Proteasomal chymotrypsin-like activity

Purified 20S proteasome (Methanosarcina thermophila, recombinant, Escherichia coli) was purchased from Calbiochem. Purified eukaryotic 20S proteasome (from rabbit) and eukaryotic 26S proteasome (from rabbit) were purchased from Sigma. Fluorogenic peptide substrates, Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chymotrypsin-like activity) and benzyloxycarbonyl, (Z)-Leu-Leu-Glu-AMC (for the proteasomal PGPH activity), were also obtained from Calbiochem, and Z-Gly-Gly-Arg-AMC (for the proteasomal trypsin-like activity) was purchased from Bachem (King of Prussia, PA). 

The chymotrypsin-like activity of purified 20S proteasome was measured as follows 2 pg of purified recombinant 20S proteasome or 0.5 pg of eukaryotic 20S proteasome or 1 pi of eukaryotic 26S proteasome was incubated with 20 pM fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chymotrypsin-like activity), for 30 min at 37°C in 200 pi of assay buffer (20 mM Tris-HCl, pH 8.0) with or without the polyphenols (5 pM). After incubation, the reaction mixture was diluted with 200 pi assay buffer followed by a measurement of the hydrolyzed 7-amido-4-methyl-coumarin (AMC) groups using a VersaFluor Fluorometer with an excitation filter of 380 mn and an emission filter of 460 nm (Bio-Rad). 

Theaflavins efficiently and specifically inhibited the proteasomal chymotrypsin-like activity of the purified mammalian 20S and 26S proteasomes in cell-free systems (figure 11.3) as well as 26S proteasomes in tumor cell extracts (table 11.1). The purified M. thermophila recombinant 20S proteasome was more sensitive to theaflavins than mammalian 20S or 26S proteasomes, possibly due to prokaryotic recombinant 20S proteasome without postmodification that forms steric hindrance that prevents theaflavins from binding to the chymotrypsin-like activity subunit of the 20S proteasome. When assaying for proteasomal chymotrypsin-like activity in human cancer cell extracts, the IC50 values of tea polyphenols were apparently increased compared... 

Recently, Banik et al. [61, 62] have explored the anticancer activity of monocyclic (3-lactam XI against leukemia and colon carcinoma cell lines and very recently, Imbach et al. [63] reported the monocyclic (3-lactam XII as the selective inhibitor of the chymotrypsin-like activity of the human 20 S proteasome (Fig. 3). 

In 2007 a series of p-lactam derivatives was designed and synthesized to inhibit the chymotrypsin-like activity of the human 20S proteasome. The most potent compounds of this new structural class of p-subunit exhibit good selectivity over the trypsin-like and post-glutamyl-peptide hydrolytic activities of the enzyme [409],... 

SI pockets [22], The three major activities, the peptidylglutamil-hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, have been assigned to the three active subunits of ySl, yS2 and yS5, respectively, based on mutational and crystal structure analyses. Furthermore, it has been claimed, based on studies with model peptides and inactivation by inhibitors, that mammalian proteasomes also contain two other peptidase activities, referred to as branched-chain amino acid-preferring and small neutral amino acid-preferring (SNAAP). 

Wojcik et al., Ubiquitin-mediated proteolysis centers in HeLa cells. Indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome, Eur. J. Cell BioL 71 (19%) 311-318. 

A series of peptidyl a-ketoaldehydes have been synthesized as putative inhibitors of the chymotrypsin-like activity of proteasome [385], The most potent peptide Z-Leu-Leu-Tyr-COCHO exhibits a K value of 3.0 nM (library 15), the lowest so far reported for tripeptidyl aldehyde-based proteasome inhibitors. A novel indanone peptide derivative (library 16 IC50 0.14 /iM) was identified as potent competitive inhibitor of the chymotrypsin-like activity of the 20S proteasome from a 400 member library [386], The SAR indicates a strong preference for lipophilic side-chains L-Leu and D-Leu at the Aai and Aa2 positions. 

Table 2.3 Inhibition of the PGPH, trypsin- and chymotrypsin-like activities of yeast 20S proteasome by maleoyl- S-alanyl-dipeptide aldehydes (/C50, pM)...

To test whether theaflavins could inhibit the chymotrypsin-like activity of the purified proteasomes, such as the recombinant 20S proteasome from M. thermophila, rabbit... 

FIGURE 11.3 Comparison of the inhibitory effect of tea polyphenols on chymotrypsin-like activity of the purified proteasomes derived from different sonrces. The chymotrypsin-like activity of the purified proteasomes recombinant prokaryotic 20S proteasome, enkaryotic 20S proteasome, and eukaryotic 26S proteasome were analyzed by incnbation with the fluogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (for chymotrypsin-like activity of the proteasome) in the presence of the tea polyphenols (5 pM) for 30 min and detected free AMC groups. Assays were performed in triplicate, and the results of representative experiments are shown as mean SD. 

Inhibition of chymotrypsin-like activity of the proteasome in different cell extracts by tea polyphenols... 

Inhibition of the Chymotrypsin-like Activity of Purified Proteasomes by Galloyl-Ester-Containing Polyphenols... 

Bortezomib (velcade) (1R)-3-methyl-l-[[(2S)-1 -oxo-3-phenyl-2-[pyrazinylcarbonyl)amino] propyl]butyl]boronic acid binds to the 20S core of the 26S proteasome and reversibly inhibits its chymotrypsin-like activity. Inhibition of the proteasome disrupts multiple signaling cascades within the cell, often leading to cell death. The most important consequences of proteasome inhibition are believed to result from downregulation of NF-kB, a key transcription factor that promotes cell survival. In a similar manner, bortezomib disrupts ubiquitin-proteasome regulation of p2I, p27, and p53, which are key regulatory proteins in the cell cycle and initiators of apoptosis. 

---