The Antitumor Drug Aclacinomycin A, Which Inhibits the Degradation of Ubiquitinated Proteins, Shows Selectivity for the Chymotrypsin-like Activity of the Bovine Pituitary 20 S Proteasome

Maria E Figueiredo-Pereira,Wei Er Chen,Jingrong Li,Osamu Johdo

doi:10.1074/jbc.271.28.16455

Maria E Figueiredo-Pereira, Wei Er Chen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.28.16455

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Abstract

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 microM (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T.(1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug. An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 microM of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 position) was inhibited only 11 and 24%, respectively. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain. The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.

Highlights

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 ␮M (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T. (1992) Biochim
Changes in the Chymotrypsin-like Activity of the 20 S Proteasome Produced by Aclacinomycin A—The effect of the cytotoxic drug on multicatalytic proteinase complex (MPC) hydrolysis of peptide bonds after hydrophobic amino acids was studied with six different substrates (Fig. 2 and Table I)
Aclacinomycin A was found to inhibit the degradation of ubiquitinated proteins in reticulocyte lysates at a step following ubiquitin-protein conjugation [1]

Summary

EXPERIMENTAL PROCEDURES

Materials—The 20 S proteasome was isolated from bovine pituitaries as described [28]. Z-LLE-NA, succinyl-LLVY-AMC, dephosphorylated ␤-casein, rabbit muscle calpain, bovine pancreas ␣-chymotrypsin and trypsin were from Sigma. Purification of the Aglycone and Sugar Moieties of Aclacinomycin A—Hydrogenolysis of the anthracycline was as described [34]. Enzyme Assays—Enzyme activities with synthetic substrates were determined as described previously, at 37 °C [8, 9]. Substrates were Z-GGL-pNA, Z-DALR-NA, and Z-LLE-NA for determination of the chymotrypsin-like, trypsin-like, and PGP activities of the 20 S proteasome, respectively. MPC hydrolysis of Z-GPALG-pAB (branched chain amino acid preferring activity) and Z-GPAGG-pAB (small neutral amino acid preferring activity) were measured as described [11]. The activity of chymotrypsin toward Z-GGL-pNA and Z-E(OtBu)AL-pNA was measured in 0.05 M Tris-HCl, pH 7.5, and trypsin activity toward Z-DALR-NA was analyzed in the presence of 1 mM CaCl2 in 0.05 M Tris-HCl, pH 8.0. All activities were expressed relative to control (vehicle alone) conditions

RESULTS

Aclacinomycin B

DISCUSSION

Additions and Corrections