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Age-related increase in the immunoproteasome content in rat hippocampus: Molecular and functional aspects
Journal of Neurochemistry
Abstract
Alterations in the proteasome activity in the CNS have been described during aging. However, a detailed study of all proteasome subunits is actually lacking. We have analyzed, in vivo, the age-related modifications in the molecular composition of hippocampal proteasomes. We found that the immunoproteasome/proteasome ratio was increased in aged hippocampus. The processing of the low-molecular-mass protein (LMP)7/beta(5i) subunit, practically absent in young hippocampus, was increased in aged animals. Among the potential factors underlying these modifications we evaluated the neuroinflammation and the transcription factor Zif268. Lipopolysaccharide (LPS)-induced neuroinflammation in young rats, up-regulated the expression of immunoproteasome subunits and increased the processing of the LMP7/beta(5i) protein. Moreover, the hydrophobicity of cellular peptides, analyzed by liquid chromatography, increased in both, young LPS-injected animals and aged rats, suggesting that immunoproteasomes including the LMP7/beta(5i) subunit could, at least in part, account for this modification. Also, the mRNA expression of the transcription factor Zif268, which down-regulates the immunoproteasome subunit LMP7/beta(5i) by binding to sequences within the promoter regions, was decreased in both, aged hippocampus and young LPS-injected animals. Finally, we found that spatial memory training in young animals, a situation in which the expression of Zif268 is increased, modified the mRNA expression of the constitutive and catalytic subunits in an opposite manner. Based on present data, we propose that the age-related increases in the content of hippocampal immunoproteasome is mostly because of neuroinflammatory processes associated to aging.
... The exact mechanisms accounting for the age-dependent decrease in the proteasome activity remain still elusive. For example, structural alterations of the proteasome, as well as reduced expression of proteasome subunits have been described (Lee et al., 1999;Bulteau et al., 2002;Chondrogianni et al., 2003;Gavilán et al., 2009a;Baraibar et al., 2012). On the other hand, the age-related increase in reactive oxygen species, mostly due to mitochondrial dysfunction and dysregulation of anti-oxidant repair mechanisms (Squier, 2001;Rottenberg and Hoke, 2017;Scialo et al., 2017), can also affect proteasome activity by oxidative damage. ...
... However, in addition to providing peptides for antigen presentation, and other immune functions, the immunoproteasome degrades nascent oxidant damaged proteins, also known as DRiPs (Seifert et al., 2010;Opitz et al., 2011), increases the cellular proportion of hydrophobic peptides (Gaczynska et al., 1996;Cascio et al., 2001;Chapiro et al., 2006 ;Paz Gavilán et al., 2006;Gavilán et al., 2009a), and regulates autophagy (Pintado et al., 2017;Karim et al., 2020). Thus, the immunoproteasome plays an important general role in the maintenance of cellular proteostasis under acute inflammation. ...
... Because aging is associated with a low grade of chronic inflammation, the modulation exerted by inflammation on cellular proteostasis might be particularly relevant in aged cells. For example, the immunoproteasome, which is not expressed in cells from young animals, is expressed in rat and human aged cells from several tissues (Ferrington et al., 2005;Mishto et al., 2006;Gavilán et al., 2007;Gavilán et al., 2009a;Wagner et al., 2017). ...
Aging is a biological and multifactorial process characterized by a progressive and irreversible deterioration of the physiological functions leading to a progressive increase in morbidity. In the next decades, the world population is expected to reach ten billion, and globally, elderly people over 80 are projected to triple in 2050. Consequently, it is also expected an increase in the incidence of age-related pathologies such as cancer, diabetes, or neurodegenerative disorders. Disturbance of cellular protein homeostasis (proteostasis) is a hallmark of normal aging that increases cell vulnerability and might be involved in the etiology of several age-related diseases. This review will focus on the molecular alterations occurring during normal aging in the most relevant protein quality control systems such as molecular chaperones, the UPS, and the ALS. Also, alterations in their functional cooperation will be analyzed. Finally, the role of inflammation, as a synergistic negative factor of the protein quality control systems during normal aging, will also be addressed. A better comprehension of the age-dependent modifications affecting the cellular proteostasis, as well as the knowledge of the mechanisms underlying these alterations, might be very helpful to identify relevant risk factors that could be responsible for or contribute to cell deterioration, a fundamental question still pending in biomedicine.
... These changes range from impairments in protein degradation associated with memory retrieval [16] to changes in resting state neural activity [33]. While decreases in protein degradation processes occur as a function of normal aging [34], impairments in protein homeostasis and degradation are primary factors underlying age-associated protein accumulation [35,36]. Importantly, protein degradation following memory retrieval is necessary for the successful formation and stabilization of long-term memory [37,38]. ...
... Zif268 is an immediate-early gene (IEG) that is necessary for the active process of memory retrieval and memory consolidation [40][41][42][43][44][45], and it also can serve as a general marker of increased neural activity [46][47][48]. Memory retrieval-related increases in zif268 are greater for aged animals than their young adult counterparts despite showing poorer recall, while baseline age differences are not typically seen in naïve controls [35,49]. Based on this, we predicted that age-related deficits in fear recall would be associated with increases in zif268 protein accumulation. ...
... In the DH ( Figure 1E), a region recruited for trace fear conditioning [21] and spatial learning [58], the ANOVA examining zif268 accumulation was again significant, F (2, 17) = 6.51, p = 0.008. As in the mPFC and the aRSC, post-hoc tests demonstrated that while 3-month-old animals differed from both 15-month-old (p = 0.03) and 22-month-old (p = 0.003) animals, these two groups did not differ from each other (p = 0. 35). No systematic changes in pRpt6 were seen in the DH ( Figure 2C), F < 1, and no group differences were observed, smallest p = 0.83. ...
Aging is associated with cognitive decline, including impairments in the ability to accurately form and recall memories. Some behavioral and brain changes associated with aging are evident as early as middle age, making the understanding of associated neurobiological mechanisms essential to aid in efforts aimed at slowing cognitive decline throughout the lifespan. Here, we found that both 15-month-old and 22-month-old rats showed impaired memory recall following trace fear conditioning. This behavioral deficit was accompanied by increased zif268 protein accumulation relative to 3-month-old animals in the medial prefrontal cortex, the dorsal and ventral hippocampi, the anterior and posterior retrosplenial cortices, the lateral amygdala, and the ventrolateral periaqueductal gray. Elevated zif268 protein levels corresponded with decreases in phosphorylation of the Rpt6 proteasome regulatory subunit, which is indicative of decreased engagement of activity-driven protein degradation. Together, these results identify several brain regions differentially impacted by aging and suggest that the accumulation of proteins associated with memory retrieval, through reduced proteolytic activity, is associated with age-related impairments in memory retention.
... In this sense, autophagy activation induced by proteasome inhibition has been described to be dependent on unfolded protein response (UPR) activation (Ding et al., 2007) or glycogen synthase kinase-3b (GSK-3b) inhibition (Gavilán et al., 2013;Parr et al., 2012;Yang et al., 2010). Because the age-related disruption in protein homeostasis makes aged neurons more vulnerable to protein accumulation (Gavilán et al., 2009a), and autophagy disruption is common to some neurodegenerative disorders, an important issue to be elucidated is to know how the 2 major proteolytic systems can interact to each other to preserve protein homeostasis. Here, we demonstrate for the first time in vivo age-related deficiencies in the crosstalk between the UPS and autophagy, with consequences on neuronal protein homeostasis. ...
... Collectively, these results supported an efficient functional crosstalk between both proteolytic systems that prevented the accumulation of ubiqutinated proteins and restored proteostasis in young rats. By contrast, aged rats were more dependent on autophagy (Gamerdinger et al., 2009), probably because of the chronic proteasome stress associated to aging (Gavilán et al., 2009a(Gavilán et al., , 2009bPaz Gavilán et al., 2006). Indeed, proteasome inhibition produced a higher and sustained elevation of the BAG3 to BAG1 ratio in aged rats, suggesting that proteins were preferentially delivered to the ALP for their degradation. ...
... Third, infected microglia disrupted neuronal autophagy activation leading to neurodegeneration (Alirezaei et al., 2008). In this line, the presence of activated microglial cells (Gavilán et al., 2007), which alter protein homeostasis in pyramidal neurons, is characteristic of aged hippocampus (Gavilán et al., 2009a;Pintado et al., 2012). Thus, we speculate that several factors such as UPR dysfunction, sustained activity of the GSK-3b, and the presence of activated microglial cells could be age-related factors contributing to the inefficient functional crosstalk between the UPS and the ALP. ...
... The is responsible for catalysis of the ATP-dependent degradation of ubiquitinated proteins (Hochstrasser 1995;Rechsteiner and Hill 2005;Jung et al. 2009). Moreover, following IFN-c stimulation or after lipopolysaccharide injection, the constitutive catalytic subunits b1, b2, and b5 are replaced by the inducible catalytic subunits b1i, b2i, and b5i, leading to immunoproteasome formation that associates with the regulatory complex PA28/11S (Gaczynska et al. 1993;Rivett et al. 2001;Gavilán et al. 2009a). Despite the fact that the exact mechanism responsible for proteasome biogenesis is not fully understood, coordinated assembly of proteasomes requires helper factors such as the mammalian proteasomeassembling chaperones (PACS) (Hirano et al. 2005(Hirano et al. , 2006 and the proteasome maturation protein (POMP) (Burri et al. 2000;Griffin et al. 2000;Witt et al. 2000;Heink et al. 2005;Fricke et al. 2007). ...
... Recent evidence suggests that immunoproteasome induction is not only relevant to the immune response but also to the preservation of protein homeostasis under stress situations (Ferrington et al. 2008;Seifert et al. 2010). In this sense, we have previously shown that immunoproteasome content is increased in aged rat hippocampus (Gavilán et al. 2009a), a situation that could be a consequence of the age-related neruoinflammatory processes (Gavilan et al. 2007;Lynch 2010) and/or of the altered protein homeostasis (Paz Gavilán et al. 2006). In this work, we wondered whether proteasome recovery after proteasome inhibition is affected by aging process. ...
... RNA extraction, reverse transcription, and real-time PCR Three hundred microliters of each homogenate was used for total RNA extraction and reverse transcription. They performed exactly as previously described (Gavilán et al. 2009a). Real-time PCR were performed in an ABI Prism 7000 sequence detector (Applied Biosystems, Madrid, Spain) using cDNA diluted in sterile water as a template. ...
Regulation of proteasome abundance to meet cell needs under stress conditions is critical for maintaining cellular homeostasis. However, the effects of aging on this homeostatic response remain unknown. In this report, we analyzed in young and aged rat hippocampus, the dynamics of proteasome recovery induced by proteasome stress. Proteasome inhibition in young rats leads to an early and coordinate transcriptional and translational up-regulation of both the catalytic subunits of constitutive proteasome and the proteasome maturation protein. By contrast, aged rats up-regulated the inducible catalytic subunits and showed a lower and shorter expression of proteasome maturation protein. This resulted in a faster recovery of proteasome activity in young rats. Importantly, proteasome inhibition highly affected pyramidal cells, leading to the accumulation of ubiquitinated proteins in perinuclear regions of aged, but not young pyramidal neurons. These data strongly suggest that age-dependent differences in proteasome level and composition could contribute to neurodegeneration induced by proteasome dysfunction in normal and pathological aging.
... In this regard, the functional changes that occur in the proteasome pool with age are of great interest and are being actively studied. Previous studies of a proteasome pool in different organs, including the central nervous system performed on aged F344BN and Wistar rats, revealed an increase in the expression of proteasomes with immune catalytic subunits [5][6][7]. Giannini et al. [8] reported that in the cerebral cortex of aging Sprague Dawley rats, the expression of immune proteasome subunits was increased and the efficiency of fluorogenic substrate hydrolysis by 20S and 26S proteasomes was decreased. At the same time, the degradation of polyubiquitinated model substrates by 26S proteasomes of old animals was slightly increased compared to those isolated from young animals. ...
