Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting-猫眼课题宝

DOI：

10.1101/025452

发表时间：

2015-08

期刊：

eLife

影响因子：

7.7

通讯作者：

中科院分区：

生物学1区

文献类型：

作者：

David Mavor;Kyle A. Barlow;Samuel Thompson;B. Barad;Alain R. Bonny;Clinton L. Cario;Garrett Gaskins;Zairan Liu;Laura Deming;S. Axen;Elena L. Cáceres;Weilin Chen;A. Cuesta;Rachel E. Gate;E. Green;Kaitlin R Hulce;Weiyue Ji;L. Kenner;Bruk Mensa;L. Morinishi;Steven M. Moss;M. Mravic;Ryan K Muir;Stefan Niekamp;C. Nnadi;Eugene Palovcak;E. Poss;T. Ross;Eugenia C. Salcedo;Stephanie K. See;Meena Subramaniam;A. Wong;Jennifer Li;K. Thorn;S. Conchúir;Benjamin P. Roscoe;E. Chow;J. Derisi;T. Kortemme;Daniel N. A. Bolon;J. Fraser

泛素化是一个重要的翻译后调控过程，可以控制蛋白质的稳定性、定位和活性。泛素是真核生物生命所必需的，并且高度保守，在酵母和人类之间只有3个氨基酸位置不同。然而，最近对酿酒酵母的深度测序研究表明，泛素对单一氨基酸突变具有高度的耐受性。为了解决这一悖论，我们假设当培养物不在富媒体条件下生长时，可容忍的替换集合将减少，并且化学诱导的生理扰动可能会揭示对泛素序列的限制。为了验证这一假设，加州大学旧金山分校一年级的研究生班采用了一种深度突变扫描程序，在存在五种小分子扰动之一的情况下，确定了泛素所有可能的单一氨基酸突变的文库的适合度图景：MG132、二硫苏糖醇(DTT)、羟基脲(HU)、咖啡因和DMSO。我们的数据显示，DTT、HU或咖啡因极大地减少了耐受取代的数量，并且这些扰动揭示了定位于疏水斑块周围区域和C末端的“共同敏化位置”。我们还显示了扰动的特异性效应，包括His68在HU中的敏化和对DTT中Lys63突变的耐受性。综上所述，我们的数据表明，化学压力降低了泛素蛋白酶体系统的缓冲效应，揭示了以前隐藏的健康缺陷。通过扩大分析的化学扰动集，可能通过其他基于课堂的经验，我们将能够进一步解决极端序列保守和实验观察到的泛素突变耐受性之间的明显二分法。最后，本研究展示了以项目实验室为基础的跨学科研究生课程的实现潜力。

Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies in S. cerevisiae indicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover “shared sensitized positions” localized to areas around the hydrophobic patch and to the C-terminus. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting

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