Activators of the 20S Proteasome

As the deletion of S5a/Rpnl0 facilitates dissociation of the RC into base and lid, it is assumed to be located at the interface this is consistent 

Rpnl3 was identified as a new subunit of the yeast 26S proteasome by mass spectrometric analysis of purified 19S cap complexes, but no homolog has been detected in other species (Verma et al., 2000). The 

N-terminal conserved domain I C-terminal in vitro Ub-binding site 

4 UCH blocks 9 dileucine repeats C2H2-finger, 2 acidic domains 

To our knowledge the existence of hybrid proteasomes has not been proved by direct visualization. When expressed in E. coli, in the absence of PA28 subunits, PA28 a subunits assemble into a heptameric particle whose crystal structure has been determined (Knowlton et al., 1997). The PA28 heptamer is a cone-shaped particle formed by bundles of a-helices it is traversed by a central channel that has a 3-nm opening on the side proximal and a 2-nm opening on the side distal to the proteasome (Knowlton etal., 1997). 

---

The 20S proteasome can exist not only as a core of 26S, but also as a separate population that cannot degrade ubiquitinated proteins. However, the 20S proteasome by itself has chymotrypsin-like, trypsin-like, and postglutamyl peptidase activities which cleave after hydrophobic, basic, and acidic residues, respectively. The peptide-hydrolyzing activity of the 20S proteasome can be modulated by an IIS regulatory cap. °°... 

Another helping hand. In eukaryotes, the 20S proteasome in conjunction with the I9S component degrades ubiquitinated proteins with the hydrolysis of a molecule of ATP. Archaea lack ubiquitin and the 26S proteasome but do contain a 20S proteasome. Some archaea also contain an ATPase that is homologous to the ATPases of the eukaryotic 19S component. This archaeal ATPase activity was isolated as a 650-kd complex (called PAN) from the archaeon Thermoplasma, and experiments were performed to determine if PAN could enhance the activity of the 20S proteasome from Thermoplasma as well as other 20S proteasomes. 

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. 

EGCG selectively inhibits the activity of topoisomerase 1 but not topoisomerase 11 in human colon cancer cell lines the doses necessary for this inhibition (10-17 pM) are lower than those required for inhibition of cell growth (IC50 = 10-90 pM). EGCG has been shown to inhibit the chymotryptic activity of the 20s proteasome in leukemic, breast cancer, and prostate cancer cell lines, leading to accumulation of p27 P and IkB, and subsequent cell cycle arrest and inhibition of NF-kB activity, respectively. 

Recently, more selective noncovalent inhibitors of proteasome have been developed. TMC-95A 15 is a potent and reversible selective inhibitor of the chymotrypsin-like, trypsinlike, and caspaselike activities of the 20S proteasome. Comparatively, TMC-95A shows no inhibition of calpain, cathepsin, or trypsin. 

Like bortezomib, MLN9708 is also a peptide boronate (Fig. 13.5) but it is orally active, shows greater tissue penetration, and has a shorter half-hfe. The drug is primarily an inhibitor of the chymotrypsin-like activity of the 20S proteasome core and, like bortezomib, it inhibits NF-kB activation and has antitumor activity in multiple myeloma and some other hematologic mahgnan-cies. Besides peptide boronates like bortezomib, other synthetic compounds tested as proteasome inhibitors include peptide aldehydes, peptide epoxyketones, and peptide vinyl sulfones. 

In the NF-kB pathway with celastrol 76, the inhibition of the iKBa degradation is due to the upstream blockage of the kinase activity and not by the direct inhibition of proteasome activity. On the contrary, direct inhibition of proteasome activity was observed with celastrol 76 and pristimerin 2 in prostate cancer cells.90-92 Both triterpene QMs directly inhibited the activity of the 20S subunits of proteasome at 2.5 iM and induced the accumulation of ubiquitinated proteins over time in cells,... 

The N-terminal extensions are removed thereby generating a new unblocked N-terminal threonine in the catalytically active yS-subunits. A small accessory protein called Umpl in yeast or proteassemblin in mammalian cells assists in the final assembly of the 20S proteasome [132], Interestingly Umpl/POMP is apparently trapped in the proteasome s central chamber and degraded upon maturation of the enzyme [133]. 

In addition to the RC there are two protein complexes, REGajS and REGy, and a single polypeptide chain, PA200, that bind the 20S proteasome and stimulate peptide hydrolysis but not protein degradation. Like the RG, proteasome activators bind the ends of the 20 S proteasome and, importantly, they can form mixed or hybrid 26S proteasomes in which one end of the 20S proteasome is associated with a 19S RC and the other is bound to a proteasome activator [147-150]. This latter property raises the possibility that proteasome activators serve to localize the 26S proteasome within eukaryotic cells. 

M. R. Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY. 

G. N. Identification, purification, and characterization of a high-molecular weight, ATP-dependent activator (PA700) of the 20S proteasome. J. 

Proteolytically active P-type subunits are synthesized in an inactive precursor form containing N-terminal extensions of variable lengths, the propeptides, which must be removed posttranslationally to allow the formation of active sites. This process is tied in with the assembly of the 20S proteasome in such a manner, that activation is delayed until assembly is complete and the active sites are sequestered from the cellular environment. Cleavage of the propeptide proceeds autocatalytically, reljdng on the active-site threonine, and the invariant glycine at position -1 appears to be the prime determinant of the cleavage site (Schmidtke et al. 1996 Seemiiller et al. 1996 Chen and Hochstrasser 1996). 

Our studies clearly differentiate NPI-0052 from other proteasome inhibitors in the speed and duration of action and the inhibition profile of the 20S proteasome. These differences and the possible off-target activities of bortezomib indicate that NPI-0052 may provide a greater therapeutic index and greater activity in diseases where bortezomib shows minimal activity.64... 

The activity of human 20S proteasome was measured by monitoring the release of 7-amino-4-methylcoumarin (AMC) from the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC. 

TMC-95A, a cydic peptide metabolite from Apiospra montagnei, is a potent competitive inhibitor of all active sites and forms characteristic hydrogen bonds with the protein backbone. The crystal structure of the yeast 20S proteasome in complex with TMC-95A indicates a non-covalent linkage to the active y -subunits the N-terminal threonine residues are not modified. The TMC-95A backbone adopts a -conformation and extends the -strand SI by the generation of an antiparallel P-sheet. This stmcture is similar to that seen with the aldehyde and epoxyketone inhibitors. An interactions of TMC-95A are formed with main-chain atoms and strictly conserved residues of the 20S proteasome. 

The structure of the 20S proteasome (Fig. 2.13) from Thermoplasma acidophilum displays four rings stacked upon each other surrounding a central cavity in which proteolysis takes place. An N-terminal threonine has been identified as an essential active site residue of the protease center. The OH-group of the threonine functions as a nucleophile during hydrolysis of the petide bond. A similar mechanism of hydrolysis has been shown for other hydrolases, which, because of this property, are now included in the family of N-terminal nucleophile hydrolases. For some /1-subunits of eucaryotes the N-terminal threonine is generated by autoproteolysis of an N-terminal prosequence. 

Orlowski, M. and Wilk, S. (2000) Catalytic activities of the 20 S proteasome, a multi-catalytic proteinase complex. Arch.Biochem.Biophys., 383, 1-16. 

Ehring B, Meyer TH, Eckerskorn C, Lottspeich F, Tampe R (1996) Effects of MHC-encodcd subunits on the peptidase and proteolytic activities of human 20S proteasomes - Cleavage of proteins and antigenic peptides. Eur J Biochem 235 404-415. 

---