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Protocol
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Biology

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

Published: September 07, 2021
doi:
10.3791/61100
, ,
1Department of Biomedical Genetics,University of Rochester Medical Center

Summary

蛋白质静力学下降是衰老的标志，促进神经退行性疾病的发作。我们概述了一种方案，通过融合到荧光报告基因的多谷氨酰胺重复序列的异源表达来量化两种不同 秀丽隐杆线虫 组织中的蛋白质稳态。该模型允许对蛋白质稳态进行快速体内遗传分析。

Abstract

在正常衰老过程中，维持蛋白质组（蛋白质稳态）的正常功能和折叠的能力下降，促进了越来越多的年龄相关疾病的发作。例如，具有聚谷氨酰胺扩增的蛋白质容易聚集，例如亨廷顿蛋白和伴随亨廷顿舞蹈症的发作。通过使用转基因 秀丽隐杆线虫（Caenorhabditis elegans） 与年龄相关的蛋白质组恶化已被广泛研究，这些秀丽隐杆线虫表达polyQ重复序列融合到黄色荧光蛋白（YFP）中。该 polyQ：：YFP 转基因动物模型通过成像荧光病灶（即蛋白质聚集体）的进行性形成以及随后由于蛋白质组塌陷而产生的运动缺陷的发作，促进了蛋白质组年龄相关衰退的直接定量。此外 ，polyQ：：YFP 转基因的表达可以由组织特异性启动子驱动，从而可以在完整的多细胞生物体的背景下评估组织内的蛋白质平衡。该模型非常适合遗传分析，因此提供了一种量化衰老的方法，与寿命测定相辅相成。我们描述了如何在衰老过程中准确测量神经元或体壁肌肉内的 polyQ：：YFP 病灶形成，以及随后行为缺陷的发作。接下来，我们将重点介绍如何使这些方法适应更高的通量，以及使用其他新兴策略进行 秀丽隐杆线虫 遗传分析的潜在未来应用。

Introduction

蛋白质稳态（蛋白质稳态）被定义为维持蛋白质组正常功能和折叠的细胞能力。蛋白质稳态的固有挑战是确保所有蛋白质都正确折叠并保持在天然构象中，其通过蛋白质大小，氨基酸组成，结构构象，稳定性，周转，表达，亚细胞区室化和修饰的不同性质进一步放大1。蛋白质平衡是通过由大约2000种独特蛋白质组成的大型蛋白质的协调作用来维持的，这些蛋白质调节蛋白质组内的适当合成，折叠，运输和降解2，3。蛋白质静膜网络的主力成分是分子伴侣的九个主要家族4。每种组织和细胞类型都优先利用分子伴侣的特定亚群，可能符合不同蛋白质组的不同需求5。

正常有机体衰老的一个标志是细胞蛋白质平衡的进行性下降和崩溃，这被认为是越来越多的年龄相关疾病发作和进展的根本基础。例如，阿尔茨海默病、帕金森病、亨廷顿病和肌萎缩性侧索硬化症（ALS）都有一个共同的特征：在每种情况下，神经变性的表现都是由遗传改变驱动的，这些改变使突变蛋白易聚集（淀粉样蛋白β/Tau，α突触核蛋白，分别是HTT，FUS / TBD – 43 / SOD-1） 6，7，8，9，10.在衰老过程中，蛋白质静剂网络的完整性和诱导性下降，这导致蛋白质毒性聚集体的积累，导致细胞功能障碍和神经变性。值得注意的是，蛋白质构象疾病不是神经元独有的，并且发生在多个组织中，如II型糖尿病，多发性骨髓瘤和囊性纤维化11，12，13，14所示。因此，阐明能够保持蛋白质平衡的机制将有助于开发有针对性的干预措施来治疗疾病并促进健康老龄化。

小土壤线虫秀丽隐杆线虫（C. elegans）在发现改变蛋白质稳态的基因和阐明途径方面发挥了重要作用。蛋白质静力学网络的许多组成部分和调节蛋白质稳态的信号转导途径在进化上是保守的。此外，秀丽隐杆线虫降低了相对于脊椎动物系统的复杂性和冗余性，使其更适合遗传分析和基因发现。秀丽隐杆线虫的其他优点导致它被广泛用作研究蛋白质稳态的模型系统，包括：强大的遗传和功能基因组学，较短的生命周期（3天）和寿命（3周），紧凑且注释良好的基因组，各种遗传突变体的可用性，以及使用荧光报告程序可视化细胞生物学中组织特异性变化的容易程度。

