Contrasting proteome biology and functional heterogeneity of the 20 S proteasome complexes in mammalian tissues

The 20 S proteasome complexes are major contributors to the intracellular protein degradation machinery in mammalian cells. Systematic administration of proteasome inhibitors to combat disease (e.g. cancer) has resulted in positive outcomes as well as adversary effects. The latter was attributed to, at least in part, a lack of understanding in the organ-specific responses to inhibitors and the potential diversity of proteomes of these complexes in different tissues. Accordingly, we conducted a proteomic study to characterize the 20 S proteasome complexes and their postulated organ-specific responses in the heart and liver. The cardiac and hepatic 20 S proteasomes were isolated from the same mouse strain with identical genetic background. We examined the molecular composition, complex assembly, post-translational modifications and associating partners of these proteasome complexes. Our results revealed an organ-specific molecular organization of the 20 S proteasomes with distinguished patterns of post-translational modifications as well as unique complex assembly characteristics. Furthermore, the proteome diversities are concomitant with a functional heterogeneity of the proteolytic patterns exhibited by these two organs. In particular, the heart and liver displayed distinct activity profiles to two proteasome inhibitors, epoxomicin and Z-Pro-Nle-Asp-H. Finally, the heart and liver demonstrated contrasting regulatory mechanisms from the associating partners of these proteasomes. The functional heterogeneity of the mammalian 20 S proteasome complexes underscores the concept of divergent proteomes among organs in the context of an identical genome.     

Exploring proteasome complexes by proteomic approaches

The ubiquitin proteasome system (UPS) represents a major pathway for intracellular protein degradation. Proteasome dependent protein quality control participates in cell cycle, immune response and apoptosis. Therefore, the UPS is in focus of therapeutic investigations and the development of pharmaceutical agents. Detailed analyses on proteasome structure and function are the foundation for drug development and clinical studies. Proteomic approaches contributed significantly to our current knowledge in proteasome research. In particular, 2-DE has been essential in facilitating the development of current models on molecular composition and assembly of proteasome complexes. Furthermore, developments in MS enabled identification of UPS proteins and their PTMs at high accuracy and high-throughput. First results on global characterization of the UPS are also available. Although the UPS has been intensively investigated within the last two decades, its functional significance and contribution to the regulation of cell and tissue phenotypes remain to be explored. This review recapitulates a variety of applied proteomic approaches in proteasome exploration, and presents an overview of current technologies and their potential in driving further investigations.     

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.     

Mammalian proteasome subpopulations with distinct molecular compositions and proteolytic activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.     

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.     

The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.     

The effect of heat shock on 20S/26S proteasomes

We have studied the consequences of heat shock on 20S/26S proteasome activity and activation, the proteasomal subunit composition, proteasome assembly, subunit mRNA stability as well as on the intracellular distribution of proteasomes. Our data show that heat shock locks 20S proteasomes in their latent inactive state and impairs further activation of the 26S proteasome by ATP. Proteasome mRNA levels are decreased after heat shock and the assembly of the proteasome complex is inhibited. Heat shock also induces a rapid reorganisation of the cellular distribution of the proteasome which appears to be connected with proteasome activity and the change of the cellular architecture after heat shock.     

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.     

The comparative analysis of extra- and intracellular proteasomes from K562 cell line

The comparative analysis of peptidase activities of extra- and intracellular proteasomes was carried out. Here we have shown that excreted proteasomes exhibit higher chymotrypsin-type and lower tripsin-like peptidase activities that cytoplasmic particles. Posttranslational modifications (PTMs) of 20S proteasomal subunits were revealed by immunoblotting techniques. We have observed the difference in PTMs of associated with enzymatic activities subunits beta2, beta5 and beta5 of extracellular and cytoplasmic proteasomes. Proteasomal subunits alpha2, 4, 7 and beta7 also had a variety of PTMs. The phosphorylation level of excreted proteasomes was lower compared to that of the intracellular ones. This observation strongly suggests the involvement of this PTM in the regulation of proteasomes excretion from cells.     

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.     

