Site-specific K63 ubiquitinomics reveals stabilization of ribosomes in response to oxidative stress
Reprograming of protein synthesis is an essential cellular process to tolerate and resist to stressing conditions. A variety of mechanisms are known to regulate translation at initiation but cells can also control proteins synthesis after the initiation checkpoint. We previously showed that K63 ubiquitin can modify ribosome proteins in response to oxidative stress. However, the mechanism by how K63 ubiquitin impacts ribosome function is entirely unknown. Here we characterized > 1000 K63 ubiquitin sites in the yeast Saccharomyces cerevisiae by mass spectrometry, and showed that many sites clustered at the head of the 40S subunit in response to H2O2. Moreover, ribosomes lacking K63 ubiquitin were depleted in proteins from the translation initiation factor eIF3, particularly Tif35 (eIF3g), which impacted ribosome stability, and protein production. Our results provided new insights on the role of K63 ubiquitin in regulating the resistance to oxidative stress via a post-initiation control of translation.
Sample Processing Protocol
Isolation of K63 ubiquinated peptides – WT and K63R cells were culture into synthetic SILAC media supplemented with light and heavy isotopes of lysine and arginine, respectively (L-Arg6 13C; L-Lys8 13C, 15N; Cambridge Isotopes). Cells were treated with 0.6 mM H2O2 for 30 min and equal number of cells were collected and lysed in buffer containing: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 20 mM Chloroacetamide, 50 nM FLAG K63-TUBE peptide, 1X EMD Millipore protease inhibitor cockatail set I. K63 ubiquitinated proteins were isolated in M2 anti-FLAG resin (Sigma), elute in glycine buffer, digested with Trypsin/Lys-C, and diglycyl-lysine peptides (GG) were isolated with Cell Signaling PTM Scan kit. Polysome proteomics – For the quantitative analysis of the polysome and monosome composition, cells were treated in the absence (1,3) of presence (2,4) of H2O2. As above, WT and K63R cells were grown into heavy and light SILAC medium, and polysome fractions were separated by a sucrose gradient. Protein was precipitated using TCA/Acetone and digested as above with Trypsin/Lys-C mixture. Samples were desalted using Hypersep spin tips (Thermo) prior to LC-MS/MS analysis Mass spectrometry analysis - Peptides were separated on a 25-cm Thermo Acclaim PepMap C18 column (75 µm ID, 2 µm particle size, 100 Å pore size) by reverse-phase chromatography with a gradient of 5–60% acetonitrile over 2.5 h, performed with an Eksigent NanoLC 2DPlus liquid chromatography system. The eluted peptides were injected in-line onto an LTQ Orbitrap Elite MS (Thermo Scientific). Data-dependent analysis was performed at a resolution of 120,000, AGC at 1e6 and maximum injection time of 100 ms with the top 20 most intense ions selected from each MS full scan. Dynamic exclusion was set to 30 s if m/z acquisition was repeated within a 30-s interval. MS2 was acquired at a resolution of 15,000, AGC at 5e4 and maximum injection time of 100ms. The K63 ubiquitinomics samples was loaded into a 50 cm Easy Spray PepMap C18 column (75 µm ID, 2 µm particle, 100 Å pore size) in-line with a Q-Exactive (Thermo Scientific) mass spectrometer using a 75-min gradient (0–40% ACN). Data dependent analysis was performed at resolution of 70,000, AGC at 1e6 and maximum injection time of 120 ms. Top 20 most intense ions selected from each MS full scan, with dynamic exclusion set to 30 s if m/z acquisition was repeated within a 30-s interval. MS2 was acquired at a resolution of 17,500, AGC at 5e4 and maximum injection time of 120ms.
Data Processing Protocol
The RAW data files were processed using MaxQuant to identify and quantify protein abundance. The spectra were matched against the yeast Saccharomyces cerevisiae database (Uniprot, 2015 release). Protein identification was performed using 10 ppm tolerance with a posterior global FDR of 1% based on the reverse sequence of the yeast FASTA file. Up to two missed trypsin cleavages were allowed, and oxidation of methionine and N-terminal acetylation were searched as variable post-translational modification and cysteine carbamidomethylation as fixed. For K63 ubiquitinated peptides, diglycyl-lysine (GlyGly(K)) was also searched as variable modification.
Back S, Gorman AW, Vogel C, Silva GM. Site-specific K63 ubiquitinomics provides insights into translation regulation under stress. J Proteome Res. 2018 PubMed: 30489083