Fig 2: Clavulanate potassium suppresses tumor growth both in vitro and in vivo by inhibiting LACTBM5L+R469K. A)The possibility of combination between LACTBM5L+R469K and p53R156P was detected by virtual screen platform. B) Cellular Thermal Shift Assay was used to detected the thermostability of LACTBM5L+R469K in vitro. C) The melting curves of LACTBM5L+R469K protein, and the data were based on the result of western blot. The grayscale of LACTBM5L+R469K 48 centigrade. D) Cellular Thermal Shift Assay was used to detected the thermostability of PSMB7 in vitro. E) The grayscale of PSMB7 protein at 69 centigrade. F) Treating U2‐OS cells with 100ug mL−1 CLA for 48 h, then detected the proteasome subunit catalytic activity. G) U2‐OS and SJSA‐1 cells were treated with different concentration CLA for 72 h, and then wild type p53 protein level was analyzed by western blot and grayscale analysis. H) U2‐OS, SJSA‐1 and HOS cells were treated with CLA for 48 h, and the inhibiting efficiency was detected by CCK‐8. I) U2‐OS, SJSA‐1 cells were treated with 1.5 mm CLA and different concentration cisplatin, and then detecting cell viability by CCK‐8. J) Photographs of resected tumors and tumor growth curves of 0.9% NaCl group, CLA group, CIS group and CLA+CIS group that using different SJSA‐1 cells injected in proximal tibia. The symbol of ## means the significant by using T test.

Fig 3: Knocking down LACTBM5L+R469K can upregulate wild type p53 protein via binding and suppressing PSMB7. A) The ubiquitination of wt‐p53 was analyzed by co‐IP and western blot. B) The wild type p53 protein level after treating by lysosome inhibitors for 24 h in control group and LACTBM5L+R469K knockdown group were detected by western blot. C) Proteasome subunits catalytic activity test kit was used to test the catalytic activity of PSMB5, PSMB6, and PSMB7. D) The wild type p53 protein level of SJSA‐1 cells after treating by PSMB5, PSMB6, and PSMB7 inhibitors (Bortezomib/Ixazomib/Leupeptin) for 24 h in control group and LACTBM5L+R469K knockdown group were detected by western blot. E) U2‐OS and SJSA‐1 cells were transfected with indicated plasmid, and then subjected to co‐IP using anti‐HA antibody follow by western blot. F) The wt‐LACTB‐HA and LACTBM5L+R469K‐HA overexpression plasmids were transfected into SJSA‐1 and U2‐OS cells, and then subjected to co‐IP using anti‐HA antibody. The protein levels of HA‐tag and PSMB7 were detected by using western blot after co‐IP.

Fig 4: EIF4EBP1 activation enhances the 26S proteasomal degradation capacity in skeletal muscle. (A) Immunoblotting of 19S (PSMD1) and constitutive 20S proteasome subunit proteins (PSMA2, PSMB5, PSMB6, and PSMB7) was performed in the soluble fraction extracted from the TA of 4‐month‐old male mice following 48-h fasting (n = 4). (B) the quantification of (A). (C) the quantification of (D). (D) Active proteasome complexes were resolved by native gel electrophoresis using proteasome lysates extracted from a pool of muscle of 4-month-old male mice under fed conditions. Native gel immunoblotting of 19S (PSMD1) and constitutive 20S proteasome subunit proteins (PSMB5, PSMB7, and PSMA2) were showed (n = 4). (E) Proteasome activity analysis in the presence of ATP was performed in lysate extracted from 4-month-old male mouse muscle under fed conditions (n = 4 for 4EPB1mt-muscle group and EIF4EBP1mt-atg7mKO group; n = 3 for control group and atg7mKO group). (F) Immunoblotting of K48 linked ubiquitinated protein and muscle-specific E3 ubiquitin ligases (FBXO32, and TRIM63) was performed in the soluble fraction extracted from TA of 4-month-old male mice following 48-h fasting (n = 3). (G) the quantification of (F). For all the quantification of immunoblotting shown in (B), (C), and (G), the relative protein expression levels were normalized to control group, and data were shown as mean. Individual points corresponded to one mouse. Ponceau S was used as a loading control. Statistical significance was determined by One-Way ANOVA with Tukey’s multiple comparison test for other proteins. Only significant differences (p < 0.05) were shown. The quantification of (A) and (F) were also shown in Table S4 and (D) was shown in Table S5.