Impact of ageing on proteasome structure and function in human lymphocytes

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major "housekeeping" proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20-63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.     

Genetic Evidence Linking Age-Dependent Attenuation of the 26S Proteasome with the Aging Process

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies'' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.Ubiquitin-conjugated, misfolded protein aggregates are observed in the brain during normal aging and in late-onset human neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, and polyglutamine diseases (e.g., Huntington''s disease or spinocerebellar ataxias) (9). Many of the mutations that cause dominantly inherited neurodegenerative diseases dramatically increase the amount of protein aggregates in vitro and in vivo, supporting the widely accepted hypothesis that proteotoxicity caused by the aggregates underlies the pathogenesis of many neurodegenerative diseases (32). Proteotoxicity can have many effects, including disruption of microtubule-dependent axonal transport (10), perturbation of membrane permeability (23), and impaired function of the ubiquitin-proteasome system (UPS) (1, 17). Aggregation-mediated toxicity has also been suggested in normal aging, because recent reports show that the impairment of autophagy in the central nervous system causes accumulation of ubiquitinated proteins and leads to neurodegenerative diseases (12, 21). These observations suggest that the continuous clearance of misfolded proteins through cellular degradation systems, including the UPS and autophagy, is important for preventing aggregation-mediated proteotoxicity both in age-related neurodegenerative diseases and in normal aging.Clinical symptoms of neurodegenerative diseases generally do not appear or progress until advanced ages, not only in sporadic forms but also in inherited forms of neurodegenerative diseases (26). These observations suggest that aggregation-mediated toxicity appears in a late-onset manner both in normal aging and in neurodegenerative diseases. Furthermore, a link between the aging process and aggregation-mediated proteotoxicity has been suggested by evidence that Huntington''s disease-associated proteotoxicity was ameliorated when the aging process slowed, that is, the life span extension via decreased insulin/insulin growth factor-1-like signaling in Caenorhabditis elegans (13, 31).A possible mechanism for the late onset of aggregation-mediated toxicity is age-related impairment of the UPS, because loss-of-function mutations in genes encoding UPS components can enhance the cytotoxicity of protein aggregation in dominantly inherited neurodegenerative diseases (4, 5, 18). In addition, an age-related decline of proteasome activity has been observed in the tissues of humans and other mammals (8) and in aged flies (36). Considering the role of the proteasome in neuroprotection and the age dependence of most neurodegenerative diseases, the age-related decline of proteasome activity could well be a key factor both in normal aging and in the late onset and/or progression of neurodegenerative diseases. However, the mechanism underlying the age-related decline of proteasome activity remains to be elucidated, and there is no direct genetic evidence showing that the age-related decline of proteasome activity causes age-related aggregation-mediated toxicity in normal aging and in age-related neurodegenerative diseases.Here, we studied the age-related decline of proteasome activity by using Drosophila melanogaster and found age-related attenuation of the 26S proteasome activity and abundance that was associated with impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we identified Rpn11, which encodes one of the lid subunits in the 19S RP, as a suppressor of the age-dependent progression of a polyglutamine-induced neurodegenerative phenotype. The overexpression of Rpn11 prevented the age-related reduction of the 26S proteasome activity, which suppressed the age-dependent accumulation of ubiquitinated proteins and extended the life span. On the other hand, the loss of function of Rpn11 enhanced the age-related reduction of 26S proteasome activity, leading to a shorter life span, a premature age-dependent accumulation of ubiquitinated proteins, and an early onset of a neurodegenerative phenotype. Our results demonstrate for the first time that the age-related reduction of the 26S proteasome activity is a key factor in the induction of certain age-related biological changes and in the increased risk for the onset or progression of neurodegenerative diseases. Our findings imply that improving the amount and/or activity of the 26S proteasome by overexpressing a lid subunit, such as Rpn11, could provide an extension to the mean life span and prevent the age-dependent onset or progression of neurodegeneration.     

Reduced proteasomal activity contributes to the accumulation of carbonylated proteins in chronic experimental autoimmune encephalomyelitis

We have recently shown that several carbonylated proteins, including glial fibrillary acidic protein, β-actin and β-tubulin, accumulate within cerebellar astrocytes during the chronic phase of myelin-oligodendrocyte glycoprotein (MOG)(35-55) peptide-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. As protein carbonyls cannot be repaired and there is less oxidative stress in chronic than in acute EAE, we hypothesized that the accumulation of carbonylated proteins in these animals may be due to a defect in the degradation of the modified proteins. Alternatively, oxidized proteins in chronic EAE mice may be more resistant to proteolysis. Using lipopolysaccharide-stimulated astrocytes and several protease inhibitors we identified the 20S proteasome as the proteolytic system responsible for the elimination of most oxidized proteins. We also discovered that the chymotrysin-like and caspase-like activities of the 20S proteasome are impaired in chronic EAE, while the amount of proteasome was unchanged. Proteasome failure in these animals was confirmed by the build-up of ubiquitinated proteins, mostly within astrocytes. In a cell-free system, carbonylated proteins from EAE mice with acute and chronic disease seem to be equally sensitive to proteasomal degradation. Altogether, the results support the notion that diminished activity of the 20S proteasome is a major contributor to the accumulation of carbonylated proteins in astrocytes of chronic EAE mice.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Altered proteasome function and subunit composition in aged muscle

