Regulation of immunoproteasome function in the lung.

Impaired immune function contributes to the development of chronic obstructive pulmonary disease (COPD). Disease progression is further exacerbated by pathogen infections due to impaired immune responses. Elimination of infected cells is achieved by cytotoxic CD8(+) T cells that are activated by MHC I-mediated presentation of pathogen-derived antigenic peptides. The immunoproteasome, a specialized form of the proteasome, improves generation of antigenic peptides for MHC I presentation thereby facilitating anti-viral immune responses. However, immunoproteasome function in the lung has not been investigated in detail yet. In this study, we comprehensively characterized the function of immunoproteasomes in the human and murine lung. Parenchymal cells of the lung express low constitutive levels of immunoproteasomes, while they are highly and specifically expressed in alveolar macrophages. Immunoproteasome expression is not altered in whole lung tissue of COPD patients. Novel activity-based probes and native gel analysis revealed that immunoproteasome activities are specifically and rapidly induced by IFNγ treatment in respiratory cells in vitro and by virus infection of the lung in mice. Our results suggest that the lung is potentially capable of mounting an immunoproteasome-mediated efficient adaptive immune response to intracellular infections.

Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver.

Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of ß5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation.

Two inhibitors of the ubiquitin proteasome system enhance long-term memory formation upon olfactory conditioning in the honeybee (Apis mellifera).

In honeybees (Apis mellifera), the proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG132) enhances long-term memory (LTM) formation. Studies in vertebrates using different inhibitors of the proteasome demonstrate the opposite, namely an inhibition of memory formation. The reason for this contradiction remains unclear. MG132 is an inhibitor of the proteasome, but also blocks other proteases. Accordingly, one possible explanation might be that other proteases affected by MG132 are responsible for the enhancement of LTM formation. We test this hypothesis by comparing the effect of MG132 and the more specific proteasome inhibitor clasto-lactacystin beta-lactone (ß-lactone). We show that these two inhibitors block the activity of the proteasome in honeybee brains to a similar extent, do not affect the animals' survival but do enhance LTM retention upon olfactory conditioning. Thus, the enhancement of LTM formation is not due to MG132-specific side effects, but to inhibition of a protease targeted by MG132 and ß-lactone, i.e. the proteasome.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties.

BACKGROUND: The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver. MATERIAL AND METHODS: 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting. RESULTS: Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits ß1i and ß5i but not their standard counterparts; all others are intermediate subtypes containing ß1 and ß5 standard- and ß1i and ß5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit ß1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing ß1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes. CONCLUSION: These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.

Trypanosoma cruzi infection down-modulates the immunoproteasome biosynthesis and the MHC class I cell surface expression in HeLa cells.

Generally, Trypanosoma cruzi infection in human is persistent and tends to chronicity, suggesting that the parasite evade the immune surveillance by down regulating the intracellular antigen processing routes. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome. However, upon IFN-γ stimulation, the catalytic constitutive subunits of the proteasome are replaced by the subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 to form the immunoproteasome. In this scenario, we analyzed whether the expression and activity of the constitutive and the immunoproteasome as well as the expression of other components of the MHC class I pathway are altered during the infection of HeLa cells with T. cruzi. By RT-PCR and two-dimensional gel electrophoresis analysis, we showed that the expression and composition of the constitutive proteasome is not affected by the parasite. In contrast, the biosynthesis of the ß1i, ß2i, ß5i immunosubunits, PA28ß, TAP1 and the MHC class I molecule as well as the proteasomal proteolytic activities were down-regulated in infected-IFN-γ-treated cell cultures. Taken together, our results provide evidence that the protozoan T. cruzi specifically modulates its infection through an unknown posttranscriptional mechanism that inhibits the expression of the MHC class I pathway components.

Expanded polyglutamine-containing N-terminal huntingtin fragments are entirely degraded by mammalian proteasomes.

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.

Poly-Ub-substrate-degradative activity of 26S proteasome is not impaired in the aging rat brain.

