20S Proteasomes

Several aryl esters of 6-chloromethyl-2-oxo-2//-l -benzopyran-3-carboxylic acid act as human Lon protease inhibitors (alternate substrate inhibitors)46 without having any effect on the 20S proteasome. Proteasomes are the major agents of protein turnover and the breakdown of oxidized proteins in the cytosol and nucleus of eukaryotic cells,47 whereas Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. The coumarin derivatives are potentially useful tools for investigating the various biological roles of Lon protease without interfering with the proteasome inhibition. 

Three other components that my laboratory has identified and partially purified from Fraction 2 of reticulocytes, termed CF1-CF3, are involved in the degradation of proteins ligated to ubiquitin [24]. These are apparently subcomplexes of the 26S proteasome, a large ATP-dependent protease complex first described by Re-chsteiner and co-workers [25], CF3 is identical to the 20S proteasome core particle [26], while CFl and CF2 may be similar to the base and lid subcomplexes of the 19S regulatory particle of the 26S proteasome, described more recently by the Finley laboratory [27], In hindsight, the reason for finding subcomplexes, rather than the complete 26S complex in Fraction 2 was technical we have routinely prepared Fraction 2 from ATP-depleted reticulocytes [20], under which conditions the 26S proteasome dissociates to its subcomplexes. We found that incubation of the three subcomplexes in the presence of ATP promotes their assembly to the 26S proteasome [24, 26]. The role of ATP in the assembly of the 26S proteasome complex remains unknown. 

Fig. 9.2. I nteraction of the 20S proteasome with other cellular components.

AAA nucleotidases share the common property of altering the conformation or association state of proteins, so it is not surprising that the RC has been shown to prevent aggregation of several denatured proteins including citrate synthase and ribonuclease A [59-61]. The chaperone activity of the RC may explain why the RC plays a role in transcription apparently in the absence of an attached 20S proteasome [62]. 

The 20S proteasome is a latent protease owing to the barrier imposed by the a-subunit rings on peptide entry. Consequently, a readily measured activity of the RC is activation of fluorogenic peptide hydrolysis by the 20S proteasome. The extent of activation is generally found to be in the range 3- to 20-fold [63]. Activation is relatively uniform for all three proteasome catalytic subunits and presumably reflects opening by the attached RC of a channel leading to the proteasome s central chamber. 

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]. 

Assembly pathways for the RC are virtually unknown. As mentioned above, the ATP-ases interact with one another and complexes containing all six S4 subfamily members have been observed following in vitro synthesis. Impaired synthesis of the yeast lid subunit Rpn6 results in the absence of the entire lid [134], so presumably lid and base subcomplexes assemble independently and associate in the final stages of RC formation cells. In mammalian cells, 26S proteasomes assemble from preformed regulatory complexes and 20S proteasomes [135]. 

S6, and SlOb) and two 20S a-subunits (C8 and C9) are known to be phosphoryl-ated. Phosphorylation appears to be particularly important for 26S proteasome assembly and stability. The kinase inhibitor staurosporine reduces 26S proteasome levels in mouse lymphoma cells [135] and interferon y results in reduced phosphorylation of 20S proteasome a-subunits and decreased 26S proteasome levels [141]. 

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. 

As the a-rings at each end of the 20S proteasome are equivalent, the 20S proteasome is capable of binding two RCs, two PA28s, two PA200s, or combinations of these components. In fact 20S proteasomes simultaneously bound to RC and PA28 or PA200 have been observed, and are called hybrid proteasomes [147, 148, 150]. 

Touitou, R. et al. A degradation signal located in the C-terminus of p21WAFl/CIPl is a binding site for the C8 alpha-subunit of the 20S proteasome. Embo J 2001, 20, 2367-75. 

Arendt, C. S. and Hoghstrasser, M. Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly. Embo J 1999, 18, 3575-85. 

Ginsburg, D. B., and Monago, f. f. Intermediates in the formation of mouse 20S proteasomes implications for the assembly of precursor beta subunits. Embo J 1997, 16, 5363-75. 

Bose, S., Steatfoed, F. L, Beoadfoot, K. 1., Mason, G. G., and Rivett, a. J. Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. Biochem J Pt 2004, 378, 177-184. 

On the sequence level, HslV shows sequence similarity with the yS-subunits of arch-aebacterial and eukaryotic proteasomes. The crystal structure of E. coli HslV confirmed that individual subunits share the Ntn-hydrolase fold with Thrl at the N-terminus as the nucleophile, just as in proteasomes. Despite these similarities, there are substantial differences between bacterial HslVU and archaebacterial and eukaryotic 20S proteasomes. In contrast to HslVU, 20S proteasomes are assembled from four rings of seven subunits each, that build up a central proteolytic chamber and two flanking antechambers. 

Currently, high-resolution EM image reconstructions for the 26S proteasome (Walz et al. 1998), but no atomic-resolution crystallographic data are available for any complex of 20S proteasomes with ATP-dependent activators. The expected assembly of PAN, the archaebacterial AAA(+) activator of proteasomes (Zwickl et al. 1999) into hexamers suggests a symmetry-mismatched complex in archaebacteria. 

As discussed above, the eubacterial HslVU is distantly related in structure to the proteasome found in archaea and eukaryotes. Surprisingly, however, the structural relationship is not reflected in the regulatory properties as will be described in Section 10.3, which focuses on structural studies of the yeast 20S proteasome and its activation, activity, and inhibition. 

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