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

Mavor D, Barlow K, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate RE, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo EC, See SK, Subramaniam M, Wong AW, Li J, Thorn KS, Conchúir SÓ, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS - Elife (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, San Francisco, San Francisco, United States.

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.

No MeSH data available.


Related in: MedlinePlus

A shared response to different chemical perturbations.(A) DMSO fitness - Caffeine fitness vs. DMSO fitness - DTT fitness. The markers are colored based on DMSO fitness - Hydroxyurea fitness. (B) DMSO fitness - Caffeine fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - DTT fitness. (C) DMSO fitness - DTT fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - Caffeine fitness.DOI:http://dx.doi.org/10.7554/eLife.15802.01610.7554/eLife.15802.017Figure 9—source data 1.Shared response mutants representing mutations that are equally perturbed by all three sensitizing perturbations.Mutants in the shared response were determined by fitting a line to the fitness scores. The distance from each point to that line was calculated. If the distance was less than 0.1 and the average Δ (DMSO - Perturbation) fitness was less than -0.2 the mutant was considered part of the shared response. E1 activity relative to WT Ub (Roscoe and Bolon, 2014) is listed and may explain the sensitization of some of the shared response mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.017Perturbation specific mutations represent alleles that are differentially affected by Caffeine, DTT and Hydroxyurea.Perturbation specific mutations were determined by fitting a line to the delta (DMSO - perturbation) fitness scores. The distance from each point to that line was calculated. If the distance was greater than 0.35 the mutant was classified as perturbation specific. Mutants with high experimental errors were deemed outliers and removed from this list.DOI:http://dx.doi.org/10.7554/eLife.15802.018Specific information regarding highlighted mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.019
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fig9: A shared response to different chemical perturbations.(A) DMSO fitness - Caffeine fitness vs. DMSO fitness - DTT fitness. The markers are colored based on DMSO fitness - Hydroxyurea fitness. (B) DMSO fitness - Caffeine fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - DTT fitness. (C) DMSO fitness - DTT fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - Caffeine fitness.DOI:http://dx.doi.org/10.7554/eLife.15802.01610.7554/eLife.15802.017Figure 9—source data 1.Shared response mutants representing mutations that are equally perturbed by all three sensitizing perturbations.Mutants in the shared response were determined by fitting a line to the fitness scores. The distance from each point to that line was calculated. If the distance was less than 0.1 and the average Δ (DMSO - Perturbation) fitness was less than -0.2 the mutant was considered part of the shared response. E1 activity relative to WT Ub (Roscoe and Bolon, 2014) is listed and may explain the sensitization of some of the shared response mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.017Perturbation specific mutations represent alleles that are differentially affected by Caffeine, DTT and Hydroxyurea.Perturbation specific mutations were determined by fitting a line to the delta (DMSO - perturbation) fitness scores. The distance from each point to that line was calculated. If the distance was greater than 0.35 the mutant was classified as perturbation specific. Mutants with high experimental errors were deemed outliers and removed from this list.DOI:http://dx.doi.org/10.7554/eLife.15802.018Specific information regarding highlighted mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.019

Mentions: To determine the elements of the shared response to HU, Caffeine and DTT, we defined 'shared sensitizing mutations' as those that were both sensitizing (delta fitness ≤ -0.2 for all perturbations) and consistent between perturbations (within 0.1 of the regression line) (Figure 9A and Figure 9—source data 1). Most of these mutations change from being mildly deleterious to being nearly upon chemical stress. For example, in DMSO Ub tolerates mutation to small hydrophobics and other polar residues at Thr7. However, chemical stresses causes mutations of small hydrophobic or charged residues at this position to be deleterious. As Thr7 is adjacent to the hydrophobic patch residue Leu8, this sensitization is likely due to non-polar substitutions disrupting Ub adaptor protein binding and poly-Ub packing (Komander and Rape, 2012). Additionally, typically destabilizing substitutions such as Proline or Tryptophan generally become more deleterious under perturbation.


