View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Dispatch R551 Giant proteases: Beyond the proteasome Tingting Yao and Robert E. Cohen Proteasomes and related proteases are thought to be Proteasomes have the remarkable ability to be highly the principal machinery responsible for intracellular selective and, at the same time, degrade an enormously protein degradation. A new class of giant proteases has diverse set of substrates. What other proteases could possi- been discovered that can augment the catabolic bly substitute for the proteasome? One clue comes from functions of proteasomes and, under some conditions, the discovery of other proteases with the potential for may even substitute for proteasomes altogether. ‘self-compartmentalization’. The active sites in protea- somes are confined to an internal cavity, and this self-com- Address: Department of Biochemistry, University of Iowa, Bowen Science Building, 51 Newton Road, Iowa City, Iowa 52242-1109, USA. partmentalizing architecture provides a unique solution to E-mail: [email protected] the problem of substrate specificity. Once a substrate enters the internal chamber, its degradation is ensured by Current Biology 1999, 9:R551–R553 access to multiple endoproteolytic sites. http://biomednet.com/elecref/09609822009R0551 © Elsevier Science Ltd ISSN 0960-9822 This strategy of limiting access to a proteolytic chamber to provide selectivity and processivity has, in fact, been Our understanding of proteolytic mechanisms of cellular exploited by several bacterial proteases as well as the regulation has advanced dramatically in the last few proteasome [1]. In Escherichia coli, the ClpP and ClpQ/HslV years. One important outcome has been the realization proteases are each composed of two oligomeric rings that that intracellular proteolysis is accomplished primarily by enclose a central cavity for proteolysis. Subunits that are only one or a few proteases. These proteases are quite members of the AAA family of ATPases [4] associate with unusual: all are large, multisubunit complexes in which these proteases — ClpA or ClpX with ClpP, and ClpY/HslU the proteolytic sites are confined to an internal cavity with ClpQ/HslV — and appear to act as chaperones that can [1,2]. The proteasome, found in all eukaryotes and also supply unfolded substrates to the proteolytic core of the some archaebacteria, is perhaps the most familiar complex. It is likely that similar concerted unfolding and example. As the destination of proteins tagged with ubiq- degradation reactions also occur when the eukaryotic 20S uitin for subsequent degradation, the proteasome has proteasome associates with its ATPase-containing 19S regu- come to be regarded as the ultimate proteolytic machine. latory complex to form the full-size 26S particle. Unexpectedly, however, some new and even bigger con- tenders have now appeared on the scene. Although the Archaebacteria offer their own examples of self-compart- functions and mechanisms of these giant proteases mentalizing proteases. In fact, the proteasome in Thermo- remain obscure, a variety of observations suggest that plasma acidophilum is the prototype of the eukaryotic core they may cooperate with proteasomes, and possibly even 20S proteasome. It is, however, another archaebacterial replace them. protease that may offer some insight into the mystery of the NLVS-adapted mammalian cells. Several years ago, in One hint of the existence of such giant, non-proteasomal a search for regulatory components of the Thermoplasma proteases came from studies reported two years ago by proteasome, Baumeister’s group [5] encountered a big Glas et al. [3]. With the aim of determining the extent to surprise, the tricorn protease. They found that, when which the proteasome is essential in eukaryotic cells, they expressed in E. coli, the 120 kDa tricorn protease polypep- challenged EL-4 lymphoma cells with a proteasome tide self-assembled to form a hexameric toroid. Electron inhibitor — N-blocked tri-leucine vinyl sulfone (NLVS), microscopy and three-dimensional image reconstruction which covalently modifies the proteasome’s catalytic β showed that three tricorn protease dimers enclose a subunits. Surprisingly, in the presence of this inhibitor, a channel that traverses the hexamer. The existence of this small proportion of the cells survived and recovered to channel, which has 2.6 nm openings into a cavity 10 nm proliferate. The frequency of survival (0.3%) was well across and up to 4.3 nm high [6], suggests that tricorn pro- above what could be expected from mutations. tease, like proteasomes, may be self-compartmentalizing. Immunoprecipitation from the adapted cells and subsequent biochemical analysis demonstrated that the The tricorn