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Cancer Research

酵母作为开发功能分析研究人类P53的机箱

Published: August 04, 2019
doi:
10.3791/59071
, , , , ,
1Mutagenesis and Cancer Prevention Unit,Ospedale Policlinico San Martino, 2Department CIBIO,University of Trento, 3LAQV/REQUIMTE, Laboratory of Microbiology, Faculty of Pharmacy,University of Porto

Summary

这里提出的四个协议,以构造和利用酵母糖糖酶报告菌株,以研究人类P53转活化潜力,其各种癌症相关突变的影响,共同表达的相互作用蛋白,和特定小分子的影响。

Abstract

发现著名的哺乳动物P53蛋白可以作为酵母S.cerevisae的转录因子(TF),使得开发不同的功能测定来研究1结合位点的影响[即反应元素(RE)]。P53转活特异性或2)TP53突变、共表达辅助因子或P53转活活性小分子的序列变异。开发了不同的基础研究和翻译研究应用。在实验中,这些方法利用了酵母模型的两个主要优点。一方面,基因组编辑的简便性,通过利用仅在特定P53-RE水平上不同的同源菌株来调查P53依赖性的序列特异性,从而快速构建定性或定量报告系统转换。另一方面,对于异位P53表达的调节系统的可用性,可以评估各种蛋白质表达的转能。本报告综述了基于色重报告基因、荧光素酶和酵母生长的广泛使用的系统,以说明其主要方法步骤,并批判性地评估其预测能力。此外,这些方法的极端多功能性可以很容易地用于研究不同的TF,包括P63和P73,它们是TP53基因家族的其他成员。

Introduction

转录是一个极其复杂的过程,涉及对染色质区域RNA聚合量的招募和调制的转录因子(TF)和辅助因子的动态、空间和时间组织,以响应特定的刺激1.大多数TF,包括人类P53肿瘤抑制剂,以称为反应元素(REs)的DNA序列的形式识别特定的cis-行为元素,这些元素由单(或多种)独特的图案+6-10核苷酸长组成。在这些主题中,个别位置可能显示不同程度的变异性2,通常用位置权重矩阵 (PWM) 或徽标 3、4 汇总。

酵母S.cerevisiae是一个合适的模型系统,通过补充测定、异位表达和功能测定来研究人类蛋白质的不同方面,即使不存在正交酵母基因56,7.由于转录系统8的基础成分的进化保存,许多人类TF(当在酵母细胞中异位表达时)可以通过工程的启动子来调节报告基因的表达。包含适当的 R。此处为人类 P53 提供的转录模型系统具有三个主要变量的特征,其效果可以调制:1) P53 的表达方式和类型,2) RE 序列控制 P53 依赖转录,3) 类型记者基因 (图 1A).

关于P53表达的方式,S.cerevisae允许选择诱导的、可抑制的或构成的促进者9,10,11。特别是,诱导的GAL1启动子允许酵母中的TF的基底(使用拉菲诺作为碳源)或变量(通过改变介质中的乳糖量)表达TF。事实上,精细可调的表达代表了一个关键的发展,不仅P53本身,但也研究其他P53家族蛋白质12,13。

关于控制P53依赖性表达的RE类型,S.cerevisae允许构建不同的报告器菌株,这些菌株在其它异源背景中具有感兴趣的RE的独特差异。这一目标是通过适应一种特别通用的基因组编辑方法,在S.cerevisiae开发,称为delitto perfetto 12,14,15,16。

此外,不同的报告基因(即URA3、HIS3和ADE2)可用于定性和定量评估S.cerevisae中人类TF的转录活动,每个基因具有特定特征, 应以适应实验需要17,18,19,20,21。这些报告基因的表达分别赋予了尿原体、组氨酸和阿丁原体。URA3报告器不允许在存在5-FOA的情况下细胞生长,因此可以反选择。ADE2报告器系统的优点是,除了营养选择外,它还允许识别表达野生型(即ADE2表达功能)或突变体(即ADE2上不起作用)的酵母细胞) P53 来自殖民地颜色。

例如,表达ADE2基因的酵母细胞在含有限制定量的阿丁鱼(2.5-5.0mg/L)的板上产生正常大小的白色菌落,而那些质量差或没有转录的细胞则在同一块板上出现较小的红色(或粉红色)殖民地。这是由于在脱胶生物合成通路(即P-ribosylamino-imidazole,以前称为氨基-依米达索核糖酸或AIR)中积累的中间体,该中间体被转换成红色颜料。基于定性颜色的ADE2报告基因已被用定量的萤火虫Photinuspyralis(LUC1)12、22所取代。最近,ADE2记者与lacZ记者进行了一个易于评分、半定量、双报机分析的合并,可以根据P53突变体的残余功能水平对其进行子分类。23.