... We studied the proteasome pool of the cerebral cortex from healthy C57BL/6 mice of four age groups: 60, 190, 380, and 720 days, and analyzed the dynamics of changes in the studied parameters throughout almost the entire life of the animals. The data we obtained on a decrease in the expression of genes of constitutive subunits and an increase in the expression of genes of immune proteasome subunits are consistent with the results obtained using other animal models [4][5][6][7][8]16]. In the lysates of the cerebral cortex of mice at the age of 720 days, compared to samples from 60-day-old animals, an increase (on average by 2-3 folds) in the amount of immune subunits β1i and β2i was revealed (Fig. 2). ...
Proteasomes are key components of the ubiquitin–proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.
... Indeed, the inhibition of iP activity was associated with decreased IFNγdependent expression of pro-inflammatory cytokines in microglia [27] and attenuation of disease progression in murine models of Alzheimer's disease [3] and experimental encephalomyelitis [28]. Accordingly, the upregulation of the iP expression over the course of Alzheimer and Huntington diseases [29,30] and in neurons of aging hippocampus [31] were reported. Nevertheless, whether the non-immunological function of iP in astrocytes, among others, in removing ROS and oxidated and poly-ubiquitinated proteins is still discussed [32,33]; notably, astrocytes do not readily present antigens in vivo [34,35]. ...
... Obtained cDNA samples were used to the gene expression analysis performed using the quantitative real-time PCR (qRT-PCR) technique for the genes encoding the β1, β1i (LMP2), β2, β2i (MECL1), β5 and β5i (LMP7). Sequences of the primers are presented in Table 1 [31,66,67]. The constitutively expressed GAPDH gene was applied as a reference gene. ...
The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer’s disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (β1 and β5) and inducible (iβ1/LMP2 and iβ5/LMP7) proteasome subunits and proteasomal activity in IL-1β-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1β-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPβ transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits’ expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1β was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.
... The replacement of constitutive proteasome subunits results primarily in an accelerated hydrolytic efficiency and altered cleavage specificity, which causes the generation of an altered and increased peptide pool (Ferrington and Gregerson, 2012;Seifert et al., 2010;Toes et al., 2001). Remarkably, low levels of IP expression were also detected in the brain (Gavilán et al., 2009;Piccinini et al., 2003), but the precise role of IPs for normal brain function remains elusive. Thus far, the induction and presence of IP subunits in the brain has been mainly associated to age and age-related neurodegenerative diseases such as Alzheimeŕs, Huntingtońs and Parkinsońs disease, or amyotrophic lateral sclerosis (Díaz-Hernández et al., 2004, 2003Gavilán et al., 2009;Jansen et al., 2014;Mishto et al., 2006;Puttaparthi and Elliott, 2005;Vilchez et al., 2014). ...
... Remarkably, low levels of IP expression were also detected in the brain (Gavilán et al., 2009;Piccinini et al., 2003), but the precise role of IPs for normal brain function remains elusive. Thus far, the induction and presence of IP subunits in the brain has been mainly associated to age and age-related neurodegenerative diseases such as Alzheimeŕs, Huntingtońs and Parkinsońs disease, or amyotrophic lateral sclerosis (Díaz-Hernández et al., 2004, 2003Gavilán et al., 2009;Jansen et al., 2014;Mishto et al., 2006;Puttaparthi and Elliott, 2005;Vilchez et al., 2014). Interestingly, IPs were also found in experimental epilepsy where β5i/ LMP7 is expressed in glia cells and also in neurons (Mishto et al., 2015). ...
Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin–proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a β5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive β5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, β5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of β5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.
... Neuroinflammatory processes associated with aging were also shown to contribute to increased immunoproteasome/proteasome ratio in aged rat hippocampus. LPS-induced neuroinflammation in young rat hippocampus leads to elevated expression of immunoproteasome subunits and enhanced processing of the β5i protein [278]. In all cases, the mRNA expression of Zif268 transcription factor, a regulator of immunoproteasome genes [279], was decreased [278]. ...
... LPS-induced neuroinflammation in young rat hippocampus leads to elevated expression of immunoproteasome subunits and enhanced processing of the β5i protein [278]. In all cases, the mRNA expression of Zif268 transcription factor, a regulator of immunoproteasome genes [279], was decreased [278]. Proteinopathies are diseases that are characterized by protein misfolding and aggregation, including desmin-related cardiomyopathy (DRC) [280,281]. ...
Aging is a natural biological process that is characterized by a progressive accumulation of macromolecular damage. In the proteome, aging is accompanied by decreased protein homeostasis and function of the major cellular proteolytic systems, leading to the accumulation of unfolded, misfolded or aggregated proteins. In particular, the proteasome is responsible for the removal of normal as well as damaged or misfolded proteins. Extensive work during last several years has clearly demonstrated that proteasome activation by either genetic means or use of compounds significantly retards aging. Importantly, this represents a common feature across evolution thereby suggesting proteasome activation to be an evolutionary conserved mechanism of aging and longevity regulation. This review article reports on the means of function of these proteasome activators and how they regulate aging in different species.
... Reverse transcription (RT) was performed using random hexamers primers, 3 μg of total RNA as template, and the High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer's recommendations as previously described. 23 Real-Time PCR. After RT, the cDNA was diluted in sterile water and used as template for the amplification by the polymerase chain reaction. ...
... For real-time RT-PCR, each specific gene product was amplified using commercial TaqMan probes using the ABI Prism 7000 sequence detector (Applied Biosystems, Madrid, Spain) as previously described. 23 The cDNA levels were determined using GAPDH as housekeeper. The amplification of the housekeeper was done in parallel with the gene to be analyzed. ...
Neuroinflammation is an important contributor to pathogenesis of age-related neurodegenerative disorders as Alzheimer or Parkinson´s disease. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for neurodegenerative processes. Flavonoids, widely distributed in the vegetable kingdom and in aliments like honey have been suggested as novel therapeutic agents for the reduction of the deleterious effects of neuroinflammation. In the present study, we investigated the potential protective effect of a honey flavonoid extract (HFE) on the production of proinflammatory mediators by lipopolysaccharide-stimulated N13 microglia. Our results show that HFE significantly inhibited the release of the proinflammatory cytokines as TNF-alpha and IL-1beta. The expressions of iNOS and the production of ROS were also significantly inhibited. Accordingly, present study demonstrates that HFE is a potent inhibitor of microglial activation and thus a potential preventive-therapeutic agent for neurodegenerative diseases involving neuroinflammation.
... TNF-a and IFN-g are main inducers of proteasome immunosubunits (Groettrup et al., 2010) and thus, the early mRNA upregulation of these two pro-inflammatory cytokines, before any neurological symptoms appeared in our 4-stage model, suggests that inflammation could play a major role in the proteasome switching described here. The proteasome also participates in neuronal survival and plasticity (Dong et al., 2008; Gavilan et al., 2009; Kwak et al., 2003 ) and its dysfunction contributes to neurocognitive disturbances as a consequence of altered protein turnover impairing synaptic function (Bingol and Sheng, 2011; Nguyen et al., 2010). Thus, the association between BDNF transcriptional blockade and proteasome inhibition, reported also in other neurological malfunctions (Dong et al., 2008; Seo et al., 2008), could be responsible for the accelerated neurological deterioration that occurs during ECM in the mouse. ...
... Thus, the association between BDNF transcriptional blockade and proteasome inhibition, reported also in other neurological malfunctions (Dong et al., 2008; Seo et al., 2008), could be responsible for the accelerated neurological deterioration that occurs during ECM in the mouse. Spatial memory training upregulates constitutive proteasome subunits and downregulates inducible ones (Gavilan et al., 2009) suggesting that the inflammatoryFig. 6 – Parasite accumulation in brain, blood and peripheral tissues. ...
The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I to IV) of the neurological progression of CM in C57BL/6 mice infected with P. berghei ANKA. Upregulation of ICAM-1, VCAM-1, E-selectin and P-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus-hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.
... Here, we show age-dependent alterations in brain immunoproteasome expression in line with previous studies. 25,26 We also show that the deficiency in immunoproteasomes was accompanied by an increase in the amount of polyubiquitinated proteins and the emergence of epilepsy and other behaviour alterations (i.e. anxiety and ataxia). ...
The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. Here, we show an age-dependent increase in polyubiquitination in the brain of wild-type mice, accompanied with induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during aging.
... iP is also involved in a wide number of inflammatory diseases, such as inflammatory bowel disease, autoimmune disease, rheumatoid arthritis, systemic lupus erythematosus, and neuroinflammation [6]. In particular, different works demonstrated that iP is expressed in neurons, glia, astrocytes, and endothelial cells of the central nervous system (CNS), and it is markedly upregulated by pro-inflammatory stimuli, such as LPS, IFN-γ, and TNF-α [7][8][9][10]. In particular, under inflammatory or stress conditions of the nervous system, the accumulation of misfolded proteins causes the production of dangerassociated molecular pattern molecules (DAMPs) which stimulate NF-kB activation and the upregulation of iP binding Toll-like receptor 9 (TLR-9) [11,12]. ...
Neuroinflammation is an inflammatory response of the nervous tissue mediated by the production of cytokines, chemokines, and reactive oxygen species. Recent studies have shown that an upregulation of immunoproteasome is highly associated with various diseases and its inhibition attenuates neuroinflammation. In this context, the development of non-covalent immunoproteasome-selective inhibitors could represent a promising strategy for treating inflammatory diseases. Novel amide derivatives, KJ3 and KJ9, inhibit the β5 subunit of immunoproteasome and were used to evaluate their possible anti-inflammatory effects in an in vitro model of TNF-α induced neuroinflammation. Differentiated SH-SY5Y and microglial cells were challenged with 10 ng/mL TNF-α for 24 h and treated with KJ3 (1 µM) and KJ9 (1 µM) for 24 h. The amide derivatives showed a significant reduction of oxidative stress and the inflammatory cascade triggered by TNF-α reducing p-ERK expression in treated cells. Moreover, the key action of these compounds on the immunoproteasome was further confirmed by halting the IkB-α phosphorylation and the consequent inhibition of NF-kB. As downstream targets, IL-1β and IL-6 expression resulted also blunted by either KJ3 and KJ9. These preliminary results suggest that the effects of these two compounds during neuroinflammatory response relies on the reduced expression of pro-inflammatory targets.
... Our results suggest age-related changes in immunoproteasome function as additional risk factor. We here show age-dependent alterations in brain immunoproteasome expression in line with previous studies (Gavilan et al, 2009;Mishto et al, 2006). We also show that the deficiency in immunoproteasomes was accompanied by an increase in the amount of polyubiquitinated proteins and the emergence of epilepsy and other behavior alterations (i.e. ...
The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly inactive in the healthy brain. Here, we show an age-dependent increase in polyubiquitination in the brain of wild-type mice, accompanied with induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout (TKO) mice deficient for LMP2, LMP7 and MECL-1), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged TKO mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, aged TKO mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during aging.
... Moreover, disturbed proteostasis in synergy with inflammation boosts the aging process as demonstrated by the discovery that pro-inflammatory cytokines cause the replacement of the canonical catalytic β subunits in the proteasome complex, leading to the assembly of the immunoproteasome, which selectively degrades proteins involved in inflammation and immune response [64,65]. On the other hand, the inhibition of the immunoproteasome prevents the expression of pro-inflammatory cytokines [66]. ...
Extracellular vesicles (EVs) are membrane-enclosed particles secreted by cells and circulating in body fluids. Initially considered as a tool to dispose of unnecessary material, they are now considered an additional method to transmit cell signals. Aging is characterized by a progressive impairment of the physiological functions of tissues and organs. The causes of aging are complex and interconnected, but there is consensus that genomic instability, telomere erosion, epigenetic alteration, and defective proteostasis are primary hallmarks of the aging process. Recent studies have provided evidence that many of these primary stresses are associated with an increased release of EVs in cell models, able to spread senescence signals in the recipient cell. Additional investigations on the role of EVs during aging also demonstrated the great potential of EVs for the modulation of age-related phenotypes and for pro-rejuvenation therapies, potentially beneficial for many diseases associated with aging. Here we reviewed the current literature on EV secretion in senescent cell models and in old vs. young individual body fluids, as well as recent studies addressing the potential of EVs from different sources as an anti-aging tool. Although this is a recent field, the robust consensus on the altered EV release in aging suggests that altered EV secretion could be considered an emerging hallmark of aging.
