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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

The structure of Ub highlighting important residues.Cartoon model of Ub (PDB 1UBQ) with important residues colored as follows: Lys48 - orange, Lys63 - light blue, Lys11 - green, other Lys residues - yellow, hydrophobic patch (Leu8, Val40, Ile 44) - red, C-terminal diGly motif (Gly75 and 76) - purple, Arg42 - pink, His68 - olive.DOI:http://dx.doi.org/10.7554/eLife.15802.014
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fig7: The structure of Ub highlighting important residues.Cartoon model of Ub (PDB 1UBQ) with important residues colored as follows: Lys48 - orange, Lys63 - light blue, Lys11 - green, other Lys residues - yellow, hydrophobic patch (Leu8, Val40, Ile 44) - red, C-terminal diGly motif (Gly75 and 76) - purple, Arg42 - pink, His68 - olive.DOI:http://dx.doi.org/10.7554/eLife.15802.014

Mentions: In DMSO only residues with well-established roles are sensitive: Arg42 (E1 activation), Ile44 (hydrophobic patch hotspot), Lys48 (essential Lys48 linked poly-Ub) and Gly75-Gly76 of the C-terminus (E1 activation) (Figure 7). The face opposite the hydrophobic patch is mostly tolerant and the protein core and residues adjacent to the sensitive residues are mostly intermediate (Figure 8B - i). When treated with Caffeine, DTT or HU, a shared set of residues become sensitive (Figure 8B ii– iv, Figure 8C). These residues are either: located adjacent to DMSO sensitive residues (e.g. Leu73, which is in the C-terminal tail); residues with important biological functions that of intermediate sensitivity in DMSO (e.g. Leu8, Val70, which are important hydrophobic patch residues); or core residues (e.g. Ile36, Leu71). These positions tolerated a small set of substitutions in DMSO but upon perturbation became only tolerant of mutations that share physical chemistry with the wild type residue.


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)

The structure of Ub highlighting important residues.Cartoon model of Ub (PDB 1UBQ) with important residues colored as follows: Lys48 - orange, Lys63 - light blue, Lys11 - green, other Lys residues - yellow, hydrophobic patch (Leu8, Val40, Ile 44) - red, C-terminal diGly motif (Gly75 and 76) - purple, Arg42 - pink, His68 - olive.DOI:http://dx.doi.org/10.7554/eLife.15802.014
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862753&req=5

fig7: The structure of Ub highlighting important residues.Cartoon model of Ub (PDB 1UBQ) with important residues colored as follows: Lys48 - orange, Lys63 - light blue, Lys11 - green, other Lys residues - yellow, hydrophobic patch (Leu8, Val40, Ile 44) - red, C-terminal diGly motif (Gly75 and 76) - purple, Arg42 - pink, His68 - olive.DOI:http://dx.doi.org/10.7554/eLife.15802.014
Mentions: In DMSO only residues with well-established roles are sensitive: Arg42 (E1 activation), Ile44 (hydrophobic patch hotspot), Lys48 (essential Lys48 linked poly-Ub) and Gly75-Gly76 of the C-terminus (E1 activation) (Figure 7). The face opposite the hydrophobic patch is mostly tolerant and the protein core and residues adjacent to the sensitive residues are mostly intermediate (Figure 8B - i). When treated with Caffeine, DTT or HU, a shared set of residues become sensitive (Figure 8B ii– iv, Figure 8C). These residues are either: located adjacent to DMSO sensitive residues (e.g. Leu73, which is in the C-terminal tail); residues with important biological functions that of intermediate sensitivity in DMSO (e.g. Leu8, Val70, which are important hydrophobic patch residues); or core residues (e.g. Ile36, Leu71). These positions tolerated a small set of substitutions in DMSO but upon perturbation became only tolerant of mutations that share physical chemistry with the wild type residue.

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