Native structure of rat liver immune proteasomes

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of “rotated” ubiquitinated proteins.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

Transferring substrates to the 26S proteasome

Ubiquitin-dependent protein degradation is not only involved in the recycling of amino acids from damaged or misfolded proteins but also represents an essential and deftly controlled mechanism for modulating the levels of key regulatory proteins. Chains of ubiquitin conjugated to a substrate protein specifically target it for degradation by the 26S proteasome, a huge multi-subunit protein complex found in all eukaryotic cells. Recent reports have clarified some of the molecular mechanisms involved in the transfer of ubiquitinated substrates from the ubiquitination machinery to the proteasome. This novel substrate transportation step in the ubiquitin-proteasome pathway seems to occur either directly or indirectly via certain substrate-recruiting proteins and appears to involve chaperones.     

The 20S/26S proteasomal pathway of protein degradation in muscle tissue

Similar to all other eukaryotic cells and tissues muscle tissue contains the proteolytic system of 20S/26S proteasomes with the 20S proteasome existing predominantly in a latent state. Unlike with the mammalian enzymein vitro transition from the latent to the activated state of the 20S proteasomes isolated from muscle of several fish species and from lobster can be achieved by heat shock. It is very likely that the activated state of the 20S proteasome corresponds to the physiologically active form of the enzyme since only that one is able to attack sarcoplasmic and myofibrillar proteins to any significant extent. As perfusion of rat hindquarters with presumptive low molecular mass activators like free fatty acids does not result in an activation of the muscle proteasome other — possibly protein activators — may serve this purposein vivo. The 26S proteasome complex may be regarded as such a proteasome/activator complex. The 26S proteasome complex has the ability to degrade protein (-ubiquitin-conjugates) by an ATP-consuming reaction. Since increased amounts of ubiquitinated proteins as well as an enhanced activity of the ATP (-ubiquitin)-dependent proteolytic system have been measured in rat muscle tissue during various catabolic conditions, it is not unlikely that this pathway is responsible for catalysis of muscle protein breakdown.Abbreviations Bz benzoyl - PGPH peptidylglutamylpeptide hydrolysing - Suc succinyl - Z benzyloxycarbonyl     

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.     

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.     

p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation

The ubiquitin–proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1–Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin–proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.     

Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats.

Flywheel-based resistance exercise (RE) attenuates muscle atrophy during hindlimb suspension. We have previously shown that protein synthesis is elevated in response to RE, but the effect on protein degradation, cell proliferation, or apoptosis was not investigated. We hypothesized that, in addition to affecting protein synthesis, RE inhibits processes that actively contribute to muscle atrophy during hindlimb suspension. Male rats were housed in regular cages (control), tail suspended for 2 wk (HS), or HS with RE every other day for 2 wk (HSRE). Although RE attenuated soleus muscle atrophy during HS, the observed fivefold elevation in apoptosis and the 53% decrease in cell proliferation observed with HS were unaffected by RE. Expression of genes encoding components of the ubiquitin-proteasome pathway of protein degradation were elevated with HS, including ubiquitin, MAFbx, Murf-1, Nedd4, and XIAP, and proteasome subunits C2 and C9. Total ubiquitinated protein was increased with HS, but proteasome activity was not different from control. RE selectively altered the expression of different components of this pathway: MAFbx, Murf-1, and ubiquitin mRNA abundance were downregulated, whereas C2 and C9 subunits remained elevated. Similarly, Nedd4 and XIAP continued to be upregulated, potentially accounting for the observed augmentation in total ubiquitinated protein with RE. Thus a different constellation of proteins is likely ubiquitinated with RE due to altered ubiquitin ligase composition. In summary, the flywheel-based resistance exercise paradigm used in this study is associated with the inhibition of some mechanisms associated with muscle atrophy, such as the increase in MAFbx and Murf-1, but not with others, such as proteasome subunit remodeling, apoptosis, and decreased proliferation, potentially accounting for the inability to completely restore muscle mass. Identifying specific exercise parameters that affect these latter processes may be useful in designing effective exercise strategies in the elderly or during spaceflight.     