... A growing number of studies have unveiled that TIMP1, HLA-DRA, and FGF2 play neuroprotective roles against brain aging through some mechanisms, such as regulation of immune activity, maintain the integrity of junctional proteins and transendothelial tightness of human brain microvessel endothelial cells, and augment of neuron survival, neurogenesis and nerve repair (Yin et al., 2017;Tang et al., 2020). SP1, which controlled most DEGs, plays an essential role in neuroinflammation of aged HC and might be related to cognitive development during brain aging (Gavilán et al., 2009;Gaur and Prasad, 2014). The roles of CD44, CD 93, and VWF in the human brain have been discussed previously. ...
Background
Given the arrival of the aging population has caused a series of social and economic problems, we aimed to explore the key genes underlying cognitively normal brain aging and its potential molecular mechanisms.
Methods
GSE11882 was downloaded from Gene Expression Omnibus (GEO). The data from different brain regions were divided into aged and young groups for analysis. Co-expressed differentially expressed genes (DEGs) were screened. Functional analysis, protein–protein interaction (PPI) network, microRNA (miRNA)-gene, and transcription factor (TF)-gene networks were performed to identify hub genes and related molecular mechanisms. AlzData database was used to elucidate the expression of DEGs and hub genes in the aging brain. Animal studies were conducted to validate the hub genes.
Results
Co-expressed DEGs contained 7 upregulated and 87 downregulated genes. The enrichment analysis indicated DEGs were mainly involved in biological processes and pathways related to immune-inflammatory responses. From the PPI network, 10 hub genes were identified: C1QC, C1QA, C1QB, CD163, FCER1G, VSIG4, CD93, CD14, VWF, and CD44. CD44 and CD93 were the most targeted DEGs in the miRNA-gene network, and TIMP1, HLA-DRA, VWF, and FGF2 were the top four targeted DEGs in the TF-gene network. In AlzData database, the levels of CD44, CD93, and CD163 in patients with Alzheimer’s disease (AD) were significantly increased than those in normal controls. Meanwhile, in the brain tissues of cognitively normal mice, the expression of CD44, CD93, and CD163 in the aged group was significantly lower than those in the young group.
Conclusion
The underlying molecular mechanisms for maintaining healthy brain aging are related to the decline of immune-inflammatory responses. CD44, CD93, and CD 163 are considered as potential biomarkers. This study provides more molecular evidence for maintaining cognitively normal brain aging.
... Equally, PSMB9 and PSME2 are upregulated in Zif268-knockout mice, 79 and reduced Zif268 expression in aged rat brain increases the immunoproteasome genes PSMB8-10, possibly contributing to the age-related induction of neuroinflammatory signals. 80 Consistent with these effects on proteasome gene expression, Zif268 overexpression suppresses proteasome activity; whereas Zif268-knock-out mice exhibit high proteasome activity in the cortex. 79 The proteasome regulates several proteins that are crucial for synaptic function such as NMDA receptor subunits, scaffolding molecules, postsynaptic density, and synaptic glutamate transporters, and thus its regulation by Zif268 provides new insight into the mechanisms sustaining the late phase of neuronal plasticity. ...
The tight regulation of proteostasis is essential for physiological cellular function. Mammalian cells possess a network of mechanisms that ensure proteome integrity under normal or stress conditions. The proteasome, being the major cellular proteolytic machinery, is central to proteostasis maintenance in response to distinct intracellular and extracellular conditions. The proteasomes are multisubunit protease complexes that selectively catalyze the degradation of short-lived regulatory proteins and damaged peptides. Different forms of the proteasome complexes comprising of different subunits and attached regulators directly affect the substrate selectivity and degradation. Thus, the proteasome participates in the turnover of a multitude of factors that control key processes that affect the cellular state, such as adaptation to environmental cues, growth, development, metabolism, signaling, senescence, pluripotency, differentiation, and immunity. Aberrations on its function are related to normal processes like aging and pathological conditions such as neurodegeneration and cancer. The past few years of research have highlighted that proteasome abundance, activity, assembly, and localization are subject to a dynamic transcriptional control that secures the continuous adaptation of the proteasome to internal or external stimuli. This review focuses on the factors and signaling pathways that are involved in the regulation of the mammalian proteasome at the transcriptional level. A comprehensive understanding of proteasome regulation has critical implications on disease prevention and treatment.
... Type-I interferons IFN-α and IFN-β also regulate the immunoproteasome expression, which was demonstrated after hepatitis C and coxsackievirus infection [63][64][65]. Tumor necrosis factor-alpha (TNF-α) also has been shown to upregulate immunoproteasome expression upon liposaccharide mediated inflammatory stimulus [66]. Induction of the immunoproteasome has also been observed after nitric oxide (NO) exposure, constituting a cytokine-independent regulatory mechanism. ...
Cell-mediated immunity is driven by antigenic peptide presentation on major histocompatibility complex (MHC) molecules. Specialized proteasome complexes called immunoproteasomes process viral, bacterial, and tumor antigens for presentation on MHC class I molecules, which can induce CD8 T cells to mount effective immune responses. Immunoproteasomes are distinguished by three subunits that alter the catalytic activity of the proteasome and are inducible by inflammatory stimuli such as interferon-γ (IFN-γ). This inducible activity places them in central roles in cancer, autoimmunity, and inflammation. While accelerated proteasomal degradation is an important tumorigenic mechanism deployed by several cancers, there is some ambiguity regarding the role of immunoproteasome induction in neoplastic transformation. Understanding the mechanistic and functional relevance of the immunoproteasome provides essential insights into developing targeted therapies, including overcoming resistance to standard proteasome inhibition and immunomodulation of the tumor microenvironment. In this review, we discuss the roles of the immunoproteasome in different cancers.
... Several studies that used non-immune cells have shown the involvement of the immunoproteasome in cellular senescence, aging, and longevity (32)(33)(34). Regarding cellular senescence, it has been reported that the TCR-mediated induction of the proteasome is impaired in senescence-associated PD-1 + CD44 high CD4 + T cells, and vice versa, the senescence phenotype is induced by proteasome inhibition (35). Although CD4 + T cells express both the constitutive proteasome and the immunoproteasome, these results indicate the involvement of proteasomes, including the immunoproteasome, in the senescence of T cells. ...
The thymus provides a microenvironment that supports the generation and selection of T cells. Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are essential components of the thymic microenvironment and present MHC-associated self-antigens to developing thymocytes for the generation of immunocompetent and self-tolerant T cells. Proteasomes are multicomponent protease complexes that degrade ubiquitinated proteins and produce peptides that are destined to be associated with MHC class I molecules. cTECs specifically express thymoproteasomes that are essential for optimal positive selection of CD8⁺ T cells, whereas mTECs, which contribute to the establishment of self-tolerance in T cells, express immunoproteasomes. Immunoproteasomes are also detectable in dendritic cells and developing thymocytes, additionally contributing to T cell development in the thymus. In this review, we summarize the functions of proteasomes expressed in the thymus, focusing on recent findings pertaining to the functions of the thymoproteasomes and the immunoproteasomes.
... Decline of proteasomal activity confirmed in various aged tissues and senescent cells (Conconi et al., 1996;Chondrogianni et al., 2003) might be related to reduction of catalytic subunit expression (Baraibar and Friguet, 2012) and proper proteasomal assembly (Tonoki et al., 2009), increased production of mutant Ubiquitin-B+1 (Ubb+1) (Fischer et al., 2009), posttranslational modification at various subunits (Tomita et al., 2018), or inhibition by protein aggregates and cross-linked proteins (Friguet and Szweda, 1997;Sitte et al., 2000b). Moreover, increased portion of i20S accumulates in tissues with ageing (Gavilán et al., 2009). ...
The field of ageing research has been rapidly advancing in recent decades and it had provided insight into the complexity of ageing phenomenon. However, as the organism-environment interaction appears to significantly affect the organismal pace of ageing, the systematic approach for gerontogenic risk assessment of environmental factors has yet to be established. This puts demand on development of effective biomarker of ageing, as a relevant tool to quantify effects of gerontogenic exposures, contingent on multidisciplinary research approach.
Here we review the current knowledge regarding the main endogenous gerontogenic pathways involved in acceleration of ageing through environmental exposures. These include inflammatory and oxidative stress-triggered processes, dysregulation of maintenance of cellular anabolism and catabolism and loss of protein homeostasis. The most effective biomarkers showing specificity and relevancy to ageing phenotypes are summarized, as well. The crucial part of this review was dedicated to the comprehensive overview of environmental gerontogens including various types of radiation, certain types of pesticides, heavy metals, drugs and addictive substances, unhealthy dietary patterns, and sedentary life as well as psychosocial stress. The reported effects in vitro and in vivo of both recognized and potential gerontogens are described with respect to the up-to-date knowledge in geroscience. Finally, hormetic and ageing decelerating effects of environmental factors are briefly discussed, as well.
... Consistently, impaired chymotrypsin-like activity was observed in aged tissues [218,219], correlated with increased HNE-modification of the β5i subunit detected in leukocyte-enriched fractions of human blood appearing around the age of 40 years [218]. Ageing has also been shown to be accompanied by an increase of inducible subunits in certain tissues, mostly due to progression of low-grade chronic inflammation [222]. ...
Proteasomal degradation provides the crucial machinery for maintaining cellular proteostasis. The biological origins of modulation or impairment of the function of proteasomal complexes may include changes in gene expression of their subunits, ubiquitin mutation, or indirect mechanisms arising from the overall impairment of proteostasis. However, changes in the physico-chemical characteristics of the cellular environment might also meaningfully contribute to altered performance. This review summarizes the effects of physicochemical factors in the cell, such as pH, temperature fluctuations, and reactions with the products of oxidative metabolism, on the function of the proteasome. Furthermore, evidence of the direct interaction of proteasomal complexes with protein aggregates is compared against the knowledge obtained from immobilization biotechnologies. In this regard, factors such as the structures of the natural polymeric scaffolds in the cells, their content of reactive groups or the sequestration of metal ions, and processes at the interface, are discussed here with regard to their influences on proteasomal function.
... Additional support for poor or only basal UPS activation in the first hours after I/R is provided by the lack of increase in the transcription of proteasome catalytic subunit by 12 h I/R, in spite of the increase in polyUb proteins at this time. The amount of polyUb proteins continued increasing over the next 48 h, but it seems unable to elicit increases in Psmβ5 transcript levels whose activity is considered mandatory for an active proteasome [63,64] and even with the transcription of proteasome subunits β1 and β2 promoted the UPS activity did seem to be activated by 48 h after I/R. ...
Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1 h of tMCAO and treated with celecoxib or vehicle 1 and 24 h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48 h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48 h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.
Graphical abstract
... Indeed, it has been shown in diverse mammalian tissues and cells that proteasome expression is reduced in older cells or individuals resulting in diminished proteasome function (reviewed in [384]). In contrast, expression of the immunoproteasomal subunits has been shown to be elevated in aging mammalian tissues such as brain [385], muscle [386], and the lung [387]. Moreover, elevated expression of immunoproteasome in fibroblast was positively correlated with organismal aging across different species [388]. ...
The proteasome is involved in the regulation of all cellular pathways and consequently plays a central role in the control of cellular homeostasis. Together with its regulators, it is at the frontline, both as an actor and as a target, in human health and when homeostasis is disturbed in disease. In this review, we aim to provide an overview of the many levels at which the functions of the proteasome and its regulators can be regulated to cope with cellular needs or are altered in pathological conditions.
... The IP is significantly up-regulated in glia and neurons, in both patients and experimental models of HD (121,139), AD (112)(113)(114)(115)(116)(117), PD (119, 120), MS (41,(98)(99)(100)(101)(102)(103)(108)(109)(110), ALS (134,140), neurotrauma (129), ischemic stroke (104,124,125), and epilepsy (38,100,126,127). In the context of PD, the induction of IP within glia and DA neurons was recently related to alphasynuclein degradation and subsequent generation of self-Ag peptides for T-cell presentation by MHC-I (79,119,132). ...
