Inside eukaryotic cells there is a massive protein complex called the proteasome whose
raison d'être is to remove unnecessary proteins by breaking them down into short peptides.
The proteasome is thus responsible for an important aspect of cellular regulation because
the timely and controlled proteolysis of key cellular factors regulates numerous biological
processes such as cell cycle, differentiation, stress response, neuronal morphogenesis,
cell surface receptor modulation, secretion, DNA repair, transcriptional regulation,
long-term memory, circadian rhythms, immune response, and biogenesis of organelles
(Glickman and Ciechanover 2002). With the multitude of substrates targeted and the myriad
processes involved, it is not surprising that aberrations in the pathway are implicated in
the pathogenesis of many diseases, including cancer.
With so many proteins to target for degradation, the activity of the proteasome is
subject to multiple levels of regulation. In the overwhelming majority of cases, selected
proteins are first “labeled” by the addition of several copies of a small protein tag
called ubiquitin and are thus targeted for degradation in the proteasome (Figure 1). The
ubiquitination of proteins is regulated through precise selection of protein substrates by
specific E3 ubiquitin ligases (Pickart 2001). These enzyme complexes each recognize a
subset of substrates and tag them by linking the carboxyl terminus of ubiquitin with an
amino group on the target protein via an amide bond (Figure 1).
Interestingly, ubiquitination is a reversible process. Even when a protein has been
tagged with ubiquitin, its fate is not sealed—specific hydrolytic enzymes called
deubiquitinases can remove the ubiquitin label intact (Figure 1). By deubiquitinating their
substrates, these enzymes compete with the proteasome, which acts on the polyubiquitined
form. In the competition between proteolysis and deubiquitination, polyubiquitinated
proteins rarely accumulate in the cytoplasm of “healthy” cells, as they are either
irreversibly degraded or deubiquitinated and rescued. It is thought that this competition
provides a certain level of stringency or quality control to the system. Based on sequence
homology, deubiquitinating enzymes were traditionally classified into two families:
ubiquitin-specific proteases (UBPs or USPs) and ubiquitin carboxy-terminal hydrolases
(UCHs). Both enzyme families are classified as cysteine proteases that employ an active
site thiol to cleave ubiquitin from its target (Kim et al. 2003; Wing 2003).
The proteasome itself is made up of a multiprotein core particle (CP) where proteolysis
occurs and a separate multiprotein regulatory particle (RP) that recognizes and prepares
substrates for degradation by the CP. A base subcomplex of the RP is pivotal in anchoring
polyubiquitin chains during this process, either directly or via auxiliary
ubiquitin-binding proteins (Lam et al. 2002; Hartmann-Petersen et al. 2003). The base
attaches to the outer surface of the CP and uses energy to unravel the substrate,
simultaneously with preparing the channel that leads into the proteolytic chamber of the CP
(Forster and Hill 2003). The lid subcomplex of the RP attaches to the base and is required
for proteolysis of ubiquitin–protein conjugates, but not of unstructured polypeptides
(Glickman et al. 1998; Guterman and Glickman 2003). The size and complexity of this
protein-eating machine hints at the exquisite controls that must rgulate its function.
An intriguing evolutionary and structural relationship between the proteasome lid and an
independent complex, the COP9 signalosome (CSN), may shed light on their respective roles
in regulated protein degradation. Both are made up of eight homologous protein subunits
that contain similar structural and functional motifs. While a lot is still unknown, the
CSN appears to mediate responses to signals (e.g., light, hormones, adhesion, nutrients,
DNA damage) in a manner that is intimately linked to the ubiquitin–proteasome system. This
is accomplished, for instance, by suppressing ubiquitin E3 ligase activity or interacting
with various components of the pathway (Bech-Otschir et al. 2002; Cope and Deshaies 2003;
Li and Deng 2003). In particular, one subunit—Csn5—moderates SCF (Skp1–cullin–F box) and
other cullin-based E3 ubiquitin ligases by removal of the ubiquitin-like Rub1/Nedd8
molecule from the cullin subunit of the ligase complex. Further analysis of the CSN will no
doubt uncover additional mechanisms whereby ubiquitin-mediated protein degradation is
controlled.
Surprisingly, the proteasome itself harbors intrinsic deubiquitination activity (Eytan
et al. 1993). Moreover, both the lid and the base contribute independently to RP
deubiquitination activity. The source of this activity has been attributed to a number of
different subunits. These include the associated cysteine proteases Ubp6/USP14 (Borodovsky
et al. 2001; Legget et al. 2002), UCH37/p37 (Lam et al. 1997; Hoelzl et al. 2000), and
Doa4/Ubp4 (Papa et al. 1999), as well as the intrinsic proteasome subunit Rpn11/POH1 (Verma
et al. 2002; Yao and Cohen 2002). The importance of these components to proteasome function
is apparent in their partially overlapping properties. In groundbreaking work, an intrinsic
“cryptic” deubiquitinating activity that is sensitive to metal chelators has been reported
for the proteasome, in addition to “classic” cysteine protease behavior (Verma et al. 2002;
Yao and Cohen 2002). This metalloprotease-like activity maps to the putative catalytic
MPN+/JAMM motif of the lid subunit Rpn11 and lies at the heart of proteasome mechanism by
linking deubiquitination with protein degradation. Notably, Rpn11 shares close homology
with Csn5, which is also responsible for proteolytic activities in its respective
complex.