Myofibrillar protein degradation is mediated through the ubiquitin-proteasome pathway. To investigate if altered proteasome activity plays a role in age-related muscle atrophy, we examined muscle size and proteasome function in young and aged F344BN rats. Significant age-related muscle atrophy was confirmed by the 38% decrease in cross-sectional area of type 1 fibers in soleus muscle. Determination of proteasome function showed hydrolysis of fluorogenic peptides was equivalent between ages. However, when accounting for the 3-fold increase in content of the 20S catalytic core in aged muscle, the lower specific activity suggests a functional loss in individual proteins with aging. Comparing the composition of the catalytic beta-subunits showed an age-related 4-fold increase in the cytokine-inducible subunits, LMP2 and LMP7. Additionally, the content of the activating complexes, PA28 and PA700, relative to the 20S proteasome was reduced 50%. These results suggest significant alterations in the intrinsic activity, the percentage of immunoproteasome, and the regulation of the 20S proteasome by PA28 and PA700 in aged muscle.     

Analysis of chain length,substitution patterns,and unsaturation of AM-404 derivatives as 20S proteasome stimulators

Proteasome-mediated degradation of proteins is a vital cellular process and is performed by the ubiquitin-dependent proteasome system (UPS) and the ubiquitin-independent proteasome system (UIPS). While both systems are necessary to maintain healthy cell function, many disease states are characterized by reduced activity of the UPS, and the UIPS cannot by itself maintain proper protein levels. It has been suggested that the 20S core particle (20S CP), the isoform of the proteasome in the UIPS that can degrade proteins without a ubiquitin tag, can be stimulated with a small molecule to assist the 20S CP to accept and hydrolyze substrates more rapidly. Several small molecule stimulators of the 20S CP have since been discovered, including AM-404, an arachidonic acid derivative. AM-404 has previously been shown to inhibit fatty acid amide hydrolase activity. We wished to evaluate what structural components of AM-404 are required to stimulate the 20S CP with the long-term goal of using this information to design a stimulator with better drug-like qualities. We synthesized numerous derivatives of AM-404, varying the chain length, substitutions, and degree of unsaturation. Through this endeavor, we obtained several molecules capable of stimulating the 20S CP to various degrees. We discovered that though chain length is important, the presence of a cis-alkene in a specific location in the aliphatic chain has the greatest impact on the ability to stimulate the 20S CP. Two of the derivatives maintain modest stimulatory activity, and have improved toxicity over AM-404.     

Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.     

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.     

Age-dependent declines in proteasome activity in the heart.

The proteasome is a major intracellular proteolytic system involved in the removal of oxidized and ubiquitinated protein and the induction of certain stress response pathways. In this study, age-dependent alterations in proteasome function were investigated to gain insight into potential factors which contribute to increased susceptibility to various forms of heart disease during aging. Proteasome activity in cellular extracts prepared from Fisher 344 rat hearts was found to decrease with age. These declines in activity were associated with a decreased 20S proteasome content and loss of specific activities. As determined by two-dimensional gel electrophoresis of purified 20S proteasome, the distribution and silver staining intensities of enzyme subunits were found to vary with age, suggesting that alterations in proteasome subunit composition and/or structure are involved in age-related declines in proteasome activity. In addition, age-dependent increases in the levels of oxidized and ubiquitinated proteins, known substrates of the proteasome, were observed. Thus, loss in proteasome function may impair the ability of myocytes to mount an appropriate response to stress, thereby enhancing the susceptibility of the aging heart to cardiovascular disease.     

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.     

Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway: ACTIVATION IN RESPONSE TO CADMIUM*

Heavy metals are known to generate reactive oxygen species that lead to the oxidation and fragmentation of proteins, which become toxic when accumulated in the cell. In this study, we investigated the role of the proteasome during cadmium stress in the leaves of Arabidopsis thaliana plants. Using biochemical and proteomics approaches, we present the first evidence of an active proteasome pathway in plants. We identified and characterized the peptidases acting sequentially downstream from the proteasome in animal cells as follows: tripeptidyl-peptidase II, thimet oligopeptidase, and leucine aminopeptidase. We investigated the proteasome proteolytic pathway response in the leaves of 6-week-old A. thaliana plants grown hydroponically for 24, 48, and 144 h in the presence or absence of 50 μm cadmium. The gene expression and proteolytic activity of the proteasome and the different proteases of the pathway were found to be up-regulated in response to cadmium. In an in vitro assay, oxidized bovine serum albumin and lysozyme were more readily degraded in the presence of 20 S proteasome and tripeptidyl-peptidase II than their nonoxidized form, suggesting that oxidized proteins are preferentially degraded by the Arabidopsis 20 S proteasome pathway. These results show that, in response to cadmium, the 20 S proteasome proteolytic pathway is up-regulated at both RNA and activity levels in Arabidopsis leaves and may play a role in degrading oxidized proteins generated by the stress.     