Regulation of the 26S proteasome activities by peptides mimicking cleavage products

The implication of the released peptides in allosteric effects during protein degradation catalyzed by the proteasome is an important question not completely resolved. We present here data showing modulation of 26S proteasome activities by peptides composed of 5 or 6 natural amino acids that mimic the products generated during protein breakdown. Several of these peptides inhibit the chymotrypsin-like activity of the Xenope 26S proteasome whereas its trypsin-like activity is enhanced. The basic peptides produced competitive inhibition of the chymotrypsin-like activity and the acidic peptides, parabolic inhibition involving two different binding sites. Our results are in agreement with a model involving hypothetical non-catalytic sites interacting with effectors to modulate the peptidase activities of the proteasome. They also suggest that allosteric effects may occur in the proteasome during protein degradation.     

Mitochondrial quality control by the ubiquitin-proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.     

Getting in and out of the proteasome

By far the best understood role of the proteasome is to remove ubiquitin-conjugated proteins from eukaryotric cells by hydrolysing them into small peptides of varying lengths. These include both misfolded/abnormal proteins, as well as 'normal' proteins that need to be rapidly removed for regulatory purposes. However, the proteasome is also present in numerous prokaryotic organisms, while ubiquitin, and the ubiquitin conjugating system, are not. The eukaryotic proteasome has been adapted to degrading proteins in a ubiquitin-dependent fashion by the addition of regulatory factors that assemble in different layers onto the proteolytic core of the proteasome, and by increasing the diversity of the core subunits as well. In addition to hydrolysing ubiquitinated proteins into amino acids, the proteasome can also proteolyse selected non-ubiquitinated proteins, process proteins, and possibly refold misfolded proteins. This review will focus on the different proteasome functions, and how these are used in the multiple regulatory roles the proteasome plays in eukaryotic cells.     

Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism.

Sepsis is associated with a pronounced catabolic response in skeletal muscle, mainly reflecting degradation of the myofibrillar proteins actin and myosin. Recent studies suggest that sepsis-induced muscle proteolysis may reflect ubiquitin-proteasome-dependent protein breakdown. An apparently conflicting observation is that the ubiquitin-proteasome pathway does not degrade intact myofibrils. Thus, it is possible that actin and myosin need to be released from the myofibrils before they can be ubiquitinated and degraded by the proteasome. We tested the hypothesis that sepsis results in disruption of Z-bands, increased expression of calpains, and calcium-dependent release of myofilaments in skeletal muscle. Sepsis induced in rats by cecal ligation and puncture resulted in increased gene expression of micro-calpain, m-calpain, and p94 and in Z-band disintegration in the extensor digitorum longus muscle. The release of myofilaments from myofibrillar proteins was increased in septic muscle. This response to sepsis was blocked by treating the rats with dantrolene, a substance that inhibits the release of calcium from intracellular stores to the cytoplasm. The present results provide evidence that sepsis is associated with Z-band disintegration and a calcium-dependent release of myofilaments in skeletal muscle. Release of myofilaments may be an initial and perhaps rate-limiting component of sepsis-induced muscle breakdown.     

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

After oxidative stress, proteins that are oxidatively modified are degraded by the 20S proteasome. However, several studies have documented an enhanced ubiquitination of yet unknown proteins. Because ubiquitination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner this raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However, we were able to confirm an increase in ubiquitinated proteins 16 h after oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide-treated cells, as well as from control cells and cells treated with lactacystin, an irreversible proteasome inhibitor, and identified some of these proteins by MALDI tandem mass spectrometry. As a result we obtained 24 different proteins that can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified, ubiquitinated proteins confirms the thesis that ubiquitination upon oxidative stress is not a random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins.     

Stress-induced polyubiquitination of proteasomal ubiquitin receptors targets the proteolytic complex for autophagic degradation

Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a “predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a “prey.”     

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.     

Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis

Machado-Joseph disease is caused by an expansion of a trinucleotide CAG repeat in the gene encoding the protein ataxin-3. We investigated if ataxin-3 was a proteasome-associated factor that recognized ubiquitinated substrates based on the rationale that (i) it is present with proteasome subunits and ubiquitin in cellular inclusions, (ii) it interacts with human Rad23, a protein that may translocate proteolytic substrates to the proteasome, and (iii) it shares regions of sequence similarity with the proteasome subunit S5a, which can recognize multiubiquitinated proteins. We report that ataxin-3 interacts with ubiquitinated proteins, can bind the proteasome, and, when the gene harbors an expanded repeat length, can interfere with the degradation of a well-characterized test substrate. Additionally, ataxin-3 associates with the ubiquitin- and proteasome-binding factors Rad23 and valosin-containing protein (VCP/p97), findings that support the hypothesis that ataxin-3 is a proteasome-associated factor that mediates the degradation of ubiquitinated proteins.