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

ABSTRACT: Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

SUBMITTER: Mavor D

PROVIDER: S-EPMC4862753 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Mavor David D Barlow Kyle K Thompson Samuel S Thompson Samuel S Barad Benjamin A BA Bonny Alain R AR Cario Clinton L CL Gaskins Garrett G Liu Zairan Z Deming Laura L Axen Seth D SD Caceres Elena E Chen Weilin W Cuesta Adolfo A Gate Rachel E RE Green Evan M EM Hulce Kaitlin R KR Ji Weiyue W Kenner Lillian R LR Mensa Bruk B Morinishi Leanna S LS Moss Steven M SM Mravic Marco M Muir Ryan K RK Niekamp Stefan S Nnadi Chimno I CI Palovcak Eugene E Poss Erin M EM Ross Tyler D TD Salcedo Eugenia C EC See Stephanie K SK Subramaniam Meena M Wong Allison W AW Li Jennifer J Thorn Kurt S KS Conchúir Shane Ó SÓ Roscoe Benjamin P BP Chow Eric D ED DeRisi Joseph L JL Kortemme Tanja T Bolon Daniel N DN Fraser James S JS

eLife 20160425

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in ...[more]

PMID: 27111525

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Project description:BACKGROUND:Pinus koraiensis is an evergreen tree species with strong cold resistance. However, the transcriptomic patterns in response to cold stress are poorly understood for P. koraiensis. In this study, global transcriptome profiles were generated for P. koraiensis under cold stress (- 20 °C) over time by high-throughput sequencing. RESULTS:More than 763 million clean reads were produced, which assembled into a nonredundant data set of 123,445 unigenes. Among them, 38,905 unigenes had homology with known genes, 18,239 were assigned to 54 gene ontology (GO) categories and 18,909 were assigned to 25 clusters of orthologous groups (COG) categories. Comparison of transcriptomes of P. koraiensis seedlings grown at room temperature (20 °C) and low temperature (- 20 °C) revealed 9842 differential expressed genes (DEGs) in the 6 h sample, 9250 in the 24 h sample, and 9697 in the 48 h sample. The number of DEGs in the pairwise comparisons of 6 h, 24 h and 48 h was relatively small. The accuracy of the RNA-seq was validated by analyzing the expression patterns of 12 DEGs by quantitative real-time PCR (qRT-PCR). In this study, 34 DEGs (22 upregulated and 12 downregulated) were involved in the perception and transmission of cold signals, 96 DEGs (41 upregulated and 55 downregulated) encoding 8 transcription factors that regulated cold-related genes expression, and 27 DEGs (17 upregulated and 10 downregulated) were involved in antioxidant mechanisms in response to cold stress. Among them, the expression levels of c63631_g1 (annexin D1), c65620_g1 (alpha-amylase isozyme 3C), c61970_g1 (calcium-binding protein KIC), c51736_g1 (ABA), c58408_g1 (DREB3), c66599_g1 (DREB3), c67548_g2 (SOD), c55044_g1 (CAT), c71938_g2 (CAT) and c11358_g1 (GPX) first increased significantly and then decreased significantly with the extension of stress time. CONCLUSIONS:A large number of DEGs were identified in P. koraiensis under cold stress, especially the DEGs involved in the perception and transmission of cold signals, the DEGs encoding TFs related to cold regulation and the DEGs removing ROS in antioxidation mechanisms. The transcriptome and digital expression profiling of P. koraiensis could facilitate the understanding of the molecular control mechanism related to cold responses and provide the basis for the molecular breeding of conifers.

Dataset Information

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

Publications

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

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