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.     

Changes in 20S proteasome activity during ageing of the LOU rat

Muscular functions decline and muscle mass decreases during ageing. In the rat, there is a 27% decrease in muscle protein between 18 and 34 months of age. We examined age-related changes in the proteasome-dependent proteolytic pathway in rats at 4, 18, 24, 29 and 34 months of age. The three best characterised activities of the proteasome (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) increased to 29 months and then decreased in the senescent animal. These variations in activity were accompanied by an identical change in the quantity of 20S proteasome measured by Western blot, whereas the S4 subunit of the 19S regulator and the quantity of ubiquitin-linked proteins remained constant. mRNA of subunits C3, C5, C9, and S4 increased in the senescent animal, but ubiquitin mRNA levels were unchanged. These findings suggest that the 20S proteasome may be partly responsible for the muscular atrophy observed during ageing in the rat.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α₇ ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr¹ residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys³³ with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys³³ in polarization of the Thr¹ N, which serves to strip a proton from the active-site Thr¹ Oγ nucleophile. Replacement of Asp⁵¹ with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.     

Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10⁻⁷ M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.     

Chaperone-mediated 26S Proteasome Remodeling Facilitates Free K63 Ubiquitin Chain Production and Aggresome Clearance

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.     

Impaired methylation as a novel mechanism for proteasome suppression in liver cells

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.     

Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome

Oxidatively modified proteins that accumulate in aging and many diseases can form large aggregates because of covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic, and threaten cell viability. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Previous work from our laboratory has shown that purified 20 S proteasome degrades oxidized proteins without ATP or ubiquitin in vitro, but there have been no studies to test this mechanism in vivo. The aim of this study was to determine whether ubiquitin conjugation is necessary for the degradation of oxidized proteins in intact cells. We now show that cells with compromised ubiquitin-conjugating activity still preferentially degrade oxidized intracellular proteins, at near normal rates, and this degradation is still inhibited by proteasome inhibitors. We also show that progressive oxidation of proteins such as lysozyme and ferritin does not increase their ubiquitinylation, yet the oxidized forms of both proteins are preferentially degraded by proteasome. Furthermore, rates of oxidized protein degradation by cell lysates are not significantly altered by addition of ATP, excluding the possibility of an energy requirement for this pathway. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26 S proteasome with ubiquitinylated substrates, our work suggests that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20 S proteasome, or the immunoproteasome, or both.     

alpha-Synuclein protofibrils inhibit 26 S proteasome-mediated protein degradation: understanding the cytotoxicity of protein protofibrils in neurodegenerative disease pathogenesis

The impaired ubiquitin-proteasome activity is believed to be one of the leading factors that contribute to Parkinson disease pathogenesis partially by causing alpha-synuclein aggregation. However, the relationship between alpha-synuclein aggregation and the impaired proteasome activity is yet unclear. In this study, we examined the effects of three soluble alpha-synuclein species (monomer, dimer, and protofibrils) on the degradation activity of the 26 S proteasome by reconstitution of proteasomal degradation using highly purified 26 S proteasomes and model substrates. We found that none of the three soluble alpha-synuclein species impaired the three distinct peptidase activities of the 26 S proteasome when using fluorogenic peptides as substrates. In striking contrast, alpha-synuclein protofibrils, but not monomer and dimer, markedly inhibited the ubiquitin-independent proteasomal degradation of unstructured proteins and ubiquitin-dependent degradation of folded proteins when present at 5-fold molar excess to the 26 S proteasome. Together these results indicate that alpha-synuclein protofibrils have a pronounced inhibitory effect on 26 S proteasome-mediated protein degradation. Because alpha-synuclein is a substrate of the proteasome, impaired proteasomal activity could further cause alpha-synuclein accumulation/aggregation, thus creating a vicious cycle and leading to Parkinson disease pathogenesis. Furthermore we found that alpha-synuclein protofibrils bound both the 26 S proteasome and substrates of the 26 S proteasome. Accordingly we propose that the inhibitory effect of alpha-synuclein protofibrils on 26 S proteasomal degradation might result from impairing substrate translocation by binding the proteasome or sequestrating proteasomal substrates by binding the substrates.