Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.     

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.     

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.     

Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.     

Degradation of Some Polyubiquitinated Proteins Requires an Intrinsic Proteasomal Binding Element in the Substrates

Lysine 48-linked polyubiquitin chains usually target proteins for 26 S proteasomal degradation; however, this modification is not a warrant for destruction. Here, we found that efficient degradation of a physiological substrate UbcH10 requires not only an exogenous polyubiquitin chain modification but also its unstructured N-terminal region. Interestingly, the unstructured N-terminal region of UbcH10 directly binds the 19 S regulatory complex of the 26 S proteasome, and it mediates the initiation of substrate translocation. To promote ubiquitin- dependent degradation of the folded domains of UbcH10, its N-terminal region can be displaced by exogenous proteasomal binding elements. Moreover, the unstructured N-terminal region can initiate substrate translocation even when UbcH10 is artificially cyclized without a free terminus. Polyubiquitinated circular UbcH10 is completely degraded by the 26 S proteasome. Accordingly, we propose that degradation of some polyubiquitinated proteins requires two binding interactions: a polyubiquitin chain and an intrinsic proteasomal binding element in the substrates (likely an unstructured region); moreover, the intrinsic proteasomal binding element initiates substrate translocation regardless of its location in the substrates.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

Autoregulation of the 26S proteasome by in situ ubiquitination

The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome''s ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.     

The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits

The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome.     

Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: Signal transduction and synaptic transmission

The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.     

Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation

Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.     

Interactions of PAN's C‐termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions

Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C‐termini a conserved hydrophobic‐tyrosine‐X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C‐terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C‐terminal HbYX motif and the 20S α‐subunits and indicates that the intersubunit pocket in the 20S undergoes an induced‐fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.     

Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised

Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.     

Evidence for an interaction between ubiquitin-conjugating enzymes and the 26S proteasome

The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.     

Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1–60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H₂O₂ resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H₂O₂ of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.