Journal: Cell reports

Article Title: Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes

doi: 10.1016/j.celrep.2022.110860

Figure Lengend Snippet: (A–C) Episomal expression of HA-tagged Rad6 reverts rad6Δ growth defect (A) and supports the accumulation of K63 ubiquitin chains under 0.6 mM H 2 O 2 as visualized by immunoblotting whole-cell lysate (B) or isolated ribosomes (C). *Rad6 bound to ubiquitin. Anti-actin was used as loading control for whole-cell lysate, and anti-uS3 was used as ribosomal loading control. (D) Immunoblot from in vitro ubiquitination assay of ribosomes isolated from rad6Δbre1Δ cells in the presence of recombinant Rad6 ubiquitination system as described in the . All lanes contain wild-type ubiquitin, ATP, energy-generation system, and DTT. (E) Accumulation of K63-linked ubiquitin chains in yeast cells depend on RAD6 and BRE1 . Immunoblot anti-K63 ubiquitin chains from ribosomes isolated from cells treated in the presence or absence of 0.6 mM H 2 O 2 for 30 min. Anti-uS3 was used as ribosomal loading control. (F) Immunoblot of in vitro reactions shows Rad6 charged with ubiquitin monomer (Ub) and K63-linked di-ubiquitin (K63 Ub 2 ). All lanes contain Rad6, E1, and ATP. (G) Immunoblot of in vitro ubiquitination assay of ribosomes with wild-type ubiquitin monomer (Ub) or K63-linked di-ubiquitin chains (K63 Ub 2 ). All lanes contain E1, Bre1, ATP, energy-generation system, DTT, and ribosomes from rad6Δ bre1Δ strain. (H) Immunoblot of in vitro ubiquitination assay of ribosomes with wild-type (WT) ubiquitin monomer or K63R mutant ubiquitin (K63R). All lanes contain E1, Bre1, ATP, energy-generation system, DTT, and ribosomes from rad6Δ bre1Δ strain.

Article Snippet: Recombinant Ubiquitin activating enzyme E1 (human) - UBA1 , Enzo Life Sciences , Cat# BML-UW9410-0050.

Techniques: Expressing, Western Blot, Isolation, In Vitro, Ubiquitin Assay, Recombinant, Mutagenesis

Journal: Cell reports

Article Title: Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes

doi: 10.1016/j.celrep.2022.110860

Figure Lengend Snippet: (A) Non-reducing immunoblot of isolated ribosomes from cells exposed to 0.6 mM H 2 O 2 for 30 min reveals a Rad6-containing high-molecular-weight complex. Anti-uS3 was used as ribosomal loading control. *Anti-Rad6 antibody unspecific band. (B) Non-reducing immunoblot of yeast cell lysate shows formation, prevention, and reduction of Rad6 high-molecular-weight complexes. Cell lysate from yeast cells expressing HA-tagged WTRad6, C88S, or C88A mutants were alkylated with 20 mM IAM, oxidized with 2 mM H 2 O 2 , or reduced with 15 mM DTT for 30 min at room temperature (RT) prior to immunoblot. *Rad6 bound to ubiquitin. (C) Formation of Rad6 disulfides in yeast cell lysate is impacted by 1 M NaCl prior to 2 mM H 2 O 2 treatment for the designated times. (D) Schematic of the denaturing coIP coupled with SILAC liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify Rad6 redox partner. Created with BioRender.com . (E) Scatterplot of MS/MS intensity and SILAC ratio of Rad6/Rad6 C88S under H 2 O 2 stress. (F) Immunoblot anti-HA (Rad6) of Uba1-myc coIP shows complex with Rad6-HA but not with Rad6 C88S , which is reduced by 15 mM DTT. *Rad6 bound to ubiquitin. (G) Immunoblot anti-myc (Uba1) shows disulfide formation upon 0.6 mM H 2 O 2 treatment for cells expressing WT Rad6 but not Rad6 C88S , which is reduced by 15 mM DTT. (H) Immunoblot of Rad6 mutants (R7A, R11A, R7A/R11A) show differential formation of Rad6-Uba1 complex in cells exposed to 0.6 mM H 2 O 2 . *Rad6 charged with ubiquitin through thioester bond. (I) Immunoblot anti-Rad6 shows that deletion of glutathione reductase ( GLR1 ) slows down the reduction of the Rad6-Uba1 complex during stress recovery after treatment with 0.6 mM H 2 O 2 for 30 min. (J) Immunoblot anti-Rad6 shows that the reversal of Rad6-Uba1 complex is impacted by glutathione balance. Cells were grown into MPD medium in the presence or absence 2.5 mM of BSO, an inhibitor of glutathione synthesis, for 3 h prior to treatment with 0.6 mM H 2 O 2 for 30 min, followed by recovery from stress for 30 min.

