Repeat sequence of Epstein-Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing

The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral Epstein-Barr virus-encoded nuclear antigen 1 protein. The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear. By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome. We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr. Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products. We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.     

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.     

Dependence of proteasome processing rate on substrate unfolding

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ～5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.     

Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing

Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.     

An unstructured initiation site is required for efficient proteasome-mediated degradation

The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.     

Structural properties of substrate proteins determine their proteolysis by the mitochondrial AAA+ protease Pim1

The protease Pim1/LON, a member of the AAA+ family of homo-oligomeric ATP-dependent proteases, is responsible for the degradation of soluble proteins in the mitochondrial matrix. To establish the molecular parameters required for the specific recognition and proteolysis of substrate proteins by Pim1, we analyzed the in organello degradation of imported reporter proteins containing different structural properties. The amino acid composition at the amino-terminal end had no major effect on the proteolysis reaction. However, proteins with an amino-terminal extension of less than 60 amino acids in front of a stably folded reporter domain were completely resistant to proteolysis by Pim1. Substrate proteins with a longer amino-terminal extension showed incomplete proteolysis, resulting in the generation of a defined degradation fragment. We conclude that Pim1-mediated protein degradation is processive and is initiated from an unstructured amino-terminal segment. Resistance to degradation and fragment formation was abolished if the folding state of the reporter domain was destabilized, indicating that Pim1 is not able to unravel folded proteins for proteolysis. We propose that the requirement for an exposed, large, non-native protein segment, in combination with a limited unfolding capability, accounts for the selectivity of the protease Pim1 for damaged or misfolded polypeptides.     

Recognition of the polyubiquitin proteolytic signal

Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. Through studies of structurally defined, polyubiquitylated model substrates, we show that tetraubiquitin is the minimum signal for efficient proteasomal targeting. The mechanism of targeting involves a simple increase in substrate affinity that is brought about by autonomous binding of the polyubiquitin chain. Assigning the proteasomal signaling function to a specific polymeric unit explains how a single ubiquitin can act as a functionally distinct signal, for example in endocytosis. The properties of the substrates studied here implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates.     

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444–1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243–13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.     

Degradation signals in ErbB-2 dictate proteasomal processing and immunogenicity and resist protection by cis glycine-alanine repeat.

ErbB-2 is ubiquitinated and degraded when dissociated from its membrane chaperone or bound by specific antibody. Reagents which induce such degradation have demonstrated antitumor activity and may impact ErbB-2 immunogenicity. To further understand ErbB-2 degradation and immunogenicity, a glycine-alanine repeat (GAr) or the reverse proline-alanine repeat (PAr) which protects certain proteins from proteasome degradation, was inserted after amino acid 5 (GAr5/PAr5) or 55 (GAr55/PAr55) of ErbB-2. When dissociated from the membrane with geldanamycin, E2-GAr5 and E2-PAr5 were not protected and still ubiquitinated and degraded by the proteasome, despite the presence of GAr. Insertional mutagenesis with GAr sequences at a.a. 55 of E2 enhanced proteasome degradation rendering E2-GAr55 and E2-PAr55 unstable on the membrane, but rescued in the cytosol by proteasome inhibitors. Immunization with E2-GAr induced antitumor immunity and CTL which lysed tumor cells expressing chimeric E2-GAr or wild-type E2 proteins, demonstrating efficient presentation through MHC I pathway. Improved understanding of the strong degradation signals in ErbB-2 may facilitate the development of anticancer agents or vaccines.     

Cleavage site selection within a folded substrate by the ATP-dependent lon protease

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.     

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.     

Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides

Accumulation of improperly folded polypeptides in the endoplasmic reticulum (ER) can trigger a stress response that leads to the export of aberrant proteins into the cytosol and their ultimate proteasomal degradation. Human cytomegalovirus encodes a type I glycoprotein, US11, that binds to nascent MHC class I heavy chain molecules and causes their dislocation from the ER to the cytosol where they are degraded by the proteasome. Examination of US11-mediated class I degradation has identified a host of cellular proteins involved in the dislocation reaction, including the cytosolic AAA ATPase p97, the membrane protein Derlin-1, and the E3 ubiquitin ligase Sel1L. However, the intermediate steps occurring between the initiation of dislocation and full extraction of the misfolded substrate into the cytosol are not known. We demonstrate that US11 itself undergoes ER export and proteasomal degradation and utilize this system to define multiple steps of US11 dislocation. Treatment of US11-expressing cells with proteasome inhibitor resulted in the accumulation of glycosylated and ubiquitinated species as well as a deglycosylated US11 intermediate. Subcellular fractionation of proteasome-inhibited US11 cells demonstrated that deglycosylated intermediates continued to be integrated within the ER membrane, suggesting that the proteasome functions in the latter steps of dislocation. The data supports a model in which US11 is modified with ubiquitin, whereas the transmembrane region is integrated in the ER membrane, and deglycosylation occurs before complete dislocation.     

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.     

What curves alpha-solenoids? Evidence for an alpha-helical toroid structure of Rpn1 and Rpn2 proteins of the 26 S proteasome

The alpha-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved alpha-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an alpha-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an "antechamber" of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.     

Protease accessibility laddering: a proteomic tool for probing protein structure

Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6xHis). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies.     

Plant ubiquitylation and proteolytic systems, the key elements in hormonal signaling pathways

The general function of the ubiquitylation systems is to conjugate ubiquitin to lysine residues within substrate proteins, thus targeting them for degradation by the proteasome. In Arabidopsis thaliana more than 1300 genes (approximately 5% of the proteome) encode components of the ubiquitin/26S proteasome pathway. Approximately 90% of these genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity to the ubiquitin/26S proteasome pathway. The plant E3 ubiquitin ligases comprise a large and diverse family of proteins or protein complexes containing either a HECT domain, a RING-finger or U-box domain. The SCF class of E3 ligases is the most thoroughly studied in plants because some of them participate in regulation of hormone signaling pathways. The role of the SCF is to ubiquitylate repressors of hormone response (auxin, gibberellins), whereas in response to ethylene, abscisic acid and brassinosteroids the SCF participate in degradation of positive regulators in the absence of the hormone.     

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.     

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.