| 10x Running Buffer | Mix: 141.65 g glycine (ThermoFisher BP381-1), 30.3 g Tizma base (Sigma-Aldrich T6066), 10 g sodium dodecyl sulfate (Sigma-Aldrich L3771), and 1 L deionized water, pH 8.8. | ||
| 12% Polyacrylamide Gels | BIO-RAD | 456-1041 | |
| 2-mercaptoethanol | Sigma-Aldrich | M3148 | |
| Anti-acetyl-Histone H3 (Lys14) Primary Antibody | MilliporeSigma | 07-353 (Lot No. 2762291) | Dilution: 1/1000 |
| Anti-acetyl-Histone H4 (Lys 16) Primary Antibody | Abcam | ab109463 (Lot No. GR187780) | Dilution: 1/2000 |
| Anti-acetyl-Histone H4 (Lys12) Primary Antibody | Abcam | ab46983 (Lot No. GR71882) | Dilution: 1/5000 |
| Anti-dimethyl-Histone H3 (Lys36) Primary Antibody | Abcam | ab9049 (Lot No. GR266894, GR3236147) | Dilution: 1/1000 |
| Anti-Histone H3 Primary Antibody | Abcam | ab24834 (Lot No. GR236539, GR174196, GR3194335) | Nuclear Loading Control; Dilution: 1/2000 |
| Anti-phospho-Histone H2B (Thr129) Primary Antibody | Abcam | ab188292 (Lot No. GR211874) | Dilution: 1/1000 |
| Anti-phospho-Histone H3 (Ser10) Primary Antibody | Abcam | ab5176 (Lot No. GR264582, GR192662, GR3217296) | Dilution: 1/1000 |
| BioPhotometer D30 | Eppendorf | 6133000010 | |
| Cell Culture Dish (100 x 20 mm) | Eppendorf | 30702118 | |
| Cell Culture Plate, 96 well | Eppendorf | 30730011 | |
| Centrifuge 5804/5804 R/5810/5810 R | Eppendorf | 22625501 | |
| Donkey Anti-Mouse IRDye 800 CW | LI-COR | 926-32212 (Lot No. C60301-05, C61116-02, C80108-05) | Dilution: 1/5000 |
| Donkey Anti-Rabbit IRDye 860 RD | LI-COR | 926-68073 (Lot No. C60217-06, C70323-06, C70601-05, C80116-07) | Dilution: 1/2500 |
| Ethanol | Sigma-Aldrich | E7023 | |
| Extra thick blot paper (filter paper) | BIO-RAD | 1703968 | |
| Galactose | Sigma-Aldrich | G0750 | Prepare 20% w/v stock solution. |
| Glucose | Sigma-Aldrich | G8270 | Prepare 20% w/v stock solution. |
| Glycerol | Sigma-Aldrich | G5516 | Prepare 50 % w/v solution. |
| Immobilon-FL Transfer Membranes | MilliporeSigma | IPFL00010 | |
| Lithium acetate dihydrate (LiAc) | Sigma-Aldrich | L4158 | Prepare a 1 M solution. |
| Loading Dye | Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue (Sigma-Aldrich B8026), 4.7 mL glycerol, 1.2 mL 0.5M Trizma base pH 6.8, 0.93 g DL-Dithiothreitol (Sigma-Aldrich D0632), and 2.1 mL deionized water. | ||
| Methanol | ThermoFisher | A412-4 | |
| Mini-PROTEAN Tetra Vertical Electrophoeresis Cell | BIO-RAD | 1658004 | |
| Multichannel pipet | Eppendorf | 2231300045 | |
| NEB Restriction Enzyme Buffer 2.1, 10x | New England Bio Labs | 102855-152 | |
| Nhe I Restriction Enzyme | New England Bio Labs | 101228-710 | |
| Nuclease Free Water | Qiagen | 129114 | |
| Odyssey Fc Imaging System | LI-COR Biosciences | 2800-03 | |
| OmniTray Cell Culture Treated w/Lid Sterile, PS (86 x 128 mm) | ThermoFisher | 165218 | |
| pAG303GAL-a-synuclein-GFP | Gift from A. Gitler | ||
| pAG303GAL-ccdB | Addgene | 14133 | |
| pAG303Gal-FUS | Addgene | 29614 | |
| pAG303GAL-TDP-43 | Gift from A. Gitler | ||
| Poly(ethylene glycol) (PEG) | Sigma-Aldrich | P4338 | Prepare a 50% w/v solution. |
| Ponceau S Stain | Sigma-Aldrich | P3504 | Mix: 0.5 g 0.1% w/w Ponceau S dye, 5 mL 1% v/v acetic acid (Sigma-Aldrich 320099), and 500 mL deionized water. |
| PowerPac Basic Power Supply | BIO-RAD | 164-5050 | |
| Raffinose pentahydrate | Sigma-Aldrich | R7630 | Prepare 10% w/v stock solution. |
| Salmon Sperm DNA | Agilent Tech | 201190 | |
| SD-His plates | Mix: 20 g Agar (Sigma-Aldrich A1296), 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids (ThermoFisher 291920), and 900 mL deionized water. | ||
| SGal-His plates | Mix: 20 g Agar, 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids, 100 mL galactose solution, and 900 mL deionized water. | ||
| Sodium dodecyl sulfate Loading Buffer | Store at -20 oC. 6X, Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue, 0.93 g DL-Dithiothreitol, 2.1 mL deionized water, 4.7 mL glycerol, and 1.2 mL 0.5 M Trizma base, pH 6.8. | ||
| Sodium hydroxide | Sigma-Aldrich | 221465 | Prepare 0.2 M solution. |
| Sucrose | Sigma-Aldrich | 84097 | Prepare 20% w/v stock solution. |
| TBS + 0.1% Tween 20 (TBST) | Mix: 100 mL 10X TBS, 1 mL Tween 20 (Sigma-Aldrich P7949), and 900 mL deionized water. | ||
| TBS Blocking Buffer | LI-COR | 927-5000 | |
| Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell | BIO-RAD | 170-3940 | |
| Transfer Buffer | Mix: 22.5 g glycine, 4.84 g Tizma base, 400 mL methanol, 1 g sodium dodecyl sulfate, and 1.6 L deionized water. | ||
| Tris-Buffered Saline (TBS) | 10X, 7.6 pH, Solution: Mix 24 g Trizma base, and 88 g sodium chloride (Sigma-Aldrich S7653). Fill to 1 L with deionized water. | ||
| WT 303 S. cerevisiae yeast | Gift from J. Shorter | ||
| Yeast Extract Peptone Dextrose (YPD) | Sigma-Aldrich | Y1375 |
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Cite This Article
Bennett, S. A., Cobos, S. N., Meykler, M., Fallah, M., Rana, N., Chen, K., Torrente, M. P. Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models. J. Vis. Exp. (145), e59104, doi:10.3791/59104 (2019).