衰老过程中蛋白质稳态的进行性衰变可以很容易地在秀丽隐杆线虫中量化。森本实验室首先证明，融合到黄色荧光蛋白（polyQ：：YFP）的聚谷氨酰胺扩增可用于量化秀丽隐杆线虫在衰老过程中的蛋白质静力下降15，16，17，18。YFP 融合至 35 个谷氨酰胺重复序列或更多次可导致与年龄相关的荧光病灶形成以及细胞病理学体征。值得注意的是，这种谷氨酰胺扩增的范围反映了亨廷顿蛋白的聚谷氨酰胺束的长度，亨廷顿舞蹈症的病理学开始在人类中观察到（通常>35 CAG重复）19。在肌肉、肠或神经元细胞中表达polyQ：：YFP的菌株已被用于证实与年龄相关的蛋白质稳态下降发生在不同的细胞和组织类型中。肌肉特异性polyQ：：YFP表达（即unc-54p：：Q35：：YFP）一直是使用最广泛的组织特异性报告基因，因为在成年期的头几天，使用简单的荧光解剖显微镜很容易量化累积的荧光病灶（图1A-1B）。此外，动物在中年时会瘫痪，因为肌肉内的蛋白质组由于报告者的蛋白质毒性作用而塌陷（图1C）。类似地，神经元蛋白平衡的年龄相关下降可以通过直接量化将动物放入液体后协调体弯曲的病灶/聚集体形成和年龄相关下降来跟踪（rgef-1p：：Q40：：YFP）（图2）。

在这里，我们提出了一个详细的方案，说明如何测量蛋白质聚集体积累的年龄依赖性进展以及秀 丽隐杆线虫中神经元和肌肉组织中多谷氨酰胺重复表达诱导的相关蛋白质毒性。我们提供了使用这些菌株和方法产生的典型结果的示例。此外，我们展示了我们如何利用这些方法来研究蛋白质静力学网络的转录调控。我们讨论了这些报告器可以与其他现有试剂轻松集成或适应更大屏幕的其他方法。

Protocol

1. 试剂的制备 选择要通过基于喂养的RNAi灭活的目标基因。购买含有感兴趣的RNAi克隆的HT115 大肠杆菌 库存20。或者，将目的基因的cDNA亚克隆到L4440质粒的多克隆位点中。注意：为了防止细菌内dsRNA的降解，请使用HT115菌株。这是一种RNase III缺陷 大肠杆菌 菌株，具有IPTG诱导的T7聚合酶活性。对于不使用基于喂养的RNAi的蛋白质稳态研究，可以使用标准NGM板上?…

Representative Results

在秀丽隐杆线虫中，聚谷氨酰胺重复模型在鉴定调节蛋白质静膜网络的基因方面起到了重要作用。例如，我们之前表明，同源域相互作用蛋白激酶（hpk-1）是一种转录辅因子，通过调节自噬和分子伴侣的表达来影响衰老过程中的蛋白质稳态31。我们发现，hpk-1的损失，无论是通过RNAi沉默还是在hpk-1（pk1393）空突变动物中，都会增加衰老过程中积累的Q35…

Discussion

衰老的特征是蛋白质稳态逐渐下降。蛋白质稳态由一个复杂的系统（蛋白质静力学网络）维持，用于蛋白质折叠、降解和翻译的协调、动态、应激响应控制。为什么蛋白质稳态在衰老过程中会失败尚不清楚，但表观基因组的衰变，应激反应的可诱导性下降以及代偿性串扰的丧失都与这种崩溃相吻合。在秀丽隐杆线虫中，由于在应激位点2，34，35处形成抑制?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢萨缪尔森实验室过去和现在的成员，感谢他们在完善这种方法和/或讨论方面提供的帮助，这些讨论有助于本文的开发。本出版物中报告的研究得到了美国国立卫生研究院国家老龄化研究所的支持，其奖项编号为RF1AG062593和R21AG064519。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。资助者在研究设计，数据收集和分析，出版决定或手稿准备方面没有任何作用。

Materials

24 Well Culture Plates Greiner Bio-One #662102
2 mL 96-well plates Greiner Bio-One #780286
600 µL 96-well plates Greiner Bio-One #786261
96-pin plate replicator Nunc 250520
Air-permeable plate seal VWR 60941-086
bacteriological agar Affymetrix/USB 10906
bacto-peptone VWR 90000-368
C. elegans RNAi clone library in HT115 bacteria- Ahringer Source Bioscience C. elegans RNAi Collection (Ahringer) See also Kamath et. al, Nature 2003.
C. elegans RNAi clone library in HT115 bacteria- Vidal Source Bioscience C. elegans ORF-RNAi Resource (Vidal) See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.
FuDR (5-Fluoro-2'-deoxyuridine) Alfa Aesar L16497
Glass microscope cover slips VWR 48404-455
Glass microscope slides VWR 160004-422
IPTG (isopropyl beta-D-1-thigalactopyranoside) Gold Bio 12481C100
Retangular non-treated single-well plate, 128x86mm Thermo-Fisher 242811
Sodium Azide, CAS #26628-22-8 Sigma-Aldrich S2002
Zeiss Axio Imager M2m microscope with AxioVision v4.8.2.0 software Zeiss unknown
Zeiss StemiSV11 M2 Bio Quad microscope Zeiss unknown
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Tags

Keywords: ProteostasisCaenorhabditis ElegansPolyglutamine Fluorescent ReporterAgingProtein Misfolding DiseasesAlzheimer’sParkinson’sHuntington’s DiseaseAmyotrophic Lateral SclerosisRNAiOP50 E.coliHypochlorite TreatmentL1 Animals
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Lazaro-Pena, M. I., Cornwell, A. B., Samuelson, A. V. Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans. J. Vis. Exp. (175), e61100, doi:10.3791/61100 (2021).
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