Article Snippet: Recombinant Ubiquitin activating enzyme E1 (human) - UBA1 , Enzo Life Sciences , Cat# BML-UW9410-0050.

Techniques: Western Blot, Isolation, Molecular Weight, Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Cell reports

Article Title: Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes

doi: 10.1016/j.celrep.2022.110860

Figure Lengend Snippet: (A) Representative immunoblot anti-Rad6 from in vitro oxidation reactions of recombinant Rad6 (100 nM) and UBA1 (100 nM) in the presence of 2 mM H 2 O 2 . Lane labels describe the sequential order of events and which protein was oxidized prior to mixing. Quantification was performed using iBright Analysis Software, and graph shows mean ± standard deviation for three biological replicates. Quantification of Rad6-E1 band was normalized by the amount present in lane 2 compared with the levels of DTT-reduced Rad6 for each sample. *p ≤ 0.005, **p ≤ 0.025 determined by Student’s t test. (B) Spectroscopic analysis of 75 μg of purified Rad6 treated in the presence or absence of 2 mM H 2 O 2 for 30 min at RT. Samples were then incubated with 100 μM 4-chloro-7-nitrobenzofurazan (NBD) for an additional 30 min, followed by removal of excess reagent by successive cycles of washes and filtration. Rad6 sulfenic acid peak is exhibited at 420 nm. (C) Close-up view of Rad6 active site (PDB: 1AYZ) highlighting its catalytic cysteine (pink) and vicinal residues (blue) as sticks. (D) Immunoblots of Rad6 mutants for amino-acid residues interacting with catalytic Cys88 show differential ubiquitination and disulfide formation. Anti-actin was used as loading control. *Rad6 charged with ubiquitin through thioester bond. (E) Immunoblots anti-Rad6, anti-K63 ubiquitin chains, and anti-ubiquitin show dynamics of Rad6-Uba1 complex and polyubiquitin chain formation as well as depletion of the pool of monomeric ubiquitin over time after cellular exposure to 0.6 mM H 2 O 2 . Anti-GAPDH was used as loading control. (F) Accumulation of Rad6-Uba1 complex and depletion of the ubiquitin pool is dose dependent. Immunoblot from Rad6-HA-expressing cells subjected to increased H 2 O 2 concentrations for 30 min. Anti-GAPDH was used as loading control. (G) Immunoblot from in vitro incubation of yeast recombinant Rad6 (100 nM)and UBA1 (100 μM) in the presence of increased ubiquitin concentration for 30 min followed by 2 mM H 2 O 2 treatment for 30 min. Proteins were also pre-incubated with the E1 inhibitor PYR-41 (75 μM) or alkylated with iodoacetamide (20 mM) for 30 min prior to exposure to H 2 O 2 . (H) Yeast cells at stationary phase do not show Rad6-Uba1 disulfide formation and have high levels of K63 ubiquitin chains. Immunoblot of cells treated with indicated H 2 O 2 concentrations for 30 min in mid-log (optical density [OD] 600 = 0.5) or late (OD 600 = 3.2) stationary phase. Anti-GAPDH was used as loading control. (I) Schematic of regulation of Rad6 activity in response to stress. Oxidation of Rad6 catalytic cysteine leads to the formation of a Rad6-Uba1 disulfide complex, which inhibits Rad6 activity and controls the levels of K63 ubiquitination in a negative feedback mechanism. Created with BioRender.com . (J) Immunoblot shows higher levels of K63-linked ubiquitin chain accumulation in Rad6 R7A/R11A cells over time. Yeast cells expressing WT Rad6 and a Rad6 R7A/R11A mutant generated through CRISPR-Cas9 method were incubated with 0.6 mM H 2 O 2 for the respective times. (K) Immunoblot from cells expressing WT Rad6 and a Rad6 R7A/R11A mutant subjected to increasing H 2 O 2 concentrations. Rad6 R7A/R11A cells show reduced formation of Rad6-Uba1 complex and higher levels of K63 ubiquitin chains. Anti-GAPDH was used as loading control.

Article Snippet: Recombinant Ubiquitin activating enzyme E1 (human) - UBA1 , Enzo Life Sciences , Cat# BML-UW9410-0050.

Techniques: Western Blot, In Vitro, Recombinant, Software, Standard Deviation, Purification, Incubation, Filtration, Expressing, Concentration Assay, Activity Assay, Mutagenesis, Generated, CRISPR

Journal: Cell reports

Article Title: Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes

doi: 10.1016/j.celrep.2022.110860

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant Ubiquitin activating enzyme E1 (human) - UBA1 , Enzo Life Sciences , Cat# BML-UW9410-0050.

Techniques: Recombinant, Protease Inhibitor, Mass Spectrometry, Mutagenesis, Expressing, Software