protease and 20S proteasomes are both ATP- proteasomes were completely assembled, yet modified independent peptidases. The tricorn protease hexamer has and inactive. These results suggested that the adaptation trypsin-like and very high chymotrypsin-like activities, involved up-regulation of one or more proteases that can whereas the archaebacterial 20S proteasome has only chy- functionally replace the proteasome in cell-cycle control motrypsin-like activity. The surprise came when it was dis- and other critical processes. covered that tricorn protease can assemble further into an R552 Current Biology, Vol 9 No 15 Figure 1 degradation [8]. Firstly, the degradation rate was found to be limited by the amount of proteasome, but not that of tricorn protease or the tricorn protease activators. Secondly, Permission to reproduce this addition of tricorn protease generated a new set of smaller figure electronically has been peptides, whereas addition of the activating factors gener- denied. ated mostly free amino acids. And thirdly, when the pro- teasome, tricorn protease and tricorn protease activating factors were combined, large amounts of free amino acids with little intermediate-sized peptides were observed. (a) Views of the tricorn protease of Thermoplasma acidophilum, Yet why should tricorn protease be assembled into a super- obtained by electron microscopy and three-dimensional reconstruction of the icosahedral capsid. The three images show views down the molecule? Tamura et al. [8] suggested that, in order to effi- three-fold (left), five-fold (middle) and two-fold (right) symmetry axes. ciently channel reaction intermediates, the capsid structure The scale bar represents 50 nm. (Adapted from [6].) (b) The TPPII acts as a scaffold that accommodates the aminopeptidase protease from murine EL-4 cells. This image is an average from 1365 activators. But so far there is no biochemical evidence for negatively-stained particles visualized by electron microscopy. The rod-like structure is 50 nm long. (Adapted from [9].) channeling, or for direct interactions between either tricorn protease and the factors or the proteasome and tricorn pro- tease. It also is not known whether the tricorn protease unprecedented 55 nm icosahedral capsid composed of 20 supermolecules have any advantage over tricorn protease hexamers (Figure 1a) [6]. This 14.6 MDa homooligomer hexamers in speeding up this catabolic pathway. An alter- appears to enclose a cavity approximately 37 nm in diame- native possibility is that the supermolecule has additional ter, large enough to accommodate a ribosome. Because this proteolytic activities which arise only in response to a cell superstructure was only observed in Thermoplasma cell stress, such as loss of proteasomal function. extracts, but not with recombinant protein, it is likely that accessory factors are required for its assembly in vivo. Returning to eukaryotes, a giant protease assembled from aminopeptidase monomers was discovered that seems to Despite its peptidase activity and the beauty of its highly- be a likely substitute for the proteasome in NLVS- ordered structure, tricorn protease by itself gives us little adapted EL-4 cells. In the original study by Glas et al. [3], indication as to its physiological role. The identification of the adapted cells displayed a remarkable increase in a aminopeptidases that act synergistically with tricorn chymotrypsin-like activity detected with the fluorogenic protease has provided evidence that tricorn protease may tripeptide substrate AAF-AMC. This activity eluted serve as one component of a complete proteolytic earlier than the proteasome upon gel filtration. Impor- pathway. Three such factors — F1, F2 and F3 — from tantly, AAF-chloromethylketone, an inhibitor of tricorn T. acidophilum have been described, each of which can protease, blocked proliferation of NLVS-adapted but not release amino acids from the unblocked amino termini of normal cells. Niedermann’s group [9] later discovered that short peptides [7,8]. Experiments by Tamura et al. [8] a large form of a protease known as tripeptidyl peptidase offered insight into how these aminopeptidases and II (TPPII) could account for the AAF-AMC hydrolyzing tricorn protease may work together. When tricorn pro- activity in the proteasome-inhibited cells. tease, aminopeptidase F2 and fluorogenic substrates were mixed in different orders, it was found that release of the TPPII is a serine peptidase that removes amino-terminal fluorophore was enhanced by F2 when the substrate was tripeptides from unblocked oligopeptides. Its substrates preincubated with tricorn protease. Thus, degradation by and inhibitors are similar to those of tricorn protease, but tricorn protease generates