Substrate selection by the proteasome through initiation regions

Proteins in the cell have to be eliminated once their function is no longer desired or they become damaged. Most regulated protein degradation is achieved by a large enzymatic complex called the proteasome. Many proteasome substrates are targeted for degradation by the covalent attachment of ubiquitin molecules. Ubiquitinated proteins can be bound by the proteasome, but for proteolysis to occur the proteasome needs to find a disordered tail somewhere in the target at which it initiates degradation. The initiation step contributes to the specificity of proteasomal degradation. Here, we review how the proteasome selects initiation sites within its substrates and discuss how the initiation step affects physiological processes.     

Proteomics of proteasome complexes and ubiquitinated proteins

Ubiquitin-proteasome-mediated protein degradation is central to the regulation of many important biological processes, including cell cycle progression, apoptosis and DNA repair. Recognition and degradation of ubiquitinated substrates by the 26S proteasome is tightly regulated to maintain normal cell growth. Disruption of the proteasomal degradation pathway has been implicated in a wide range of human diseases. Although the ubiquitin-proteasome system has been intensively investigated, many key questions remain unanswered in regard to its components and regulation of its activities. A key step towards a full understanding of the pathway is to investigate the proteasome complex subunit composition, heterogeneity, post-translational modifications, assembly, proteasome interaction networks and degradation substrates. Mass spectrometry-based proteomic approaches have been successfully applied for unraveling the details of the proteasome complexes and their substrates in an unprecedented fashion. An overview of the current knowledge of the proteasomal degradation pathway based on mass spectrometry approaches is presented.     

Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.     

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.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Structure of the human 26S proteasome: subunit radial displacements open the gate into the proteolytic core

The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.     

Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel

The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.     

Ubiquitin-specific protease 25 functions in Endoplasmic Reticulum-associated degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome.     

Ubiquitin receptor proteins hHR23a and hPLIC2 interact

Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated. In previous work, we determined UBL/UBA domain interactions to promote intramolecular interactions in hHR23a that are abrogated with the addition of either ubiquitin or the proteasome component S5a. In yeast, we determined the hHR23a ortholog (Rad23) to interact with another UBL/UBA family member (Ddi1) and to bind a common tetraubiquitin chain. Here, we use NMR spectroscopy to reveal that hHR23a interacts with hPLIC2 via UBL/UBA domain interactions and to map their binding surfaces. In addition, we demonstrate that these two proteins associate in mammalian cells. Intriguingly, inhibition of the proteasome mitigates hHR23a/hPLIC2 interaction.     

20S proteasome prevents aggregation of heat-denatured proteins without PA700 regulatory subcomplex like a molecular chaperone

The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation.     

The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

The 26S proteasome Rpn10 gene encoding splicing isoforms: evolutional conservation of the genomic organization in vertebrates

Recognition of polyubiquitinated substrates by the 26S proteasome is a key step in the selective degradation of various cellular proteins. The Rpn10 subunit of the 26S proteasome can bind polyubiquitin conjugates in vitro. We have previously reported the unique diversity of Rpn10, which differs from other multiple proteasome subunits, and that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated alternative splicing. To determine whether such alternative splicing mechanisms occur in other species, we searched for Rpn10 isoforms in databases and in our original PCR products. Here we report the genomic organization of the Rpn10 gene in lower vertebrates and provide evidence for the competent generation of distinct forms of Rpn10 by alternative splicing through evolution.     

The N-end rule pathway: from recognition by N-recognins, to destruction by AAA+proteases

Intracellular proteolysis is a tightly regulated process responsible for the targeted removal of unwanted or damaged proteins. The non-lysosomal removal of these proteins is performed by processive enzymes, which belong to the AAA+superfamily, such as the 26S proteasome and Clp proteases. One important protein degradation pathway, that is common to both prokaryotes and eukaryotes, is the N-end rule. In this pathway, proteins bearing a destabilizing amino acid residue at their N-terminus are degraded either by the ClpAP protease in bacteria, such as Escherichia coli or by the ubiquitin proteasome system in the eukaryotic cytoplasm. A suite of enzymes and other molecular components are also required for the successful generation, recognition and delivery of N-end rule substrates to their cognate proteases. In this review we examine the similarities and differences in the N-end rule pathway of bacterial and eukaryotic systems, focusing on the molecular determinants of this pathway.     

Identification of substrates of the Mycobacterium tuberculosis proteasome

The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.     

Characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control

Approximately 30% of polypeptides synthesized by mammalian cells are degraded with a half-life of <10 min by proteasomes. These rapidly degraded polypeptides (RDPs) constitute the bulk of proteasome substrates and are the principal source of viral and self-peptide ligands for major histocompatibility complex class I molecules. Here we provide evidence that approximately 75% of RDPs are degraded by the standard ubiquitin 26 S proteasome system and that their degradation is regulated by modulating Hsc70 activity in cells. Surprisingly, the remaining approximately 25% of RDPs are degraded without ubiquitylation by 20 S proteasomes independently of 19 S regulators and in a manner that is largely unaffected by modulating Hsc70 activity. This latter pathway is utilized for generating an antigenic peptide from viral-defective ribosomal products. The dichotomy in the behavior of RDPs points to a novel quality control level for nascent proteins that is independent of the well established Hsc70-ubiquitin 26 S proteasome pathway.     

Functional asymmetries of proteasome translocase pore

Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber. The aromatic (Ar) residue of the Ar-Φ loop in all six Rpts of S. cerevisiae is tyrosine; this amino acid is thought to have important functional contacts with substrate. Six yeast strains were constructed and characterized in which Tyr was individually mutated to Ala. The mutant cells were viable and had distinct phenotypes. rpt3, rpt4, and rpt5 Tyr/Ala mutants, which cluster on one side of the ATPase hexamer, were substantially impaired in their capacity to degrade substrates. In contrast, rpt1, rpt2, and rpt6 mutants equaled or exceeded wild type in degradation activity. However, rpt1 and rpt6 mutants had defects that limited cell growth or viability under conditions that stressed the ubiquitin proteasome system. In contrast, the rpt3 mutant grew faster than wild type and to a smaller size, a defect that has previously been associated with misregulation of G1 cyclins. This rpt3 phenotype probably results from altered degradation of cell cycle regulatory proteins. Finally, mutation of five of the Rpt subunits increased proteasome ATPase activity, implying bidirectional coupling between the Ar-Φ loop and the ATP hydrolysis site. The present observations assign specific functions to individual Rpt proteins and provide insights into the diverse roles of the axial loops of individual proteasome ATPases.     

20S proteasomes have the potential to keep substrates in store for continual degradation

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.