The wealth of recent evidence about a bi-directional communication between nerve- and immune- cells revolutionized the traditional concept about the brain as an “immune-privileged” organ while opening novel avenues in the pathophysiology of CNS disorders. In fact, altered communication between the immune and nervous system is emerging as a common hallmark in neuro-developmental, neurodegenerative, and neuro-immunological diseases. At molecular level, the ubiquitin proteasome machinery operates as a sentinel at the crossroad between the immune system and brain. In fact, the standard proteasome and its alternative/inducible counterpart, the immunoproteasome, operate dynamically and coordinately in both nerve- and immune- cells to modulate neurotransmission, oxidative/inflammatory stress response, and immunity. When dysregulations of the proteasome system occur, altered amounts of standard- vs. immune-proteasome subtypes translate into altered communication between neurons, glia, and immune cells. This contributes to neuro-inflammatory pathology in a variety of neurological disorders encompassing Parkinson's, Alzheimer's, and Huntingtin's diseases, brain trauma, epilepsy, and Multiple Sclerosis. In the present review, we analyze those proteasome-dependent molecular interactions which sustain communication between neurons, glia, and brain circulating T-lymphocytes both in baseline and pathological conditions. The evidence here discussed converges in that upregulation of immunoproteasome to the detriment of the standard proteasome, is commonly implicated in the inflammatory- and immune- biology of neurodegeneration. These concepts may foster additional studies investigating the role of immunoproteasome as a potential target in neurodegenerative and neuro-immunological disorders.
... Subcellular fractionation. The nuclear and the cytosolic fractions were obtained from the 400 μL homogenate by sequential centrifugation steps as previously described 27 . The homogenized sample was first cleaned by centrifugation at 500 g for 10 min, and the supernatant recentrifuged at 600 g for 10 min. ...
Proteostasis alteration and neuroinflammation are typical features of normal aging. We have previously shown that neuroinflammation alters cellular proteostasis through immunoproteasome induction, leading to a transient decrease of proteasome activity. Here, we further investigated the role of acute lipopolysaccharide (LPS)-induced hippocampal neuroinflammation in cellular proteostasis. In particular, we focused on macroautophagy (hereinafter called autophagy) and endoplasmic reticulum-associated protein degradation (ERAD). We demonstrate that LPS injection induced autophagy activation that was dependent, at least in part, on glycogen synthase kinase (GSK)-3β activity but independent of mammalian target of rapamycin (mTOR) inhibition. Neuroinflammation also produced endoplasmic reticulum (ER) stress leading to canonical unfolded protein response (UPR) activation with a rapid activating transcription factor (ATF) 6α attenuation that resulted in a time-dependent down-regulation of ERAD markers. In this regard, the time-dependent accumulation of unspliced X-box binding protein (XBP) 1, likely because of decreased inositol-requiring enzyme (IRE) 1α-mediated splicing activity, might underlie in vivo ATF6α attenuation. Importantly, lactacystin-induced activation of ERAD was abolished in both the acute neuroinflammation model and in aged rats. Therefore, we provide a cellular pathway through which neuroinflammation might sensitize cells to neurodegeneration under stress situations, being relevant in normal aging and other disorders where neuroinflammation is a characteristic feature.
... Binding of the 11S-regulator significantly enhances proteolysis by gate-opening and the selectivity of i20S towards oxidized substrates provides enhanced capacity during phases of oxidative stress. Also, in long-lived species (rodents and primates) a higher basal expression of i20S was shown compared to short-lived ones [74], while expression of the immunoproteasome as well increases with age (shown in rats [75]) and its induction via IFN-γ is reduced [76]. Since aging is also accompanied by both enhanced protein oxidation and thus, also an intracellular accumulation of oxidatively damaged proteins, expression of i20S may be a counteracting cellular antioxidative response in order to preserve proteostasis. ...
The production of reactive species is an inevitable by-product of metabolism and thus, life itself. Since reactive species are able to damage cellular structures, especially proteins, as the most abundant macromolecule of mammalian cells, systems are necessary which regulate and preserve a functional cellular protein pool, in a process termed “proteostasis”. Not only the mammalian protein pool is subject of a constant turnover, organelles are also degraded and rebuild. The most important systems for these removal processes are the “ubiquitin-proteasomal system” (UPS), the central proteolytic machinery of mammalian cells, mainly responsible for proteostasis, as well as the “autophagy-lysosomal system”, which mediates the turnover of organelles and large aggregates.
Many age-related pathologies and the aging process itself are accompanied by a dysregulation of UPS, autophagy and the cross-talk between both systems. This review will describe the sources and effects of oxidative stress, preservation of cellular protein- and organelle-homeostasis and the effects of aging on proteostasis in mammalian cells.
... 20S core proteasome is formed by four seven-member rings; the outer rings contain seven different α-subunits, and the inner rings contain seven different β-subunits. [3] Three of the β-subunits (β1, β2, and β5) are known to possess protease activity. In addition to seven constitutively synthesized β-subunits, vertebrates have three inducible β-immunosubunits: low molecular weight proteins LMP2 (β1i) and LMP7 (β5i) and multicatalytic endopeptidase-like complex 1 MECL-1 (β2i), which could replace constitutive β1, β2, and β5 subunits and form immunoproteasomethe complex with alternative specificity of protein cleavage. ...
Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2′-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.
... Long-lived species showed higher basal Immunoproteasome levels and activity compared to short-lived animals (Pickering et al., 2015). This finding is supported by studies showing that although the Immunoproteasome expression increases with age (Husom et al., 2004, Ferrington et al., 2005, Gavilán et al., 2009, its inducibility by γ-interferon is diminished (Stratford et al., 2006). This is accompanied by a concurrent age-related accumulation of oxidized proteins within senescent cells. ...
The Immunoproteasome has traditionally been viewed primarily for its role in peptide production for antigen presentation by the major histocompatibility complex, which is critical for immunity. However, recent research has shown that the Immunoproteasome is also very important for the clearance of oxidatively damaged proteins in homeostasis, and especially during stress and disease. The importance of the Immunoproteasome in protein degradation has become more evident as diseases characterized by protein aggregates have also been linked to deficiencies of the Immunoproteasome. Additionally, there are now diseases defined by mutations or polymorphisms within Immunoproteasome-specific subunit genes, further suggesting its crucial role in cytokine signaling and protein homeostasis (or "proteostasis"). The purpose of this review is to highlight our growing understanding of the importance of the Immunoproteasome in the management of protein quality control, and the detrimental impact of its dysregulation during disease and aging.
... This function is essential for many cellular processes such as transcription, cell cycle progression, differentiation, apoptosis, and the control of stress and immune responses [1,2]. The eukaryotic 26S proteasome consists of the multicatalytic 20S core proteasome associated with regulatory complexes such as PA700/19S or PA28/11S [3]. The 20S core proteasome is formed by a and b subunits. ...
The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2'-O-methyl modifications into nuclease-sensitive sites. This led to 90−95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases.
... In this comparison we included only Old World The cell lines available to us vary somewhat in both passage number and in the age of the donor animal (summarized in Supplemental Table 1). Several groups have reported elevations of immunoproteasome with organismal aging in muscle, retina, and hippocampus tissue derived from mice and rats (29,(38)(39)(40) as well as with continued passage of primary cells in culture (41). To reduce artifacts of this kind, we used cell lines at the lowest practical passage number, with only 1 cell line used at a passage above 20. ...
There is large variation in lifespan among different species, and there is evidence that modulation of proteasome function may contribute to longevity determination. Comparative biology provides a powerful tool for identifying genes and pathways that control the rate of aging. Here, we evaluated skin-derived fibroblasts and demonstrate that among primate species, longevity correlated with an elevation in proteasomal activity as well as immunoproteasome expression at both the mRNA and protein levels. Immunoproteasome enhancement occurred with a concurrent increase in other elements involved in MHC class I antigen presentation, including β-2 microglobulin, (TAP1), and TAP2. Fibroblasts from long-lived primates also appeared more responsive to IFN-γ than cells from short-lived primate species, and this increase in IFN-γ responsiveness correlated with elevated expression of the IFN-γ receptor protein IFNGR2. Elevation of immunoproteasome and proteasome activity was also observed in the livers of long-lived Snell dwarf mice and in mice exposed to drugs that have been shown to extend lifespan, including rapamycin, 17-α-estradiol, and nordihydroguaiaretic acid. This work suggests that augmented immunoproteasome function may contribute to lifespan differences in mice and among primate species.
... In higher eukaryotes, there are three 11S isoforms, called PA28␣, , and ␥ (or REG␣, , and ␥) (Rechsteiner and Hill, 2005). Despite the role of immunoproteasomes in antigen presentation, recent data implicate this proteasome type in adaptation to oxidative stress by selectively degrading oxidized proteins (Pickering et al., 2010), in aging possibly as a response to chronic inflammation (Gavilan et al., 2009;Mishto et al., 2006) and in longevity (Rodriguez et al., 2012). ...
Aging is a natural process accompanied by a progressive accumulation of damage in all constituent macromolecules (nucleic acids, lipids and proteins). Accumulation of damage in proteins leads to failure of proteostasis (or vice versa) due to increased levels of unfolded, misfolded or aggregated proteins and, in turn, to aging and/or age-related diseases. The major cellular proteolytic machineries, namely the proteasome and the lysosome, have been shown to dysfunction during aging and age-related diseases. Regarding proteasome it is well established that it can be activated either through genetic manipulation or through treatments with natural or chemical compounds that eventually result to extension of lifespan or deceleration of the progression of age-related diseases. This review article focuses on proteasome activation studies in several species and cellular models and their effects on aging and longevity. Moreover, it summarizes findings regarding proteasome activation in the major age-related diseases as well as in progeroid syndromes.
Copyright © 2014. Published by Elsevier B.V.
... This procedure allows the isolation of total RNA, DNA and protein fractions from a single sample. Reverse transcription (RT) was performed using random hexamers primers, 3 μg of total RNA as a template and the High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer's recommendations as previously described [19]. ...
Grape pomace, a winemaking industry by-product, is a rich source of bioactive dietary compounds. Using proteases we have developed an enzymatic process for obtaining a water-soluble extract (GP-EE) that contains biomolecules such as peptides, carbohydrates, lipids and polyphenols in soluble form. Of especial interest is its high polyphenol content (12 %), of which 77 % are flavonoids and 33 % are phenolic acids. The present study evaluates in vitro the potential anti-inflammatory effect of GP-EE by monitoring the expression of inflammatory molecules on N13 microglia cells stimulated with lipopolysaccharide (LPS). GP-EE decreases the mRNA levels of the inflammatory molecules studied. The molecules under study were as follows: inducible nitric oxide synthase (iNOS), tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), the ionized calcium binding adaptor molecule-1(Iba-1) and the Toll like receptor-4 (TLR-4), as well as the iNOS protein level in LPS-stimulated microglia. Our findings suggest that, as a result of its ability to regulate excessive microglial activation, GP-EE possesses anti-inflammatory properties. Therefore, acting as a chemopreventive agent, it may be of therapeutic interest in neurodegenerative diseases involving neuroinflammation. We can, therefore, propose GP-EE as a useful natural extract and one that would be beneficial to apply in the field of functional foods.
... Subcellular fractionation of lung cells to determine the expression of nuclear and cytoplasmic NF-kB p65 and cytoplasmic IkBa from lung tissues was performed by sequential density centrifugation. 43 The lungs were snap frozen in liquid nitrogen 20 hours after CLP or sham operation and homogenized in ice-cold PBS (pH 7.4), supplemented with protease inhibitor cocktail (Roche Diagnostics). All steps were conducted at 4 C. ...
Acute lung injury (ALI) secondary to sepsis is a complex syndrome associated with high morbidity and mortality. We report that aminoprocalcitonin (NPCT), an endogenous peptide derived from the prohormone procalcitonin, plays a critical role in the development of ALI during severe sepsis and is a suggested risk factor for sepsis morbidity and mortality. Lethal sepsis was induced in rats by cecal ligation and puncture (CLP). Two hours after CLP, an i.p. injection of 200 μg/kg of anti-rat NPCT antibody was followed by continuous infusion of anti-NPCT (16 μg per hour) via a minipump for 18 hours. Samples were harvested 20 hours after CLP. High expressions of the CALCA gene, procalcitonin, and NPCT were detected in the lung tissue of rats with severe sepsis. Immunoneutralization of NPCT decreased pulmonary levels of CALCA, procalcitonin, and NPCT; reduced lung inflammation and injury, neutrophil infiltration, and bacterial invasion; and improved survival in sepsis. Anti-NPCT treatment also suppressed sepsis-induced inflammatory cytokine expression, cytoplasmic degradation of the inhibitor of NF-κB, IκBα, and nuclear NF-κB translocation in lung tissues. Therapeutic benefits of anti-NPCT were also associated with increased pulmonary levels of the anti-inflammatory cytokine IL-10. These data support a pathogenic role for NPCT in sepsis and suggest NPCT as a potential new target for clinical prevention and treatment of ALI in severe sepsis.
