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Genetics

演示序列比对以预测跨物种敏感性的工具，用于快速评估蛋白质保存

Published: February 10, 2023
doi:
10.3791/63970
, , , , , , , ,
1Office of Research and Development, Center for Computational Toxicology and Exposure, Scientific Computing and Data Curation Division,U.S. Environmental Protection Agency, 2Oak Ridge Institute for Science and Education, 3Swenson College of Science and Engineering, Department of Biology,University of Minnesota Duluth, 4University of Wisconsin-Madison Aquatic Sciences Center, 5General Dynamics Information Technology,Research Triangle Park, 6Office of Research and Development, Center for Computational Toxicology and Exposure, Great Lakes Toxicology and Ecology Division,U.S. Environmental Protection Agency

Summary

在这里，我们提出了一个协议，以利用最新版本的美国环境保护署序列比对来预测跨物种敏感性（SeqAPASS）工具。该协议演示了在线工具的应用，以快速分析蛋白质保存，并提供可定制且易于解释的跨物种化学敏感性预测。

Abstract

美国环境保护署跨物种敏感性预测序列比对（SeqAPASS）工具是一种快速，免费提供的在线筛选应用程序，允许研究人员和监管机构推断跨物种的毒性信息。对于模型系统中的生物靶标，如人类细胞、小鼠、大鼠和斑马鱼，可以使用各种化学物质的毒性数据。通过评估蛋白质靶标保守性，该工具可用于将从此类模型系统生成的数据外推到数千种缺乏毒性数据的其他物种，从而预测相对内在化学敏感性。该工具的最新版本（版本 2.0-6.1）包含新功能，允许快速合成、解释和使用数据以进行发布以及演示质量的图形。

这些功能包括可定制的数据可视化和全面的摘要报告，旨在汇总SeqAPASS数据，以便于解释。本文描述了指导用户提交作业、浏览各种级别的蛋白质序列比较以及解释和显示结果数据的协议。重点介绍了 SeqAPASS v2.0-6.0 的新功能。此外，还描述了使用此工具专注于转甲状腺素蛋白和阿片受体蛋白质保存的两个用例。最后，讨论了SeqAPASS的优势和局限性，以定义该工具的适用范围，并强调跨物种外推的不同应用。

Introduction

传统上，毒理学领域严重依赖使用全动物测试来提供化学安全评估所需的数据。此类方法通常成本高昂且需要大量资源。然而，由于目前使用的化学品数量众多，新化学品的开发速度很快，全球范围内都认识到需要更有效的化学品筛选方法1，2。这种需求以及由此产生的从动物试验的范式转变导致了许多新方法的开发，包括高通量筛选测定、高通量转录组学、二代测序和计算建模，这些都是有前途的替代测试策略3，4。

评估可能受化学品暴露影响的物种多样性的化学安全性一直是一个持久的挑战，不仅要进行传统的毒性测试，还要使用新的方法。比较和预测毒理学的进步为理解不同物种的相对敏感性提供了框架，计算方法的技术进步继续提高这些方法的适用性。在过去的十年中，已经讨论了几种策略，这些策略利用现有的基因和蛋白质序列数据库以及特定化学分子靶点的知识，以支持跨物种外推的预测方法，并增强典型模式生物以外的化学安全性评估5，6，7，8。

为了将科学转化为行动，在这些预测毒理学基础研究的基础上，优先考虑化学测试工作并支持决策，创建了美国环境保护署序列比对以预测跨物种敏感性（SeqAPASS）工具。该工具是一个公共且免费提供的基于Web的应用程序，它使用不断扩展的蛋白质序列信息的公共存储库来预测物种多样性的化学敏感性9。基于可以通过评估该化学品的已知蛋白质靶标的保守性来确定物种对特定化学物质的相对内在敏感性的原理，该工具可快速比较具有已知敏感性的物种的蛋白质氨基酸序列与具有现有蛋白质序列数据的所有物种。该评估通过三个级别的分析完成，包括（1）伯爵氨基酸序列，（2）功能结构域和（3）关键氨基酸残基比较，每个级别都需要更深入地了解化学 – 蛋白质相互作用，并在敏感性预测中提供更高的分类分辨率。SeqAPASS的一个主要优势是，用户可以根据感兴趣的化学-蛋白质或蛋白质-蛋白质相互作用的可用信息量，通过添加额外的证据线来定制和完善他们的评估。

