Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.     

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Although cellular proteins conjugated to K48‐linked Ub chains are targeted to proteasomes, proteins conjugated to K63‐ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48‐ and K63‐ubiquitinated substrates similarly. Therefore, we investigated why K63‐ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63‐ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48‐ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48‐ or K63‐ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal‐lysosomal pathway.     

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.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins

Long glutamine sequences (polyQ) occur in many cell proteins, and several neurodegenerative diseases result from expansion of these sequences. PolyQ-containing proteins are degraded by proteasomes, whose three active sites prefer to cleave after hydrophobic, basic, or acidic residues. We tested whether these particles can digest a polyQ chain. Eukaryotic 26S and 20S proteasomes failed to cut within stretches of 9-29Q residues in peptides. While digesting a myoglobin Q(35) fusion protein, the proteasomes spared the polyQ sequence. In contrast, archaeal proteasomes, whose 14 active sites are less specific, rapidly digested such polyQ repeats. Therefore, when degrading polyQ proteins, eukaryotic proteasomes must release aggregation-prone polyQ-containing fragments for further hydrolysis by unidentified peptidases. In polyQ diseases, such polyQ sequences (38-300Qs) exceed the lengths of normal proteasome products (2-25 residues). Occasional failure of these long undegradable sequences to exit may interfere with proteasome function and help explain why longer polyQ expansions promote early disease onset.     

Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes

PA28 is a gamma-interferon-induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP-ubiquitin-dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with 'singly capped' 26S (i.e. 19S-20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no significant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28alphabeta or PA28alpha generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.     

Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.     

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.     

The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis (Kleijnen et al., 2000). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2-proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAA receptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.     

Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly‐ubiquitinated by the proteasome‐associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat‐shock or arsenite treatment, when poly‐ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin‐conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.     

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.     

Proteasomes: protein and gene structures.

Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides. These particles have a latent multicatalytic proteinase activity. Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin. This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells. We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution. Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene. Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell. In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.     

A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S proteasomes to specific cellular compartments.     

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

Proteasomes cleave at multiple sites within polyglutamine tracts: activation by PA28gamma(K188E)

Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95-104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28gamma(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ(10)RR or fluorescein-HPHQ(10)RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ(10)RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin.     

Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core alpha1 (psmA), beta (psmB), and alpha2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either alpha1 or alpha2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either beta alone or alpha1 and alpha2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either alpha1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of alpha1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.     

Rab5a-mediated localization of claudin-1 is regulated by proteasomes in endothelial cells

Tight junctions composed of transmembrane proteins, including claudin, occludin, and tricellulin, and peripheral membrane proteins are a major barrier to endothelial permeability, whereas the role of claudin in the regulation of tight junction permeability in nonneural endothelial cells is unclear. This study demonstrates that claudin-1 is dominantly expressed and depletion of claudin-1 using small interfering RNA (siRNA) increased tight junction permeability in EA hy.926 cells, indicating that claudin-1 is a crucial regulator of endothelial tight junction permeability. The ubiquitin-proteasome system has been implicated in the regulation of endocytotic trafficking of plasma membrane proteins. Therefore, the involvement of proteasomes in the localization of claudin-1 was investigated by pharmacological and genetic inhibition of proteasomes using a proteasome inhibitor, N-acetyl-Leu-Leu-Nle-CHO, and siRNA against the β?-subunit of the 20S proteasome, respectively. Claudin-1 was localized at cell-cell contact sites in control cells. Claudin-1 was localized in the cytoplasm in association with Rab5a and EEA-1, a marker of early endosome, following inhibition of proteasomes. Depletion of Rab5a using siRNA reversed the localization of claudin-1 induced by inhibition of proteasomes. These data suggest that proteasomes regulate claudin-1 localization at the plasma membrane, which changes upon proteasomal inhibition to a Rab5a-mediated endosomal localization.     

Stable ubiquitination of human T-cell leukemia virus type 1 tax is required for proteasome binding

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukemia and HTLV-1-associated myelopathy. Adult T-cell leukemia development is mainly due to the ability of the viral oncoprotein Tax to promote T-cell proliferation, whereas the appearance of HTLV-1-associated myelopathy involves the antigenic properties of Tax. Understanding the events regulating the intracellular level of Tax is therefore an important issue. How Tax is degraded has not been determined, but it is known that Tax binds to proteasomes, the major sites for degradation of intracellular proteins, generally tagged through polyubiquitin conjugation. In this study, we investigated the relationship between Tax, ubiquitin, and proteasomes. We report that mono- and polyubiquitinated Tax proteins can be recovered from both transfected 293T cells and T lymphocytes. We also show that lysine residues located in the carboxy-terminal domain of Tax are the principal targets of this process. Remarkably, we further demonstrate that mutation of lysine residues in the C-terminal part of Tax, which massively reduces Tax ubiquitination, impairs proteasome binding, and conversely, that a Tax mutant that binds poorly to this particle (M22) is faintly ubiquitinated, suggesting that Tax ubiquitination is required for association with cellular proteasomes. Finally, we document that comparable amounts of ubiquitinated species were found whether proteasome activities were inhibited or not, providing evidence that they are not directly addressed to proteasomes for degradation. These findings indicate that although it is ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitin-proteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.