... However, in this study by Zheng et al., an equal expression of s-and i-proteasome subunits has been described in control mouse brain, contrasting with other studies on rodents and humans which reported a faint expression of i-proteasome in young/adult brains [21,24,25]. Furthermore, Zheng et al. reported no differences in the i-proteasome expression by comparing young and old control mouse brains, which is in contrast to studies on other mammals such as rats [21,26] and humans [27], but in agreement with a study conducted on nonhuman primates [28]. ...
The ubiquitin-proteasome system is the major intracellular molecular machinery for protein degradation and maintenance of protein homeostasis in most human cells. As ubiquitin-proteasome system plays a critical role in the regulation of the immune system, it might also influence the development and progression of multiple sclerosis (MS). Both ex vivo analyses and animal models suggest that activity and composition of ubiquitin-proteasome system are altered in MS. Proteasome isoforms endowed of immunosubunits may affect the functionality of different cell types such as CD8(+) and CD4(+) T cells and B cells as well as neurons during MS development. Furthermore, the study of proteasome-related biomarkers, such as proteasome antibodies and circulating proteasomes, may represent a field of interest in MS. Proteasome inhibitors are already used as treatment for cancer and the recent development of inhibitors selective for immunoproteasome subunits may soon represent novel therapeutic approaches to the different forms of MS. In this review we describe the current knowledge on the potential role of proteasomes in MS and discuss the pro et contra of possible therapies for MS targeting proteasome isoforms.
... Therefore, the age-dependent increment in intermediate-type and immuno-proteasomes might belong to those cellular mechanisms aimed to balance the consequences of the impaired antioxidative capacity in aging liver (Hussong et al. 2010;Suh et al. 2004;Stio et al. 1994). Since an age-related increase in immuno-subunits was also observed in other tissues, e.g., in skeletal muscle (Husom et al. 2004) and brain (Gavilan et al. 2009;Mishto et al. 2006), we suggest that part of the mechanism to keep up proteostasis during aging is the formation of immunoproteasomes (Mishto et al. 2003). ...
Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only β5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.
... Similarly, subunit b1i was detected in human hippocampus in non-demented elderly, but not in young controls [8] . An agerelated increase in immunoproteasome content was also described for rat hippocampus [35] and muscle [7]. Our Coomassie-stained 2D-gels revealed that standard b-subunits were still predominant, however, small amounts of the immuno-subunits b1i, b2i and b5i appeared in 20S proteasomes from aged cerebellum. ...
Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young) and 24 month old (aged) rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process.
... I-proteasome is also present, albeit in low abundance under basal conditions, in cells outside the immune system, including neurons of the retina and brain, skeletal muscle and epithelial cells of the retina [5][6][7]. When these cells are exposed to various stressors, such as inflammatory cytokines, disease, or oxidative stress, i-proteasome is significantly upregulated [8][9][10][11]. In addition to its rapid induction, assembly of the nascent i-proteasome core particle is four times faster than the standard core and conversely, i-proteasome's half-life is substantially shorter [12]. This highly dynamic adjustment in i-proteasome content permits its rapid response to environmental challenges. ...
Immunoproteasome is a protease abundant in immune cells and also present, albeit at lower concentrations, in cells outside the immune system. Recent evidence supports a novel role for the immunoproteasome in the cellular stress response potentially through regulation of NFkappaB signaling, which is the primary response to multiple stressors. The current study tests whether the Classical or Alternative Pathways are regulated by immunoproteasome following chronic TNFalpha exposure in cultured retinal pigment epithelial cells isolated from wild-type mice and mice deficient in one (LMP2, L2) or two (LMP7 and MECL-1, L7M1) immunoproteasome subunits. Assays were performed to assess the expression of NFkappaB responsive genes, the content and activity of NFkappaB transcription factors (p65, p50, p52, cRel, RelB), and expression and content of regulatory proteins (IkappaBalpha, A20, RPS3). Major findings include distinct differences in expression of NFkappaB responsive genes in both KO cells. The mechanism responsible for the altered gene expression could not be established for L7M1 since no major differences in NFkappaB transcription factor content or activation were observed. However, L2 cells exhibited substantially higher content and diminished activation of NFkappaB transcription factors associated with the Alternative Pathway and delayed termination of the Classical Pathway. These results provide strong experimental evidence supporting a role for immunoproteasome in modulating NFkappaB signaling.
... In agreement with this, a recent study suggested that the immunoproteasome may remove oxidized proteins that accumulate after IFN-induced oxidative stress [4]. A previous study has also reported that the ratio of the immunoproteasome is increased in the aged brain [7]. It is further reported that immunoproteasome expression is Abbreviations used: AGE, advanced glycation-end product; DTT, dithiothreitol; FBS, fetal bovine serum; Fru, D-fructose; G, glyoxal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Glc, D-glucose; IFN, interferon; Jak, Janus kinase; MAPK, mitogen-activated protein kinase; MG, methylglyoxal; NF-κB, nuclear factor κB; RAGE, receptor for AGEs; Rib, D-ribose; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription. 1 To whom correspondence should be addressed (email tilman.grune@uni-jena.de). ...
AGEs (advanced glycation-end products) accumulate during aging and several pathologies such as Alzheimer's disease and diabetes. These protein products are known to inhibit proteolytic pathways. Moreover, AGEs are known to be involved in the activation of immune responses. In the present study we demonstrate that AGEs induce the expression of immunoproteasomal subunits. To elucidate a molecular basis underlying the observed effects we were able to demonstrate an activation of the Jak2 (Janus kinase 2)/STAT1 (signal transducer and activator of transcription 1) pathway. Inhibition of Jak2 by AG-490 and STAT1 by specific siRNA (small interfering RNA) abolished AGEinduced expression of immunoproteasomal subunits. Furthermore, silencing of RAGE (receptor for AGEs) revealed that AGE-induced up-regulation of the immunoproteasome is mediated by a RAGE signalling process. Thus we have described for the first time that the signalling pathway of Jak2 and STAT1 activated by AGEs via RAGE is involved in the induction of the immunoproteasome.
Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes were revealed, as well as an increased expression of their individual forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in the cerebral cortex samples from C57BL/6 mice at the age of 60, 190, 380, and 720 days. In addition, the content of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis under native conditions were characterized. Compared with samples from young animals, in the cerebral cortex of animals aged 720 days the following changes in the expression patterns of proteasome genes were revealed: a decrease in the expression of PSMB5 gene encoding the constitutive proteasome subunit β5; activation of genes encoding immune subunits β5i and β1i. In clarified tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, decreased chymotrypsin-like activity and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity were also revealed. Analysis of the activity of native complexes in the tissues of old animals revealed decreased chymotrypsin-like activity of both 26S and 20S proteasomes containing the β5i subunit. Based on the data obtained, it can be assumed that changes in the pool of non-constitutive proteasomes reflect aging-associated adaptive processes in mouse brain.
In the nervous system, proteasomes are important for proteolysis and cellular homeostasis of neurons and glial cells and for brain health. Proteasome function declines with age in many tissues, including the nervous system, and this decline affects many of the nervous system processes important to brain health and may be related to age‐related cognitive decline. Therefore, we analyzed the factors that contribute to this decline in function using the brain of mice from different months of life. Peptidase activity of proteasomes in crude extracts decreased with aging, while ubiquitinated proteins increased with aging. Additionally, there was a tendency for the number of subunits that form proteasomes to decrease slightly with age. On the other hand, ump1, which is required for proteasome formation, accumulated with age. Therefore, analysis of proteasome dynamics in each month revealed that proteasome formation decreased with aging. This study suggests that with aging, not only 20S proteasome function but also 26 proteasome function decreases, the decline in proteasome function is due to the lack of proteasome formation, the PA28‐20S‐PA700 complex, which is involved in immunity, increases in the brain, and one factor in this lack of proteasome formation is that the proteins called UMP1.
Nature is an important source of bioactive compounds and has continuously made a large contribution to the discovery of new drug leads. Particularly, plant-derived compounds have long been identified as highly interesting in the field of ageing research and senescence. Many plants contain bioactive compounds that have the potential to influence cellular processes and provide health benefits. Among them, Piper alkaloids have emerged as interesting candidates in the context of age-related diseases and particularly senescence. These compounds have been shown to display a variety of features, including antioxidant, anti-inflammatory, neuroprotective, and other bioactive properties that may help counteracting the effects of cellular ageing processes. In the review, we will put the emphasis on piperlongumine and other related derivatives, which belong to the Piper alkaloids, and whose senomodulating potential has emerged during the last several years. We will also provide a survey on their potential in therapeutic perspectives of age-related diseases.
The proteasome is a large multi-subunit protease responsible for the degradation and removal of oxidized, misfolded, and polyubiquitinated proteins. The proteasome plays critical roles in nervous system processes. This includes maintenance of cellular homeostasis in neurons. It also includes roles in long-term potentiation via modulation of CREB signaling. The proteasome also possesses roles in promoting dendritic spine growth driven by proteasome localization to the dendritic spines in an NMDA/CaMKIIα dependent manner. Proteasome inhibition experiments in varied organisms has been shown to impact memory, consolidation, recollection and extinction. The proteasome has been further shown to impact circadian rhythm through modulation of a range of ‘clock’ genes, and glial function. Proteasome function is impaired as a consequence both of aging and neurodegenerative diseases. Many studies have demonstrated an impairment in 26S proteasome function in the brain and other tissues as a consequence of age, driven by a disassembly of 26S proteasome in favor of 20S proteasome. Some studies also show proteasome augmentation to correct age-related deficits. In amyotrophic lateral sclerosis Alzheimer’s, Parkinson’s and Huntington’s disease proteasome function is impaired through distinct mechanisms with impacts on disease susceptibility and progression. Age and neurodegenerative-related deficits in the function of the constitutive proteasome are often also accompanied by an increase in an alternative form of proteasome called the immunoproteasome. This article discusses the critical role of the proteasome in the nervous system. We then describe how proteasome dysfunction contributes to brain aging and neurodegenerative disease.
The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.
The key to a healthy mammalian cell lies in properly functioning proteolytic machineries called proteasomes. The proteasomes are multisubunit complexes that catalyze the degradation of unwanted proteins and also control half-lives of key cellular regulatory factors. Aberrant proteasome activity is often associated with human diseases such as cancer and neurodegeneration, and so an in-depth understanding of how it is regulated has implications for potential disease interventions. Transcriptional regulation of the proteasome can dictate its abundance and also influence its function, assembly, and location. This ensures proper proteasomal activity in response to developmental cues and to physiological conditions such as starvation and oxidative stress. In this review, we highlight and discuss the roles of the transcription factors that are involved in the regulation of the mammalian proteasome.
Aging is a biological process accompanied by gradual increase of damage in all cellular macromolecules, i.e., nucleic acids, lipids, and proteins. When the proteostasis network (chaperones and proteolytic systems) cannot reverse the damage load due to its excess as compared to cellular repair/regeneration capacity, failure of homeostasis is established. This failure is a major hallmark of aging and/or aggregation-related diseases. Dysfunction of the major cellular proteolytic machineries, namely the proteasome and the lysosome, has been reported during the progression of aging and aggregation-prone diseases. Therefore, activation of these pathways is considered as a possible preventive or therapeutic approach against the progression of these processes. This chapter focuses on UPS activation studies in cellular and organismal models and the effects of such activation on aging, longevity and disease prevention or reversal.