第一个版本于2016年发布，允许用户以简化的方式评估伯代氨基酸序列和功能结构域，以预测化学敏感性，并且包含最少的数据可视化功能（表1）。个体氨基酸差异已被证明是化学 – 蛋白质相互作用跨物种差异的重要决定因素，这会影响物种的化学敏感性10，11，12。因此，开发了后续版本以考虑对直接化学相互作用很重要的关键氨基酸13。为了响应利益相关者和用户的反馈，该工具每年发布一次版本，增加了新功能，旨在满足研究人员和监管界应对跨物种外推挑战的需求（表1）。2020 年推出的 SeqAPASS 5.0 版本带来了以用户为中心的功能，这些功能包括数据可视化和数据合成选项、外部链接、汇总表和报告选项以及图形功能。总体而言，该版本的新属性和功能改进了数据合成、外部数据库之间的互操作性以及预测跨物种敏感性的数据解释的便利性。

Protocol

1. 入门 注意：此处介绍的协议侧重于工具实用程序和关键功能。有关方法、特性和组件的详细说明，请参见网站上的综合用户指南（表1）。 表 1：SeqAPASS 工具的演变。 从初始部署添加到 SeqAPASS 工具的功能和更新列表。缩写：seqAPASS = 序列比对以预测跨物种敏感性;ECOTOX = 生态毒素学知识库。 <a href="https://www.jove.com/files…

Representative Results

为了演示SeqAPASS工具的应用并突出新功能，描述了两个案例研究，代表了蛋白质保守预测不同物种（人甲状腺素转运蛋白）的化学敏感性存在差异并且没有差异（μ阿片受体[MOR]）的情况。这些例子中的第一个涉及蛋白质序列/结构比较，以预测不良结果途径的适用性领域（AOP，定义见 表2 ），而第二个则侧重于开发与废水中存在的阿片类药物的跨物种敏感性相关的研究假设。这些案例?…

Discussion

人们普遍认识到，对足够的物种进行经验测试以捕获可能暴露于毒理学相关化学物质的生物体的基因组、表型、生理和行为多样性是不可行的。SeqAPASS的目标是最大限度地利用现有和不断扩展的蛋白质序列和结构数据，通过分子水平的比较，帮助和告知从测试生物体到数百或数千种其他物种的化学毒性数据/知识的外推。SeqAPASS工具旨在通过简化和快速的分析来降低科学家，风险评估人员和监管机?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢Daniel L. Villeneuve博士（美国环保署计算毒理学和暴露中心）和Jon A. Doering博士（路易斯安那州立大学环境科学系）对手稿的早期草稿提供评论。这项工作得到了美国环境保护署的支持。本文中表达的观点是作者的观点，不一定反映美国环境保护署的观点或政策，提及的商品名称或商业产品也不表示联邦政府的认可。

Materials

Spreadsheet program N/A N/A Any program that can be used to view and work with csv files (e.g. Microsoft Excel, OpenOffice Calc, Google Docs) can be used to access data export files.
Basic computing setup and internet access N/A N/A SeqAPASS is a free, online tool that can be easily used via an internet connection. No software downloads are required.
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Tags

Sequence AlignmentProtein ConservationSeqAPASSCross-species ExtrapolationToxicity PredictionBiological PathwayProtein TargetProtein AccessionWeb-based InterfaceSpecies SensitivityChemical Susceptibility

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Vliet, S. M. F., Hazemi, M., Blatz, D., Jensen, M., Mayasich, S., Transue, T. R., Simmons, C., Wilkinson, A., LaLone, C. A. Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation. J. Vis. Exp. (192), e63970, doi:10.3791/63970 (2023).
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