Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.     

Ubiquitin-independent proteolytic functions of the proteasome

The discovery of the 20S proteasome (multicatalytic proteinase complex) was followed by the recognition that this multisubunit macromolecule is the proteolytic core of the 26S proteasome. Most of the research on extralysosomal proteolysis has concentrated on the role of the 26S proteasome in the ubiquitin-dependent proteolytic pathway. However, little attention has been directed toward the possible involvement of the proteasome in ubiquitin-independent proteolysis. In the past few years, many publications have provided evidence that both the 20S proteasome and the 26S proteasome can degrade some proteins in an ubiquitin-independent manner. Furthermore, it is becoming clear that demonstration of ubiquitin-protein conjugates after exposure of cells to proteasome inhibitors does not eliminate the possibility that the same protein can also be degraded by the proteasome without ubiquitination. The possible mechanisms of degradation of an unmodified protein by the 20S proteasome are discussed. These include targeting, protein unfolding, and opening of the gated channel to the catalytic sites. It is reasonable to assume that in the future the number of proteins recognized as substates of the ubiquitin-independent pathway will continue to increase, and that the metabolic significance of this pathway will be clarified.     

Proteasomal AAA-ATPases: structure and function

The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1-6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S 'gate', and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome.     

APC/C and SCF: controlling each other and the cell cycle

Regulated protein degradation has emerged as a key recurring theme in multiple aspects of cell-cycle regulation. Importantly, the irreversible nature of proteolysis makes it an invaluable complement to the intrinsically reversible regulation through phosphorylation and other post-translational modifications. Consequently, ubiquitin-protein ligases, the protagonists of regulated protein destruction, have gained prominence that compares to that of the cyclin-dependent kinases (Cdks) in driving the eukaryotic cell-cycle clock. This review will focus on the two main players, the related ubiquitin-protein ligases APC/C and SCF, and how they control cell-cycle progression. I will also try to delineate the regulation and interplay of these destruction mechanisms, which are intricately connected to the kinase network as well as to extrinsic signals. Moreover, cell-cycle ubiquitin-protein ligases are themselves subject to proteolytic control in cis as well as in trans. Finally, a careful comparison of the functions and regulation of APC/C and SCF shows that, in certain aspects, their logic of action is fundamentally different.     

Impact of the N-terminal amino acid on targeted protein degradation

The N-terminus of any protein may be used as a destabilization signal for targeted protein degradation. In the eukaryotic cytosol, the signal - the so-called N-degron--is recognized for degradation by (i) the N-end rule, a well-described degradation process involving epsilon-ubiquitination; or (ii) N-terminal ubiquitination, a more recently described pathway. Dedicated E3 ubiquitin ligases known as N-recognins then act on the protein. The proteolytic pathways involve ATP-dependent chambered proteases, such as the 26S proteasome in the cytosol, which generate short oligopeptides. The N-terminus of the polypeptide chain is also important for post-proteasome degradation by specific aminopeptidases, which complete peptide cleavage to generate free amino acids. Finally, in each compartment of the eukaryotic cell, N-terminal methionine excision creates a variety of N-termini for mature proteins. It has recently been shown that the N-terminal methionine excision pathway has a major impact early in targeted protein degradation.