bortezomib treatment recovery  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants

doi: 10.1101/2024.09.08.611708

Figure Lengend Snippet: (A) CESAR has a conserved CUE domain. Protein domain architecture of MpCESAR. The CUE and ELKS-Rab6-interacting/CAST family member 1 (ELKS) domains are highlighted in purple and green, respectively. A schematic representation of the conservation-mapped AF2-predicted model for the CUE domain shown in Fig. S19A is highlighted. (B) MpCESAR binds ubiquitin in vitro . S. cerevisiae (Sc) VPS9 was used as positive control. (C-D) MpCESAR CUE domain is necessary for ubiquitin binding. MpCESAR ΔCUE =MpCESAR 47–987 . Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST or anti-MBP antibodies. (E) MpCESAR CUE binds different ubiquitin chain linkages with similar affinities. Halo-tagged MpCESAR CUE coupled to HaloLink resin was incubated with tetra-ubiquitin (Ub4) of the indicated linkage types. The captured materials were separated on 4-12% SDS-PAGE gel and silver stained. The asterisk indicates non-specific bands from Halo-MpCESAR CUE which have a similar electrophoretic mobility as K63-Ub4 chains. (F-G) Proteotoxic stress enhances AtCESAR2 association with TUBEs. 5-d-old A. thaliana seedlings expressing GFP-EV (GFP) or AtCESAR2-GFP in wild -type (Col-0) background were incubated in liquid ½ MS medium with 1% sucrose for 4h at 21°C (Control, C) or 37°C (Heat stress, HS) followed by 4h recovery phase at 21°C (panel F) or for 1h at 21°C in DMSO (D)-supplemented or 5 µM Bortezomib (B)-supplemented media followed by 1h recovery phase in fresh media (panel G) and used for co-immunoprecipitation. Plant lysates were incubated with Magne® HaloTag ® Beads conjugated with HaloTag-TUBE. Input and bound proteins were detected by immunoblotting using the respective antibodies as indicated. Total protein loading control was analysed by Amidoblack (AB) staining. Immunoblotting for bait is shown in Fig. S19E (related to ) and Fig. S19F (related to ). (H-I) Proteotoxic stress enhances AtCESAR1 association with TUBEs. 5-d-old A. thaliana seedlings expressing mCherry-EV (mCherry) or AtCESAR1-mCherry in wild -type (Col-0) background were incubated in liquid ½ MS medium with 1% sucrose for 4h at 21°C (Control, C) or 37°C (Heat stress, HS) followed by 4h recovery phase at 21°C (panel H) or for 1h at 21°C in DMSO (D)-supplemented or 5 µM Bortezomib (B)-supplemented media followed by 1h recovery phase in fresh media (panel I) and used for co-immunoprecipitation. Plant lysates were incubated with Magne® HaloTag ® Beads conjugated with HaloTag-TUBE. Input and bound proteins were detected by immunoblotting using the respective antibodies as indicated. Total protein loading control was analysed by Amidoblack (AB) staining. Immunoblotting for bait is shown in Fig. S19G (related to ) and Fig. S19H (related to ). (J-K) HS increases CESAR localization to the insoluble fraction. 5-d-old A. thaliana seedlings expressing either GFP-EV (GFP) or AtCESAR2-GFP (panel J), either mCherry-EV (mCherry) or AtCESAR1-mCherry (panel K) in wild -type (Col-0) background were incubated in liquid ½ MS medium with 1% sucrose for 4h at 21°C (Control, C) or 37°C (Heat stress, HS) followed by 4h recovery phase at 21°C. Soluble and pellet fractions were separated by centrifugation and normalized before immunoblotting using the respective antibodies as indicated. Total protein loading control was analysed by Amidoblack (AB) staining. (L) CESAR partitioning to the insoluble fraction is reversible. 5-d-old A. thaliana seedlings expressing AtCESAR1-mCherry in wild -type (Col-0) background were incubated in liquid ½ MS medium with 1% sucrose for 4h at 37°C followed by a 4 - hour recovery phase at 21°C. Soluble (input) and pellet fractions were separated by centrifugation, and pellet fraction was further sonicated in a water bath before immunoblotting using the respective antibodies as indicated. (M) TUBE interactors are differentially enriched in atg5 and cesar1 cesar2 . Protein abundance pattern represented by a heatmap as (Log2 (PSM+0.1) – mean PSM value per protein) for the 16 and 13 proteins belonging to clusters 8 and 3 in Fig. S20B, respectively. A full caption of the heatmap is shown in Fig. S20C. Rows were clustered using Euclidean distance and resulting dendrograms are omitted from the figures. (N) Insoluble proteins accumulate in the pellet fraction upon heat stress recovery. Dot plot representing protein abundance as Log2 (Fold change) for the ratio of two pairwise comparisons, cesar1 cesar2 vs. WT (x-axis) and atg5 vs. WT (y-axis). Dot size is mapped to reflect significant enrichment in atg5 represented as – Log10 (Adjusted P value). Enriched proteins in cesar1 cesar2 vs. WT or atg5 vs. WT are colored in purple and yellow, respectively. (O) Comparison between atg5 and cesar1 cesar2 pellet-enriched fractions. Venn diagram of the two overlapping pairwise comparisons in comparing proteins enriched in atg5 (yellow circle) or cesar1 cesar2 (purple circle) pellet fractions. (P-R) Analysis of protein features in the pellet-enriched fractions of atg5 and cesar1 cesar2 . Boxplots representing protein size (P), isoelectric point (Q) and relative aromaticity (R) of the indicated genotypes. TAIR10 proteome was used as a reference. Horizontal white lines within the boxes indicate the median and the top and bottom edges of the boxes represent the 3 rd and 1 st quartile, respectively. Vertical colored dashed lines indicate 1.5x the interquartile range. P values derived from pairwise comparisons by Wilcoxon-Mann-Whitney test are shown. ns, not significant. (S) Enriched proteins in cesar1 cesar2 pellets are highly hydrophobic. Line plots representing the hydrophobicity profiles along the protein sequence of pellet-enriched proteins in the indicated genotypes. The solid line represents the mean hydrophobicity and the shaded are represents the standard deviation. TAIR10 proteome was used as a reference. Protein hydrophobicity levels as defined by the Kyle-Doolittle scale is shown on the y-axis and proteins as defined by percentiles of its total sequence length are shown on the x-axis.

