Fig. 4

Downregulated proteasome pathway and activity in DKO zebrafish, which lead to MG-H1 accumulation and form a feedback loop in larvae. (A) Heat map showed relative mRNA expression levels of genes from the proteasome pathway gene set. Higher and lower expressions are displayed in orange and blue, respectively. n = 5 clutches with 20 larvae in each group. (B–C) Proteasome activity (chymotrypsin-like) was decreased in DKO larvae and muscle compared to WT, GLO1KO and ALDH3A1KO zebrafish, n = 6. (D) Proteasome activity (chymotrypsin-like) was significantly reduced in larvae treated with 1 mM MG-H1 compared to the untreated larvae, and it was significantly increased in larvae co-treated with 1 mM MG-H1 and 10 mM CAR. n = 4. (E) Protein-bound MG-H1 levels were increased in larvae treated with 7.5 μM BOR compared to the untreated larvae. n = 4. (F) Graphical overview of the feedback loop between MG-H1 and proteasome activity. Figure was created with BioRender.com. (G) Crystal structure of the β5 subunit of proteasome in zebrafish (gray) in complex with MG-H1 (green). Identified sites are highlighted in red. Hydrogen bonds (black dashed lines), salt bridges (yellow dashed lines) and hydrophobic (gray dashed lines) between MG-H1 and the amino acids are shown. The bars indicate mean ± SD values. Statistical analysis was applied by Student's t-test and one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MG-H1, methylglyoxal-derived hydroimidazolone 1; CAR, L-Carnosine; BOR, bortezomib.