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

Mavor D, Barlow K, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate RE, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo EC, See SK, Subramaniam M, Wong AW, Li J, Thorn KS, Conchúir SÓ, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS - Elife (2016)

A shared response to different chemical perturbations.(A) DMSO fitness - Caffeine fitness vs. DMSO fitness - DTT fitness. The markers are colored based on DMSO fitness - Hydroxyurea fitness. (B) DMSO fitness - Caffeine fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - DTT fitness. (C) DMSO fitness - DTT fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - Caffeine fitness.DOI:http://dx.doi.org/10.7554/eLife.15802.01610.7554/eLife.15802.017Figure 9—source data 1.Shared response mutants representing mutations that are equally perturbed by all three sensitizing perturbations.Mutants in the shared response were determined by fitting a line to the fitness scores. The distance from each point to that line was calculated. If the distance was less than 0.1 and the average Δ (DMSO - Perturbation) fitness was less than -0.2 the mutant was considered part of the shared response. E1 activity relative to WT Ub (Roscoe and Bolon, 2014) is listed and may explain the sensitization of some of the shared response mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.017Perturbation specific mutations represent alleles that are differentially affected by Caffeine, DTT and Hydroxyurea.Perturbation specific mutations were determined by fitting a line to the delta (DMSO - perturbation) fitness scores. The distance from each point to that line was calculated. If the distance was greater than 0.35 the mutant was classified as perturbation specific. Mutants with high experimental errors were deemed outliers and removed from this list.DOI:http://dx.doi.org/10.7554/eLife.15802.018Specific information regarding highlighted mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.019
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Related In: Results  -  Collection

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fig9: A shared response to different chemical perturbations.(A) DMSO fitness - Caffeine fitness vs. DMSO fitness - DTT fitness. The markers are colored based on DMSO fitness - Hydroxyurea fitness. (B) DMSO fitness - Caffeine fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - DTT fitness. (C) DMSO fitness - DTT fitness vs. DMSO fitness - Hydroxyurea fitness. The markers are colored based on DMSO fitness - Caffeine fitness.DOI:http://dx.doi.org/10.7554/eLife.15802.01610.7554/eLife.15802.017Figure 9—source data 1.Shared response mutants representing mutations that are equally perturbed by all three sensitizing perturbations.Mutants in the shared response were determined by fitting a line to the fitness scores. The distance from each point to that line was calculated. If the distance was less than 0.1 and the average Δ (DMSO - Perturbation) fitness was less than -0.2 the mutant was considered part of the shared response. E1 activity relative to WT Ub (Roscoe and Bolon, 2014) is listed and may explain the sensitization of some of the shared response mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.017Perturbation specific mutations represent alleles that are differentially affected by Caffeine, DTT and Hydroxyurea.Perturbation specific mutations were determined by fitting a line to the delta (DMSO - perturbation) fitness scores. The distance from each point to that line was calculated. If the distance was greater than 0.35 the mutant was classified as perturbation specific. Mutants with high experimental errors were deemed outliers and removed from this list.DOI:http://dx.doi.org/10.7554/eLife.15802.018Specific information regarding highlighted mutants.DOI:http://dx.doi.org/10.7554/eLife.15802.019
Mentions: To determine the elements of the shared response to HU, Caffeine and DTT, we defined 'shared sensitizing mutations' as those that were both sensitizing (delta fitness ≤ -0.2 for all perturbations) and consistent between perturbations (within 0.1 of the regression line) (Figure 9A and Figure 9—source data 1). Most of these mutations change from being mildly deleterious to being nearly upon chemical stress. For example, in DMSO Ub tolerates mutation to small hydrophobics and other polar residues at Thr7. However, chemical stresses causes mutations of small hydrophobic or charged residues at this position to be deleterious. As Thr7 is adjacent to the hydrophobic patch residue Leu8, this sensitization is likely due to non-polar substitutions disrupting Ub adaptor protein binding and poly-Ub packing (Komander and Rape, 2012). Additionally, typically destabilizing substitutions such as Proline or Tryptophan generally become more deleterious under perturbation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, San Francisco, San Francisco, United States.

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.

No MeSH data available.


Related in: MedlinePlus