By defining a new family of putative metalloproteases that includes a proteasomal
subunit, a CSN subunit, and additional proteins from all domains of life, the MPN
+ /JAMM motif garnered great attention. The trademark of the MPN
+ /JAMM motif is a consensus sequence E—HxHx
(7) Sx
(2) D that bears some resemblance to the active site of zinc
metalloproteases. Members of this family were predicted to be hydrolytic enzymes, some of
which are specific for removal of ubiquitin or ubiquitin-like domains from their targets
(Maytal-Kivity et al. 2002; Verma et al. 2002; Yao and Cohen 2002).
In a further development, two independent groups determined the molecular structure of
an MPN
+ /JAMM protein from an archaebacterium (Ambroggio et al. 2003; Tran et
al. 2003). The structures identify a zinc ion chelated to the two histidines and the
aspartic residue of the MPN
+ /JAMM sequence. The fourth ligand appears to be a water molecule
activated through interactions with the conserved glutamate to serve as the active site
nucleophile. Overall, this protein certainly has properties consistent with a
metallohydrolase and can serve as the prototype for the deubiquitinating enzymes in its
class. This revelation adds an all-new enzymatic activity and, with it, an additional layer
of regulation to the ubiquitin–proteasome system.
Now that it is evident that the proteasome contains a member of a novel metalloprotease
family, a fundamental question can be raised: why does a proteolytic enzyme like the
proteasome need auxiliary proteases for hydrolysis of ubiquitin domains? At first glance,
the delegation of tasks between the proteolytic subunits of the proteasome (situated in the
proteolytic core particle) and the auxiliary deubiquitinating enzymes (situated in the
regulatory particle) is clear-cut: the latter cleave between ubiquitin domains, while the
core proteolytic subunits process the target protein itself (Figure 1). However, this still
does not explain the mechanistic rational for finding deubiquitination within the
proteasome itself. In principle, deubiquitination could be used for (1) recycling of
ubiquitin, (2) abetting degradation by removal of the tightly folded highly stable globular
ubiquitin domain, or (3) mitigating degradation by removal of the ubiquitin anchor, without
which the substrate is easily released and rescued. There is evidence that recycling of
ubiquitin by the proteasome is indeed a crucial feature of deubiquitination in proper
cellular maintenance (Legget et al. 2002). Distinguishing between options 2 and 3, however,
depends to a large extent on the delicate balance between the two proteolytic activities
associated with the proteasome: proteolysis and deubiquitination (Figure 2).
Once bound to the proteasome, a polyubiquitinated substrate can be unfolded by the RP
and irreversibly translocated into the CP. It has been proposed that long polyubiquitin
chains commit a substrate to unfolding and degradation by the proteasome, whereas short
chains are poor substrates because they are edited by deubiquitinating enzymes, resulting
in premature substrate release (Eytan et al. 1993; Lam et al. 1997; Thrower et al. 2000;
Guterman and Glickman 2003). Extended polyubiquitin chains could slow down chain
disassembly, thereby allowing ample time for unfolding and proteolysis of the substrate
(Figure 2). Interestingly, both “trimming” and “shaving” deubiquitinating activities are
associated with the proteasome, though the exact contribution of the various
proteasome-associated deubiquitinating enzymes to each of these distinct activities has yet
to be elucidated. It is expected that in order to obtain efficient proteolysis of the
target, shaving of chains at their proximal ubiquitin should be slower than the rate of
trimming at the distal moiety. As an outcome of this requirement, longer polyubiquitin tags
would be preferential substrates for degradation by the proteasome. Thus, the uniqueness of
ubiquitin as a label for degradation may lie in its being a reversible tag.
Deubiquitinases, such as Rpn11, serve as proofreading devices for reversal of fortune at
various stages of the process, right up to the final step before irreversible degradation
by the proteasome.
Identifying Rpn11 and Csn5 as members of a novel class of metallohydrolases immediately
elevates them into promising “drugable” candidates. Undoubtedly, the molecular structures
deciphered by the groups of Deshaies (Ambroggio et al. 2003) and Bycroft (Tran et al. 2003)
will focus efforts to design novel site-specific inhibitors of the ubiquitin–proteasome
pathway. While Csn5 is thought to impede the action of ubiquitin ligases through shaving
cullins from their Rub1/Nedd8 modification (and possibly also by deubiquitinating
substrates bound to the cullins), the outcome of Rpn11 inhibition will depend largely on
whether Rpn11 participates primarily in shaving substrates from their chains, promoting
release and rescue, or in trimming the polyubiquitin tag, allowing for proteolysis quality
control (Figure 2).