A subset of 26S proteasomes is activated at critically low ATP concentrations and contributes to myocardial injury during cold ischemia

Molecular mechanisms leading to myocardial injury during warm or cold ischemia are insufficiently understood. Although proteasomes are thought to contribute to myocardial ischemia-reperfusion injury, their roles during the ischemic period remain elusive. Because donor hearts are commonly exposed to prolonged global cold ischemia prior to cardiac transplantation, we evaluated the role and regulation of the proteasome during cold ischemic storage of rat hearts in context of the myocardial ATP content. When measured at the actual tissue ATP concentration, cardiac proteasome peptidase activity increased by 225% as ATP declined during cold ischemic storage of hearts in University of Wisconsin (UW) solution for up to 48 h. Addition of the specific proteasome inhibitor epoxomicin to the UW solution inhibited proteasome activity in the cardiac extracts, significantly reduced edema formation and preserved the ultrastructural integrity of the cardiomyocyte. Utilizing purified 20S/26S proteasome enzyme preparations, we demonstrate that this activation can be attributed to a subset of 26S proteasomes which are stable at ATP concentrations far below physiological levels, that ATP negatively regulates its activity and that maximal activation occurs at ATP concentrations in the low μmol/L range. These data suggest that proteasome activation is a pathophysiologically relevant mechanism of cold ischemic myocardial injury. A subset of 26S proteasomes appears to be a cell-destructive protease that is activated as ATP levels decline. Proteasome inhibition during cold ischemia preserves the ultrastructural integrity of the cardiomyocyte.     

Activity-Based Imaging Probes of the Proteasome

Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors—bortezomib and carfilzomib—have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes.     

Stalled proteasomes are directly relieved by P97 recruitment

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.     

Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis

Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.     

Structure of a Proteasome Pba1-Pba2 Complex: IMPLICATIONS FOR PROTEASOME ASSEMBLY,ACTIVATION, AND BIOLOGICAL FUNCTION*

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.     

Proteasome-associated proteins: regulation of a proteolytic machine

The proteasome is a compartmentalized, ATP-dependent protease composed of more than 30 subunits that recognizes and degrades polyubiquitinated substrates. Despite its physiological importance, many aspects of the proteasome's structural organization and regulation remain poorly understood. In addition to the proteins that form the proteasome holocomplex, there is increasing evidence that proteasomal function is affected by a wide variety of associating proteins. A group of ubiquitin-binding proteins assist in delivery of substrates to the proteasome, whereas proteasome-associated ubiquitin ligases and deubiquitinating enzymes may alter the dynamics of ubiquitin chains already associated with the proteasome. Some proteins appear to influence the overall stability of the complex, and still others have the capacity to activate or inhibit the hydrolytic activity of the core particle. The increasing number of interacting proteins identified suggests that proteasomes, as they exist in the cell, are larger and more diverse in composition than previously assumed. Thus, the study of proteasome-associated proteins will lead to new perspectives on the dynamics of this uniquely complex proteolytic machine.     

Archaeal proteasomes effectively degrade aggregation-prone proteins and reduce cellular toxicities in mammalian cells

The 20 S proteasome is a ubiquitous, barrel-shaped protease complex responsible for most of cellular proteolysis, and its reduced activity is thought to be associated with accumulations of aberrant or misfolded proteins, resulting in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis, spinal and bulbar muscular atrophy, Parkinson disease, and Alzheimer disease. The 20 S proteasomes of archaebacteria (archaea) are structurally simple and proteolytically powerful and thought to be an evolutionary precursor to eukaryotic proteasomes. We successfully reproduced the archaeal proteasome in a functional state in mammalian cells, and here we show that the archaeal proteasome effectively accelerated species-specific degradation of mutant superoxide dismutase-1 and the mutant polyglutamine tract-extended androgen receptor, causative proteins of familial amyotrophic lateral sclerosis and spinal and bulbar muscular atrophy, respectively, and reduced the cellular toxicities of these mutant proteins. Further, we demonstrate that archaeal proteasome can also degrade other neurodegenerative disease-associated proteins such as alpha-synuclein and tau. Our study showed that archaeal proteasomes can degrade aggregation-prone proteins whose toxic gain of function causes neurodegradation and reduce protein cellular toxicity.