荧光报告机,如EGFP(增强的绿色荧光蛋白)或DsRed(迪斯科索玛sp.红色荧光蛋白)也被用于定量评估与所有可能的误感突变相关的变活活性。TP53编码序列24.最后,P53等位基因表达的可调启动子与RE和/或报告基因的异源酵母菌株相结合的机会,导致开发一个数据矩阵,生成癌症相关和生殖系的精细分类突变P53等位电25,26,27 。

上述方法用于测量P53蛋白的转录活性。然而,在酵母S.cerevisiae28和Schizosaccharis pombe29中野生型P53的表达可能导致生长迟缓,这与细胞周期阻滞28、30或细胞死亡31。在这两种情况下,酵母生长抑制由高P53表达触发,并与参与细胞生长的内源酵母基因的潜在转录调制相关。支持这一假设,功能丧失突变体P53 R273H在以与野生型P5332相似的水平表示时,不会干扰酵母细胞的生长。相反,在酵母中表达的有毒突变体P53 V122A(已知比野生型P53的转录活性更高)比野生型P533产生更强的生长抑制作用。

此外,证明人类MDM2能够抑制人类P53转录活性在酵母,促进其普及和随后的降解33。因此,人类MDM2和MDMX抑制P53诱导酵母生长抑制的能力被证明为32,34。在另外一项研究中,建立了P53转录活性与反应性表达水平之间的相关性,在酵母32中的ACT1基因上鉴定了假定的P53 RE上游。始终,行为蛋白表达由野生型P53增强,P53 V122A更增强,但突变P53 R273H更增强。相反,P53的活性素表达在P53抑制剂MDM2、MDMX或pifithrin-α(P53转录活性的小分子抑制剂)的共存在中减少,与基于酵母生长测定的结果一致。重要的是,这些结果建立了P53诱导的生长抑制与其在酵母中的活性程度之间的相关性,也用于识别和研究调节P53功能的小分子28,34,35.

Protocol

1. 构建含有特定RE(yAFM-RE或yLFM-RE)的ADE2或LUC1报告酵母菌株 在GAL1促进剂下,可分得 yAFM-ICORE 或 yLFM-ICORE 菌株 12、14(ICORE = I,ISce-I 内分糖;CO = 计数器可选URA3;RE = 记者KanMX4授予卡那霉素耐药性;表 1)从15%甘油储存在-80°C在YPDA琼脂板上(表2)。让它在30°C下生长2-3天。 从新鲜板(不超过3周)取?…

Representative Results

建造ADE2或LUC1记者 酵母菌株 Delitto perfetto 方法12、14、15、16已经过改造,可实现 P53 报告酵母菌株的构造 (图1B)。该方法采用?…

Discussion

基于酵母的检测已被证明对研究P53蛋白功能的各个方面是有用的。这些测定对于评估P53对RE目标位点的变异性(包括功能多态性评价)特别敏感。彩色检测器的使用以及荧光酶测定的小型化,可产生成本效益和相对可扩展的测定。此外,生长抑制试验可能适用于化学库筛选,通过测量吸收性自动定量酵母细胞活力。虽然由于ABC运输机的细胞壁和作用而限制渗透性被认为是使用酵母进行药物筛选的一个限制…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢欧洲联盟(FEDER基金POCI/01/0145/FEDER/007728通过竞争歌剧因素方案)和国家基金(FCT/MEC,发展基金和埃杜卡奥省)下伙伴关系协议 PT2020 UID/QUI/50006/2019 和项目 (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581。FCT研究金:SFRH/BD/96189/2013(S. Gomes)。这项工作得到了意大利都灵的Compagnia S. Paolo(项目 2017.0526)和卫生部的支持(项目 5×1000、2015 和 2016;2016 年当前研究)。我们深切感谢特雷莎·洛佩斯-阿里亚斯·黑山博士(特伦托大学实验科学教学实验室)协助进行录像。

Materials

L-Aspartic acid SIGMA 11189
QIAquick PCR Purification Kit QIAGEN 28104
L-Phenylalanine SIGMA 78019
Peptone BD Bacto 211677
Yeast ex+A2:C26tract BD Bacto 212750
Difco Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate BDTM 233520
Lithium Acetate Dihydrate SIGMA 517992
Bacteriological Agar Type A Biokar Diagnostics A1010 HA
G418 disulfate salt SIGMA A1720
Ammonium Sulfate SIGMA A2939
L-Arginine Monohydro-chloride SIGMA A5131
Adenine Hemisulfate Salt SIGMA A9126
Passive Lysis Buffer 5x PROMEGA E1941
Bright-Glo Luciferase Assay System  PROMEGA E2620
5-FOA Zymo Research F9001
D-(+)-Galactose SIGMA G0750
L-Glutamic acid SIGMA G1251
Dextrose  SIGMA G7021
L-Histidine SIGMA H8125
L-Isoleucine SIGMA I2752
L-Lysine SIGMA L1262
L-Leucine SIGMA L8000
L-Methionine SIGMA M2893
PEG SIGMA P3640
D-(+)-Raffinose Pentahydrate SIGMA R0250
L-Serine SIGMA S4500
L-Tryptophan SIGMA T0271
L-Threonine SIGMA T8625
Uracil SIGMA U0750
L-Valine SIGMA V0500
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Tags

YeastP53TranscriptionReporter GeneAdenine-2Firefly LuciferaseLithium AcetateTransformationPCRSanger SequencingTransactivationColor-based Assay

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Monti, P., Bosco, B., Gomes, S., Saraiva, L., Fronza, G., Inga, A. Yeast As a Chassis for Developing Functional Assays to Study Human P53. J. Vis. Exp. (150), e59071, doi:10.3791/59071 (2019).
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