Article Snippet: For autophagic flux upon Bortezomib treatment recovery, 5-d-old seedlings were transferred to fresh liquid 1⁄2 MS medium with 50 μM Bortezomib (Santa Cruz; CAS 179324-69-7; dissolved in DMSO) or an equivalent volume of DMSO and incubated for 2 hours under control conditions.

Techniques: Ubiquitin Proteomics, In Vitro, Positive Control, Binding Assay, Recombinant, Western Blot, Incubation, SDS Page, Staining, Expressing, Control, Immunoprecipitation, Centrifugation, Sonication, Quantitative Proteomics, Comparison, Derivative Assay, MANN-WHITNEY, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants

doi: 10.1101/2024.09.08.611708

Figure Lengend Snippet: (A) cesar1 cesar2 plants are not hypersensitive to carbon or nitrogen starvation. 9-d-old A. thaliana seedlings of the indicated genotypes were grown in ½ MS + MES + 1% sucrose for 9 days, followed by either 4 days of carbon starvation (-C, left) or 6 days of nitrogen starvation (-N, right), respectively. 13-d-old and 15-d-old seedlings are shown in the images. Representative images of 3 independent biological replicates (see Fig. S22A) per genotype are shown. Scale bar, 1 cm. (B) cesar1 cesar2 plants are sensitive to HS. A. thaliana plants of the indicated genotypes were grown on soil under a 8-h light/16-h dark photoperiod at 21°C for 3 weeks before incubation at 21°C (Control) or 37°C (Heat stress, HS) for 3 days without watering, followed by a 18day recovery at 21°C, after which they were imaged. 42-d-old plants are shown in the images. Representative images per genotype are shown. Scale bar, 1 cm. (C) Rosette area quantification and statistical analysis. Left panel, rosette areas were measured for each plant and normalized to the maximum rosette area value of wild-type (Col-0) at 21°C. Ordinary-one-way Anova with Tukey’s multiple comparisons test was performed to assess the differences in the normalized Rosette Area between genotypes at 37°C (Heat stress, HS). ***, Adjusted P value < 0.001 (0.0003 for WT vs atg5 ; 0.0006 for WT vs cesar1 cesar2 ); *, Adjusted P value < 0.05 (0.0121 for atg5 vs nbr1 ; 0.0219 for cesar1 cesar2 vs nbr1 ), non-significant differences are not shown. Right panel, size factor difference between the normalized rosette area for the indicated genotypes. (D) cesar1 cesar2 plants are sensitive to proteasome inhibition. A. thaliana seedlings of the indicated genotypes were grown in 1% agar ½ MS + MES + 1% sucrose plates with either DMSO (left plate) or 3.75 µM Bortezomib (right plate) for 18 days before imaging. Representative images of 6 independent biological replicates are shown (n = 40 seeds per genotype per replicate). Scale bar, 1 cm. (E) Quantification of the survival rate of seedlings grown in Bortezomib-containing plates. Normalized survival rate for the replicate of Bortezomib plate assays shown in . Survival rate of each replicate was calculated by dividing the number of seedlings that showed a phenotype of size bigger than 0.3 cm and green colour by the total number of seeds sown per genotype (40) and normalized to the highest survival value for the wild-type (Col-0) background of all the 6 biological replicates (n = 240, each dot represents a replicate with 40 seeds per genotype per plate)). Ordinary One-way Anova with Tukey’s multiple comparisons test was performed to assess the differences between the survival rates of the different phenotypes. ****, Adjusted P value < 0.0001; *, Adjusted P value < 0.05 (0.0322). Non-significant differences are not shown.

Article Snippet: For autophagic flux upon Bortezomib treatment recovery, 5-d-old seedlings were transferred to fresh liquid 1⁄2 MS medium with 50 μM Bortezomib (Santa Cruz; CAS 179324-69-7; dissolved in DMSO) or an equivalent volume of DMSO and incubated for 2 hours under control conditions.

Techniques: Incubation, Control, Inhibition, Imaging