The 20S proteasome is a multicatalytic proteinase catalysing the degradation of the majority of intracellular proteins. Thereby it is involved in almost all basic cellular processes, which is facilitated by its association with various regulator complexes so that it appears in different disguises like 26S proteasome, hybrid-proteasome and others. The 20S proteasome has a cylindrical structure built up by four stacked rings composed of α- and β-subunits. Since the three active site-containing β-subunits can all or in part be replaced by immuno-subunits, three main subpopulations exist, namely standard-, immuno- and intermediate-proteasomes. Due to posttranslational modifications or/and genetic variations all α- and β-subunits occur in multiple iso- or proteoforms. This leads to the fact that each of the three subpopulations is composed of a variety of 20S proteasome subtypes. This review summarizes the knowledge of proteasome subtypes in mammalian cells and tissues and their possible biological and medical relevancy.
Formation of the central nervous system in ontogeny and its functioning in adult mammals are controlled by the universal ubiquitin-proteasome proteolytic system. The aim of the present work was to study the dynamics of immune proteasome expression as compared with the dynamics of chymotrypsin-like and caspase-like activities of the proteasome (ChLA and CLA) and expression of the transcription factor Zif268 in selected brain structures (cortex, hippocampus, and brainstem) in embryonic (days E19 and E21 of embryonic development) and early postnatal (postnatal days P1, P3, P4, P5, P7, and P15) development in rats. ChLA and CLA were determined in clarified homogenates of rat brain structures using commercial fluorogenic oligopeptide substrates Suc-LLVY-AMC and Z-LLG-AMC, respectively. Levels of constitutive (β1 and β5) and immune (LMP7 and LMP2) proteasome subunits, as well as those of proteasome activators PA700 and PA28 and the transcription factor Zif268, were evaluated by Western blotting. Increased expression of the immune subunit LMP7 in the cerebral cortex and hippocampus was observed during the period of active formation of the biochemical mediator structure of neurons (P7-P15). Increased ChLA and CLA were registered in the cortex during this period. The content of the immune subunit LMP2 was significantly elevated in all brain structures between P7 and P15. The content of the constitutive proteolytic subunit β1 in all structures was lower on P4 than on P1 and increased to the same level as on P1 by P15. However, the expression level of the constitutive proteolytic β5 subunit of the proteasome in the cortex, hippocampus, and brainstem increased starting from E21 and reached maximal values at P3, P5 and P1, respectively, with a sharp decline in all structures by P7. Expression of the immune subunit LMP2 and the constitutive subunit β1 in all structures increased sharply around P15 concomitantly to the increase in the content of the immune subunit LMP7. Moreover, a positive correlation of increased expression of the activator PA28 and the constitutive β5 subunit in the hippocampus during P3-P5 and in the brain stem during P1-P5 has been demonstrated. Elevation of proteasome CLA and expression levels of immune proteasome subunits LMP2 and LMP7 during days P7-P15 of postnatal development in the brain regions investigated were related to the expression of the transcription factor Zif268. Immune proteasomes probably play an important role in the regulation of key biochemical processes during early ontogenesis of the central nervous system and are necessary for the emergence and maintenance of synaptic plasticity in rat brain structures analyzed.
1. Abstract 2. Introduction 3. Structure, assembly, and biochemical properties 4. Expression and regulation 5. Immune functions 6. Anti-oxidative stress function 7. Immunoproteasome and diseases 7.1. Cancer 7.2. Autoimmune diseases 7.3. Neurodegenerative diseases 7.4. Cardiovascular diseases 8. Conclusion 9. Acknowledgements 10. References
Previous data has shown that prior history of immune challenge may affect central and behavioural responses to subsequent immune challenge, either leading to exaggerated responses via priming mechanisms or lessened responses via endotoxin tolerance. In this set of experiments we have examined how previously lipopolysaccharide (LPS)-induced sepsis shapes the response to subsequent treatment with lower dose LPS. After treatment with LPS (5 mg/kg) or saline mice were allowed to recover for 3-4 months before being challenged with a lower dose of LPS (100 μg/kg) for assessment of sickness behaviours. Performance on the open field test and the tail suspension test was assessed, and no evidence was found that prior sepsis altered sickness or depressive-like behaviour following LPS treatment. We then examined the responsiveness of the circadian system of mice to LPS. We found that in control animals, LPS induced a significant phase delay of the behavioural rhythm and that this was not the case in post-septic animals (4-6 weeks after sepsis), indicating that prior sepsis alters the responsivity of the circadian system to subsequent immune challenge. We further assessed the induction of the immediate early genes c-Fos and EGR1 in the hippocampus and the suprachiasmatic nucleus (SCN; the master circadian pacemaker) by LPS in control or post-septic animals, and found that post-septic animals show elevated expression in the hippocampus but not the SCN. These data suggest that previous sepsis has some effect on behavioural and molecular responses to subsequent immune challenge in mice.
The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive
role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates
were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced.
These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the
concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.
The multicatalytic and multisubunit proteasomal complexes have been implicated in the processing of antigens to peptides presented by class I major histocompatibility complex molecules. Two structural complexes of this proteinase, 20 S and 26 S proteasomes, have been isolated from cells. By analyzing in vivo assembly of the proteasomal complexes we show that the 20 S proteasomal complexes are irreversibly assembled via 15 S assembly intermediates containing unprocessed beta-type subunits. The 20 S proteasomes further associate reversibly with proteasome activators PA28 or pre-existing ATPase complexes to form 26 S proteasomal complexes. Our findings that not all of the 20 S proteasomal complexes are assembled into 26 S proteasomal complexes within cells and that all of PA28 and ATPase complexes are associated with 20 S proteasomes strongly suggest that all proteasomal complexes coexist within cells. We further demonstrate that 26 S proteasomal complexes are predominantly present in the cytoplasm and a significant portion of the 20 S proteasomal complexes is associated with the endoplasmic reticulum membrane. Taken together, our findings suggest that depending upon their associated regulatory components, 26 S and 20 S-PA28 proteasomal complexes serve different housekeeping functions within the cells, while they degrade antigens in a cooperative manner in antigen processing.
The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.
The presentation of intracellular proteins to the immune system requires their degradation to small peptides that then become associated with major histocompatibility complex (MHC) class I molecules. The generation of these peptides may involve the 20S or 26S proteasome particles, which contain multiple proteolytic activities including distinct sites that preferentially cleave small peptides on the carboxyl side of hydrophobic, basic or acidic residues. Degradation of most cell proteins requires their conjugation to ubiquitin before hydrolysis by the 26S proteasome. This large complex contains the 20S proteasome as its proteolytic core. This ubiquitin-dependent proteolytic pathway is implicated in MHC class I presentation. gamma-Interferon (gamma-IFN), a stimulator of antigen presentation, induces a subclass of proteasomes that contain two MHC-encoded subunits, LMP2 and 7 (refs 5-10). Here we show that gamma-interferon alters the peptidase activities of the 20S and 26S proteasomes without affecting the rates of breakdown of proteins or of ubiquitinated proteins. By enhancing the expression of MHC genes, gamma-IFN increases the proteasomes' capacity to cleave small peptides after hydrophobic and basic residues but reduces cleavage after acidic residues. Moreover, proteasomes of mutants lacking LMP subunits show decreased rates of cleavage after hydrophobic and basic residues. Thus, gamma-IFN and expression of these MHC genes should favour the production by proteasomes of the types of peptides found on MHC class I molecules, which terminate almost exclusively with hydrophobic or basic residues.
Most antigenic peptides presented on major histocompatibility complex class I molecules are generated by proteasomes. Interferon-gamma, which stimulates antigen presentation, induces new proteasome beta-subunits LMP2 and LMP7, which replace the homologous beta-subunits Y (delta) and X (epsilon). As a result, the capacity of the proteasome to cleave model peptides increases after hydrophobic and basic residues and falls after acidic residues. To clarify the function of these subunits, we examined the effects of overexpressing subunits X (delta) and Y (epsilon). Transfection of the Y gene into HeLa cells stimulated the proteasomal cleavage after acidic residues without altering other peptidase activities. This effect was proportional to the amount of the Y subunits and opposite to the effect of its homolog, LMP2. Y appears to promote cleavages after acidic residues. Furthermore, in mutants lacking the LMP genes (in contrast to wild-type cells), interferon-gamma treatment increased the proteasome content of Y subunits and enhanced postacidic cleavages. Transfection with cDNA for the X subunit reduced hydrolysis after hydrophobic and basic residues, an effect opposite to transfection of LMP2 and LMP7. Surprisingly, transfection of X increased the amounts not only of X, but also of Y, while decreasing LMP2 content. Thus, the loss of the Y subunit upon interferon-gamma treatment or LMP2 transfection accounts for the suppression of postacidic cleavages, and the loss of X contributes to the increased hydrolysis after hydrophobic and basic residues. These adaptations should favor the production of the kinds of peptides that are presented on major histocompatibility complex class I molecules.
The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.
Knowledge about the sizes of peptides generated by proteasomes during protein degradation is essential to fully understand their degradative mechanisms and the subsequent steps in protein turnover and generation of major histocompatibility complex class I antigenic peptides. We demonstrate here that 26 S and activated 20 S proteasomes from rabbit muscle degrade denatured, nonubiquitinated proteins in a highly processive fashion but generate different patterns of peptides (despite their containing identical proteolytic sites). With both enzymes, products range in length from 3 to 22 residues, and their abundance decreases with increasing length according to a log-normal distribution. Less than 15% of the products are the length of class I presented peptides (8 or 9 residues), and two-thirds are too short to function in antigen presentation. Surprisingly, these mammalian proteasomes, which contain two "chymotryptic," two "tryptic," and two "post-acidic" active sites, generate peptides with a similar size distribution as do archaeal 20 S proteasomes, which have 14 identical sites. Furthermore, inactivation of the "tryptic" sites altered the peptides produced without significantly affecting their size distribution. Therefore, this distribution is not determined by the number, specificity, or arrangement of the active sites (as proposed by the "molecular ruler" model); instead, we propose that proteolysis continues until products are small enough to diffuse out of the proteasomes.
We have investigated the age-dependent modifications in the expression of eight different subunits of the gamma-aminobutyric acid, type A (GABA(A)) receptor (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2S, and gamma2L) and all four subunits of the alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1-4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 +/- 0.54) in the mRNA levels of the short version of the gamma2 subunit in parallel with a similar increase in the gamma2 subunit protein (aged/adult ratio, 2.90 +/- 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different alpha subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the alpha1 subunit mRNA (aged/adult ratio, 1.26 +/- 0.18), in parallel with a similar increase on the alpha1 protein (aged/adult ratio, 1. 27 +/- 0.12) and in the alpha1 incorporated to the assembled GABA(A) receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 +/- 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABA(A) receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rats.
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.
Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).
It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)
Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.
The idea that new memories undergo a time-dependent consolidation process after acquisition has received considerable experimental support. More controversial has been the demonstration that established memories, once recalled, become labile and sensitive to disruption, requiring "reconsolidation" to become permanent. By infusing antisense oligodeoxynucleotides into the hippocampus of rats, we show that consolidation and reconsolidation are doubly dissociable component processes of memory. Consolidation involves brain-derived neurotrophic factor (BDNF) but not the transcription factor Zif268, whereas reconsolidation recruits Zif268 but not BDNF. These findings confirm a requirement for BDNF specifically in memory consolidation and also resolve the role of Zif268 in brain plasticity, learning, and memory.
The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.
The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.
At advanced stages, Alzheimer's disease (AD) is characterized by an extensive neuronal loss. However, the early neurodegenerative deficiencies have not been yet identified. Here we report an extensive, selective and early neurodegeneration of the dendritic inhibitory interneurons (oriens-lacunosum moleculare, O-LM, and hilar perforant path-associated, HIPP, cells) in the hippocampus of a transgenic PS1xAPP AD model. At 6 months of age, from 22 different pre- and postsynaptic mRNA markers tested (including GABAergic, glutamatergic and cholinergic markers), only the expression of somatostatin (SOM) and NPY neuropeptides (O-LM and HIPP markers) displayed a significant decrease. Stereological cell counting demonstrated a profound diminution (50-60%) of SOM-immunopositive neurons, preceding the pyramidal cell loss in this AD model. SOM population co-expressing NPY was the most damaged cell subset. Furthermore, a linear correlation between SOM and/or NPY deficiency and Abeta content was also observed. Though the molecular mechanism of SOM neuronal loss remains to be determined, these findings might represent an early hippocampal neuropathology. Therefore, SOM and NPY neuropeptides could constitute important biomarkers to assess the efficacy of potential early AD treatments.
