Abstract
The ubiquitin proteasome–proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.
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References
Adams J. Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer. Curr Opin Chem Biol. 2002;6:493–500.
Arbiser JL et al. Naturally occurring proteasome inhibitors from mate tea (Ilex paraguayensis) serve as models for topical proteasome inhibitors. J Invest Dermatol. 2005;125:207–12.
Brignole C et al. Effect of bortezomib on human neuroblastoma cell growth, apoptosis, and angiogenesis. J Natl Cancer Inst. 2006;98:1142–57.
Canfield SE, Zhu K, Williams SA, McConkey DJ. Bortezomib inhibits docetaxel-induced apoptosis via a p21-dependent mechanism in human prostate cancer cells. Mol Cancer Ther. 2006;5:2043–50.
Cardozo C, Michaud C. Proteasome-mediated degradation of tau proteins occurs independently of the chymotrypsin-like activity by a nonprocessive pathway. Arch Biochem Biophys. 2002;408:103–10.
Chauhan D, Hideshima T, Anderson KC. Proteasome inhibition in multiple myeloma: therapeutic implication. Annu Rev Pharmacol Toxicol. 2005;45:465–76.
Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat Biotechnol. 2000;18:538–43.
Efuet ET, Keyomarsi K. Farnesyl and geranylgeranyl transferase inhibitors induce G1 arrest by targeting the proteasome. Cancer Res. 2006;66:1040–51.
Feling RH et al. Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora. Angew Chem Int Ed Engl. 2003;42:355–7.
Furet P et al. Entry into a new class of potent proteasome inhibitors having high antiproliferative activity by structure-based design. J Med Chem. 2004;47:4810–3.
Golab J et al. Synergistic antitumor effects of a selective proteasome inhibitor and TNF in mice. Anticancer Res. 2000;20:1717–21.
Kisselev AF, Kaganovich D, Goldberg AL. Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active Sites of the 20S proteasome: evidence for peptide-induced channel opening of the alpha-rings. JBC. 2002;277:22260–70.
Kisselev AF, Callard A, Goldberg AL. Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate. J Biol Chem. 2006;281:8582–90.
Koguchi Y et al. TMC-89A and B, new proteasome inhibitors from streptomyces sp. TC 1087. J Antibiot (Tokyo). 2000;53:967–72.
Lindsten K et al. A transgenic mouse model of the ubiquitin/proteasome system. Nat Biotechnol. 2003;21(8):897–902.
Luker GD, Pica CM, Song J, Luker KE, Piwnica-Worms D. Imaging 26S proteasome activity and inhibition in living mice. Nat Med. 2003;9:969–73.
Moravec RA et al. Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format. Anal Biochem. 2009;387:294–304.
Rechsteiner M, Hill CP. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol. 2005;15(1):27–33.
Richardson PG et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003;348:2609–17.
Vinitsky A, Cardozo C, Sepp-Lorenzino L, Michaud C, Orlowski M. Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. J Biol Chem. 1994;269:29860–6.
Zaarur N, Gabai VL, Porco Jr JA, Calderwood S, Sherman MY. Targeting heat shock response to sensitize cancer cells to proteasome and Hsp90 inhibitors. Cancer Res. 2006;66:1783–91.
Acknowledgements
This work was supported in part by a National Institute Grant 5R01CA087548 to K.K. and by an NIH K01 training grant CA105066 to E.T. E. We thank Dr. Kelly Hunt for providing the velcade.
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Keyomarsi, K., Efuet, E.T. & Bui, T.N. Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells. Cell Biol Toxicol 27, 123–131 (2011). https://doi.org/10.1007/s10565-010-9175-1
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DOI: https://doi.org/10.1007/s10565-010-9175-1