The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100(209-217) (ITDQVPSFV) and peptide tyrosinase369-377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2(336-344) (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.
Most forms of neuronal plasticity are associated with induction of the transcription factor Zif268 (Egr1/Krox24/NGF-IA). In a genome-wide scan, we obtained evidence for potential modulation of proteasome subunit and regulatory genes by Zif268 in neurons, a finding of significance considering emerging evidence that the proteasome modulates synaptic function. Bioinformatic analysis indicated that the candidate proteasome Zif268 target genes had a rich concentration of putative Zif268 binding sites immediately upstream of the transcriptional start sites. Regulation of the mRNAs encoding the psmb9 (Lmp2) and psme2 (PA28beta) proteasome subunits, along with the proteasome-regulatory kinase serum/glucocorticoid-regulated kinase (SGK) and the proteasome-associated antigen peptide transporter subunit 1 (Tap1), was confirmed after transfection of a neuronal cell line with Zif268. Conversely, these mRNAs were upregulated in cerebral cortex tissue from Zif268 knock-out mice relative to controls, confirming that Zif268 suppresses their expression in the CNS. Transfected Zif268 reduced the activity of psmb9, SGK, and Tap1 promoter-reporter constructs. Altered psmb9, SGK, and Tap1 mRNA levels were also observed in an in vivo model of neuronal plasticity involving Zif268 induction: the effect of haloperidol administration on striatal gene expression. Consistent with these effects on proteasome gene expression, increased Zif268 expression suppressed proteasome activity, whereas Zif268 knock-out mice exhibited elevated cortical proteasome activity. Our findings reveal that Zif268 regulates the expression of proteasome and related genes in neuronal cells and provide new evidence that altered expression of proteasome activity after Zif268 induction may be a key component of long-lasting CNS plasticity.
Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3′- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity.
Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.
The proteasome is a multisubunit protease complex with an apparent sedimentation coefficient of 20S. Two types of regulatory
complexes, named PA700 and PA28, bind to both ends of the cylindrical 20S proteasome to form the dumbbell-like and football-like
proteasomes, respectively. The former complex, named the 26S proteasome, is a eukaryotic ATP-dependent protease and appears
to be well organized as a large complex of 2 MDa, consisting of approximately 40 polypeptides, to facilitate rapid proteolysis.
It is assumed to be a protein “death machine”, destroying a variety of cellular proteins that have acquired a specific degradation
signal(s) such as a multiubiquitin chain. Recently data on in vivo substrates for the ubiquitin-proteasome pathway have been
accumulating rapidly, implying its involvement in many biologically important processes, such as cell-cycle regulation, signal
transduction, protein quality control, and the immune response. The newly-identified PA28 family proteins are inducible by
interferons, and may cooperate with the 26S proteasome or play additional roles. Since the proteasome is capable of catalyzing
breakdown of proteins not only irreversibly, but also rapidly and timely, it is thought to be a new regulatory system for
biological reactions in eukaryotes.
The non-essential mouse proteasome beta-type subunits LMP2 and LMP7 are thought to connect proteasomes to the MHC class I antigen processing pathway. Both subunits are synthesized as proproteins. We have studied the processing of both subunits, correlated with the maturation of 20 S proteasomes in mouse T cells. Our data show that proteasome assembly occurs via 13-16 S precursor complexes which possess a protein pattern distinct from that of 20 S proteasomes. Both LMP2 and LMP7 proproteins are processed within these preproteasome complexes and only their processed forms become part of active 20 S proteasomes. Our data show that the maturation and assembly of 20 S proteasomes via precursor particles is a translation-dependent gradual process, that processing of subunit proproteins takes place in these 13-16 S complexes and that subunit processing and proteasome formation occur together.
The T cell arm of the immune system of higher vertebrates is specific for antigenic peptides bound to cell surface major histocompatibility complex (MHC) molecules. These peptides are derived from two distinct pathways of antigen processing. The class I, or endogenous pathway, utilizes proteasomes and the ubiquitin system for protein degradation, with subsequent transport of the resulting peptides into the lumen of the endoplasmic reticulum by a specific peptide transporter, called TAP. The expression of distinct proteasome subsets is regulated by the cytokine gamma interferon (IFN-gamma). The class II, or exogenous pathway, utilizes the endosomal and lysosomal pathways for protein degradation, and a number of immune-specific accessory molecules including the class-II associated Invariant chain (Ii) and MHC-encoded HLA-DM (H2-DM in mouse) molecules.
Recent studies of the 20S proteasome from Thermoplasma acidophilum have uncovered some fundamental new properties of its catalytic mechanism. Unlike conventional proteases, 20S and 26S proteasomes degrade protein substrates in a highly processive fashion. They cleave a protein substrate to small peptides before attacking another substrate molecule. This processive behavior is an inherent feature of the 20S particle not requiring cofactors or ATP hydrolysis. Recently, we have described a proteasome-like particle, HslVU, in Escherichia coli. HslVU is a two-component ATP-dependent protease composed of the proteasome-related peptidase HslV (beta-subunit) and the ATPase HslU. In active HslVU complex, cleavage of small peptides and proteins requires the presence of ATP. EM analysis revealed that HslV and HslU are both ring-shaped particles and that the active HslVU complex is a cylindrical four-ring structure, composed of HslV, a two-ring dodecamer, sandwiched between HslU rings. Elucidation of its mode of action may help us understand the role of ATP in function of the 26S proteasome. Several proteasome-specific inhibitors have been recently identified which block the function of proteasome in vivo. These agents have proven very useful to clarify the intracellular function of the proteasome. In mammalian cells, both the rapid degradation of short-lived regulatory proteins and of abnormal polypeptides and the slower degradation of long-lived proteins are blocked by these agents. Thus, in mammalian cells, the proteasome is the site for the degradation of most cell proteins. In contrast, in budding yeast, proteasome inhibitors block the degradation of short-lived proteins but not the breakdown of long-lived proteins, which can be blocked by inhibitors of vacuolar proteases. The inhibition of proteasome function in yeast and mammalian cells, presumably by causing an accumulation of unfolded proteins, triggers the expression of heat shock proteins and concomitantly increases cell resistance to high temperature and various toxic insults.
S100beta is an astrocyte-derived protein with paracrine and autocrine effects on neurons and glia. Brain S100beta expression increases progressively with age, and this increased expression has been implicated as a factor underlying the increasing risk of Alzheimer's disease that accompanies aging. Senescence acceleration-prone (SAMP) mice are a group of inbred strains that provide animal models of aging and of various age-related disease processes in the brain and peripheral tissues. One of these strains, the osteopenic SAMP6, has not been previously associated with central nervous system alterations. We used Northern and Western immunoblot analysis and immunohistochemical labeling to examine S100beta expression in brains of SAMP6 mice. Cerebral tissue levels of S100beta and of S100beta mRNA were 2.2-fold and 1.6-fold those of senescence-resistant (control) mice at 4 months of age (p < 0.05 in each case), and were 3.7-fold and 1.9-fold those of control mice at 6 months of age (p < 0.01 in each case). In contrast, levels of glial fibrillary acidic protein (GFAP) in cerebral hemispheres were not different from those of controls. Image analysis of immunohistochemical preparations showed increased numbers and immunoreactive intensity of S100beta-immunoreactive astrocytes in both the hippocampus and cerebral cortex of SAMP6 mice at 4 months of age (p < 0.05 or better in each case). These increases were greater in the hippocampus than in the cerebral cortex. In contrast, increases in numbers of GFAP immunoreactive astrocytes were noted only in the hippocampus. Our finding of increased S100beta gene expression in brains of SAMP6 mice mirror age-associated increases in S100beta expression in human brain and suggest that SAMP6 may provide insights into age-associated brain alterations and diseases.
Most cellular proteins are targeted for degradation by the proteasome, a eukaryotic ATP-dependent protease, after they have been covalently attached to ubiquitin (Ub) in the form of a poly Ub chain functioning as a degradation signal. The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (called the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations, to form enzymatically active proteasomes. The large assembled proteasome acts as a protein-destroying machine responsible for the selective breakdown of numerous ubiquitinylated cellular proteins and certain nonubiquitinylated proteins. To date, proteolysis mediated by the Ub-proteasome pathway has been shown to be involved in a wide variety of biologically important processes, such as the cell cycle, apoptosis, metabolism, signal transduction, immune response and protein quality control, implying that it functions as a previously unrecognized regulatory system for determining the final fate of protein factors involved in these biological reactions.
Chronic overexpression of the neurite growth-promoting factor S100beta has been implicated in the pathogenesis of neuritic plaques in Alzheimer's disease. Such plaques are virtually universal in middle-aged Down's syndrome, making Down's a natural model of Alzheimer's disease. We determined numbers of astrocytes overexpressing S100beta, and of neurons overexpressing beta-amyloid precursor protein (beta-APP), and assayed for neurofibrillary tangles in neocortex of 20 Down's syndrome patients (17 weeks gestation to 68 years). Compared to controls, there were twice as many S100beta-immunoreactive (S100beta+) astrocytes in Down's patients at all ages: fetal, young, and adult (p = 0.01, or better, in each age group). These were activated (i.e., enlarged), and intensely immunoreactive, even in the fetal group. There were no neurofibrillary changes in fetal or young Down's patients. The numbers of S100beta+ astrocytes in young and adult Down's patients correlated with the numbers of neurons overexpressing beta-APP (p < 0.05). Our findings are consistent with the idea that conditions--including Down's syndrome--that promote chronic overexpression of S100beta may confer increased risk for later development of Alzheimer's disease.
Antigen processing by MHC class I molecules begins with the generation of peptides by proteolytic breakdown of proteins. IFN-gamma upregulates gene expression of several proteasomal subunits as well as the proteasome regulator PA28; this implicated their role in antigen degradation. Crystallographic, mutational and biochemical studies contributed to our understanding of the basic principles of proteasomal protein degradation and the consequences of IFN-gamma induction for proteasome function. In addition, nonproteasomal mechanisms seem to be involved in antigen degradation. Leucine aminopeptidase, which is also upregulated by IFN-gamma, was shown to collaborate with the proteasome for epitope production and unknown proteases seem to compensate for the loss of proteasomal degradation in the presence of proteasome inhibitors. Thus, a rather complex picture emerges for the rules governing peptide production in the presence or absence of IFN-gamma.
The 26 S proteasome is a large eukaryotic protease complex acting in ubiquitin-mediated degradation of abnormal and many short-lived, regulatory proteins. Its cylinder-shaped 20 S proteolytic core consists of two sets, each of seven different alpha and beta-type subunits arranged into two outer alpha-rings surrounding two inner beta-rings. The beta-rings form a central chamber with a total of six proteolytically active centers located in the beta1, beta2 and beta5 subunits. Activation of these subunits occurs during late assembly stages through intramolecular precursor autolysis removing propeptides attached to Thr1, which then serves as N-terminal nucleophile in substrate hydrolysis. This maturation entails intermolecular cleavage of propeptides residing in two of the non-active beta-type subunits, beta6 and beta7. In yeast, deletion of the beta5/Pre2 propeptide was shown to be lethal by preventing assembly of the core particle, while its expression as a separate entity restored growth. We investigated the role of the yeast beta1/Pre3, beta2/Pup1 and beta7/Pre4 propeptides by expressing the mature subunit moieties without propeptides as C-terminal fusions to ubiquitin. In all cases, viable strains could be generated. Deletion of the beta1/Pre3 and beta7/Pre4 propeptides did not affect cell growth, but deletion of the beta2/Pup1 propeptide led to poor growth, which was partially restored by co-expression of the free propeptide. Gain of proteolytic activity of beta1/Pre3 and beta2/Pup1 was abolished or drastically reduced, respectively, if their respective propeptides were not N-terminally bound. We detected N -alpha-acetylation at Thr1 of beta1/Pre3 as cause for its inactivation. Thus, one role for the propeptides of active beta-type subunits might be to protect the mature subunits catalytic Thr1 alpha-amino group from acetylation. The beta2/Pup1 propeptide was, in addition, required for efficient 20 S proteasome maturation, as revealed by the accumulation of beta7/Pre4 precursor and intermediate processing forms upon expression of mature beta2/Pup1. Finally, growth phenotypes resulting from expression of active site mutated beta-type subunits uncoupled from their propeptides allowed us to deduce the hierarchy of the importance of individual subunit activities for proteasomal function as follows: beta5/Pre2>beta2/Pup1>/=beta1/Pre3.
Oxidative stress may contribute to the cellular alterations, which occur as the result of aging, and the nervous system is particularly vulnerable to aging associated oxidative injury. The multicatalytic proteasome (MCP) is responsible for the majority of protein degradation and is sensitive to oxidative stress. To determine if MCP activity is altered during aging, studies were conducted in multiple tissues from aged Fisher 344 rats. Analysis of heart, lung, kidney, and liver revealed decreased MCP activity in 12, 24, and 28 month old rats, compared with 3 week or 3 month old animals. The spinal cord, hippocampus, and cerebral cortex demonstrated age dependent decreases in MCP activity, but at no timepoint was MCP activity decreased in either the brain stem or cerebellum. Oxidative injury and the lipid oxidation product 4-hydroxynonenal caused decreased MCP activity in neural PC6 cells, while application of MCP inhibitors was sufficient to induce cell death in neural PC6 cells. Together, these data indicate a role for MCP inhibition in cellular dysfunction associated with aging and oxidative injury.
Neuron death and neuron degeneration occur in the CNS during the course of aging. Although multiple cellular alterations transpire during the aging process, those that mediate age-associated neuron death have not been identified. Recent evidence implicates oxidative stress as a possible means of neuron death and neuron degeneration during aging. In the present study, we demonstrate a marked decrease in multicatalytic proteasome activity in the spinal cord of Fisher 344 rats at 12, 24 and 28 months, compared with spinal cord tissue from 3-week- and 3-month-old animals. Application of oxidative injury (FeSO(4)) or the lipid peroxidation product 4-hydroxynonenal decreases multicatalytic proteasome activity in a time- and dose-dependent manner in a motor neuron cell line. Loss of multicatalytic proteasome activity occurs before the loss of multicatalytic proteasome immunoreactivity, with FeSO(4)- and 4-hydroxynonenal-mediated decreases ameliorated by the application of a cell permeable form of the antioxidant glutathione. Application of multicatalytic proteasome inhibitors, but not inhibitors of lysosomal proteases, induced neuron death that was attenuated by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone or N-acetyl-Asp-Glu-Val-Asp-Cho (aldehyde). Together, these data suggest that multicatalytic proteasome inhibition occurs during aging of the spinal cord, possibly as the result of oxidative stress, and that multicatalytic proteasome inhibition may be causally related to neuron death.
The proteasome is an essential part of our immune surveillance mechanisms:
by generating peptides from intracellular antigens it provides peptides that
are then 'presented' to T cells. But proteasomes ? the waste-disposal
units of the cell ? typically do not generate peptides for antigen presentation
with high efficiency. How, then, does the proteasome adapt to serve the immune
system well?
Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.
The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.
The immediate-early gene Zif268 is a member of the Egr family of inducible transcription factors. Data from gene expression studies have suggested that this gene may play a critical role in initial triggering of the genetic machinery that has long been considered a necessary mechanism for maintenance of the later phases of LTP and also for the consolidation or stabilization of long-lasting memories. Until recently, however, the data supporting this assumption have been based primarily on circumstantial evidence, with no direct evidence to suggest that Zif268 is required for long-lasting synaptic plasticity and memory. In this report, we review our own data using Zif268 mutant mice; we show that although the early phase of dentate gyrus LTP is normal in these mice, the later phases are not present, and the ability of the mice to maintain learned information over a 24-h period is deficient. In addition, we present new information showing a task-dependent gene dosage effect in Zif268 heterozygous mice. We show that spatial learning is particularly sensitive to reduced levels of Zif268, as one-half of the complement of Zif268 in heterozygous mice is insufficient to maintain spatial long-term memories.
The immediate early genes (IEGs) are activated rapidly and transiently in response to a multitude of stimuli. The zif268 belongs to a category of regulatory IEGs that activate downstream target genes and is considered to be a triggering mechanism to activate the genomic response in neurons. Several studies have shown that zif268 mRNA is upregulated during different forms of associative learning, and following tetanic stimulation that induces long-lasting LTP. To date, there is a general consensus that zif268 activation may constitute a critical mechanism for the encoding of long-lasting memories, however this is based on relatively few studies. Given the fact that zif268 can be activated by a number of different types of stimuli, it becomes important to determine exactly how it may be implicated in memory. Examination of the current literature suggests that zif268 is necessary in the processing of several types of memory, however, it is not entirely clear what aspects of memory zif268 may be implicated in. Here, we review the existing literature and emphasise that understanding the signalling pathways that lead to activation of the IEGs and the downstream targets of these genes will advance our understanding of how functional activation of zif268 may be implicated in processing long-term memories.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Zif268 is a transcription regulatory protein, the product of an immediate early gene. Zif268 was originally described as inducible in cell cultures; however, it was later shown to be activated by a variety of stimuli, including ongoing synaptic activity in the adult brain. Recently, mice with experimentally mutated zif268 gene have been obtained and employed in neurobiological research. In this review we present a critical overview of Zif268 expression patterns in the naive brain and following neuronal stimulation as well as functional data with Zif268 mutants. In conclusion, we suggest that Zif268 expression and function should be considered in a context of neuronal activity that is tightly linked to neuronal plasticity.
Proteasomes perform the majority of proteolysis that occurs in the cytosol and nucleus of eukaryotic cells and, thereby, perform crucial roles in cellular regulation and homeostasis. Isolated proteasomes are inactive because substrates cannot access the proteolytic sites. PA28 and PA200 are activators that bind to proteasomes and stimulate the hydrolysis of peptides. Several protein inhibitors of the proteasome have also been identified, and the properties of these activators and inhibitors have been characterized biochemically. By contrast, their physiological roles--which have been reported to include production of antigenic peptides, proteasome assembly and DNA repair--are controversial. In this article, we briefly review the biochemical data and discuss the possible biological roles of PA28, PA200 and proteasome inhibitors.
Aging represents the main risk factor to develop Alzheimer disease (AD) and protein aggregation constitutes a pathological hallmark thought to be involved in the etiology of this disease. Here, we show that, in basal conditions, the expression of chaperones calnexin, protein disulfide isomerase (PDI) and Grp78 was decreased in aged hippocampus, whereas the protein ubiquitination increased, suggesting the existence of age-related deficits in the systems involved in the defense against unfolded proteins. Interestingly, when cellular stress was induced by intra-hippocampal lactacystin injection, the aged rats were less efficient than young animals in alleviating the protein accumulation and, as an important factor, did not induce the expression of chaperones as young animals. However, the expression of the pro-apoptotic factor CHOP/GADD153 was induced and caspase-12 was activated in stressed aged rats but not in young animals. Current results demonstrated that unfolding protein response (UPR) is not correctly activated in aged rat hippocampus. Consequently, the up-regulation of apoptotic pathway mediators is increased in aged rats. Results might provide further understanding of the pathogenic mechanisms of age-related neurodegenerative disorders.
The proteasome system is a central component of a cascade of proteolytic processing steps required to generate antigenic peptides presented at the cell surface to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The nascent protein pool or DRiPs (defective ribosomal products) appear to represent an important source for MHC class I epitopes. Owing to the destructive activities of aminopeptidases in the cytosol, at most 1% of the peptides generated by the ubiquitin-proteasome system seems to be made available to the immune system. Interferon-gamma (IFN-gamma) helps to override these limitations by the formation of immunoproteasomes, the activator complex PA28, and the induction of several aminopeptidases. Both immunoproteasomes and PA28 use cleavage sites already used by constitutive proteasomes but with altered and in some cases dramatically enhanced frequency. Therefore, two proteolytic cascades appear to have evolved to provide MHC class I epitopes. The 'constitutive proteolytic cascade' is designed to efficiently degrade proteins to single amino acid residues, allowing only a small percentage of peptides to be presented at the cell surface. In contrast, the IFN-gamma-controlled proteolytic cascade generates larger amounts of appropriate antigenic peptides, assuring more peptides to overcome the proteolytic restrictions of the constitutive system, thereby enhancing MHC class I antigen presentation.
Protein aggregation is a pathologic hallmark of familial amyotrophic lateral sclerosis caused by mutations in the Cu, Zn superoxide dismutase gene. Although SOD1-positive aggregates can be cleared by proteasomes, aggregates have been hypothesized to interfere with proteasome activity, leading to a vicious cycle that further enhances aggregate accumulation. To address this issue, we measured proteasome activity in transgenic mice expressing a G93A SOD1 mutation. We find that proteasome activity is induced in the spinal cord of such mice compared to controls but is not altered in uninvolved organs such as liver or spleen. This induction within spinal cord is not related to an overall increase in the total number of proteasome subunits, as evidenced by the steady expression levels of constitutive alpha7 and beta5 subunits. In contrast, we found a marked increase of inducible beta proteasome subunits, LMP2, MECL-1 and LMP7. This induction of immunoproteasome subunits does not occur in all spinal cord cell types but appears limited to astrocytes and microglia. The induction of immunoproteasome subunits in G93A spinal cord organotypic slices treated with TNF-alpha and interferon-gamma suggest that certain cytokines may mediate such responses in vivo. Our results indicate that there is an overall increase in proteasome function in the spinal cords of G93A SOD1 mice that correlates with an induction of immunoproteasomes subunits and a shift toward immunoproteasome composition. These results suggest that increased, rather than decreased, proteasome function is a response of certain cell types to mutant SOD1-induced disease within spinal cord.
In this study, we investigated the presence and role of immunoproteasome and its LMP2 subunit polymorphism at codon 60 in Alzheimer's disease (AD). Immunoproteasome was present in brain areas such as hippocampus and cerebellum and localized in neurons, astrocytes and endothelial cells. A higher expression of immunoproteasome was found in brain of AD patients than in brain of non-demented elderly, being its expression in young brain negligible or absent. Furthermore, AD affected regions showed a partial decrease in proteasome trypsin-like activity. The study of LMP2 polymorphism (R/H) showed that it does not influence LMP2 expression (neither the mRNA nor mature protein) in brain tissue. However, control brain areas of AD patients carrying the RR genotype showed an increased proteasome activity in comparison with RH carriers. To test whether this effect of the genotype might be related to AD onset we performed a genetic study, which allowed us to exclude an association of LMP2 codon 60 polymorphism with AD onset, despite its influence on the proteasome activity in human brain.
Protein metabolism contributes in the regulation of gut barrier function, which may be altered during inflammatory states. There are three major proteolytic pathways in mammalian cells: lysosomal, Ca(2+)-activated and ubiquitin-proteasome. The regulation of proteolytic activities during inflammation remains unknown in intestine. Intestinal epithelial cells, HCT-8, were stimulated by IL-1beta, IFNgamma and TNFalpha each alone or in combination (Cytomix). Proteolytic activities were assessed using fluorogenic substrates and specific inhibitors, protein expressions by Western blot. Lysosomal and Ca(2+)-activated pathways were not significantly altered by any treatment. In contrast, the activity of ubiquitin-proteasome system was stimulated by IFNgamma and Cytomix (155, 160 versus 100, P<0.05, respectively) but remained unaffected by IL-1beta and TNFalpha. Free ubiquitin expression, but not ubiquitinated proteins, was enhanced by IFNgamma and Cytomix. The expression of proteasome 20S alpha1 subunit, a constitutive proteasome 20S subunit, was not altered, beta5 subunit expression was weakly decreased by Cytomix and inducible beta5i subunit expression was markedly increased in response to IFNgamma and to Cytomix (202, 206 versus 100, P<0.05, respectively). In conclusion, lysosomal, Ca(2+)-activated and constitutive proteasome activities were not affected by IL-1beta, IFNgamma and TNFalpha alone or in combination, in HCT-8 cells. These results suggest that IFNgamma, but not IL-1beta and TNFalpha, increases immunoproteasome, which might contribute to enhanced antigen presentation during inflammatory bowel diseases.