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.

Driving forces of proteasome-catalyzed peptide splicing in yeast and humans.

Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site ß subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H(2)(18)O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.

Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted ß-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90ß, of which one corresponds to a previously described phosphorylation site.

Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells.

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin-proteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC-ESI-MS. Taking the results of all experiments together, no quantitative changes were observed for the α- and ß-subunits of the 20S proteasome except for subunit α7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (α7a) and up-regulation of the more basic protein species (α7b) during apoptosis. The difference in these two α7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in α7a, whereas these modifications were missing in α7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit α7 after induction of apoptosis.

Multiple cardiac proteasome subtypes differ in their susceptibility to proteasome inhibitors.

AIMS: The proteasome is the proteolytically active core of the ubiquitin-proteasome system, which regulates vital processes and which can cause various diseases when it malfunctions. Therefore, the proteasome has become an attractive target for pharmaceutical interventions. Inhibition of the cardiac proteasome by specific proteasome inhibitors has been shown to attenuate cardiac hypertrophy and ischaemia reperfusion injury of the heart. We have resolved the cardiac proteasome into its subtypes and have addressed the key question of how proteasome inhibitors affect single cardiac proteasomal subtypes. METHODS AND RESULTS: The 20S proteasome from rat heart was dissected into three different subpopulations (groups I-III), each comprising 4-7 different subtypes. The major group (group II) comprises standard proteasome subtypes; the two minor subpopulations (groups I and III) contain intermediate proteasome subtypes. All subtypes exhibit chymotrypsin-, trypsin-, and caspase-like activity but to different degrees. We have tested the effect of two common proteasome inhibitors on the chymotrypsin-like activity of all subtypes: 20-30 nmol/L MG132 caused 50% inhibition of all subtypes from groups I and II, whereas 100 nmol/L was necessary to affect group III subtypes to the same extent. However, another inhibitor, bortezomib (VELCADE), already used clinically, inhibited 50% of the activity of group III proteasome subtypes even below 20 nmol/L, a concentration showing almost no effect on group I and II proteasome subtypes. The caspase-like activity of group II proteasome subtypes was not affected by MG132 and was inhibited by bortezomib only at concentrations above 100 nmol/L. CONCLUSION: These data show that different inhibitors have differential inhibitory effects on the various cardiac proteasome subtypes. Different cardiac subtypes are inhibited by the same dose of proteasome inhibitor to a different extent.

Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome.

RATIONALE: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown. OBJECTIVES: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS. METHODS: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (+/-epoxomicin), I(125) albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques. MEASUREMENTS AND MAIN RESULTS: Proteasome concentrations in patients with ARDS were markedly increased (1,069 +/- 1,194 ng/ml) in comparison to healthy subjects (60.8 +/- 49.8; P < 0.001), ALI (154 +/- 43; P = 0.006), and sarcoidosis (97.6 +/- 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 +/- 8.8 pkat/mg; BZ-VGR-AMC, 1.8 +/- 3.1; Suc-LLE-AMC, 1 +/- 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification. CONCLUSIONS: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.

The cell-penetrating peptide octa-arginine is a potent inhibitor of proteasome activities.

Oligo-arginines are cell-penetrating peptides and find use as carriers for transportation of various membrane-impermeable biopharmaceuticals into target cells. We have found that oligo-arginines of a length of 4-10 amino acids, but especially (Arg)(8), are able to inhibit the major intracellular proteolytic system, the proteasome, with mixed-type inhibition characteristics. The IC(50) values of (Arg)(8) for the proteasomal chymotrypsin-like and caspase-like activities are approximately 100 and 200 nM, respectively. The inhibition of the trypsin-like activity never exceeds 50% even at micromolar concentrations. (Arg)(8) also inhibits 20S proteasome/PA28 complexes as well as 26S proteasomes, although with a decreased efficiency. Due to its cell membrane-penetrating capability, incubation of HeLa cells in the presence of (Arg)(8) resulted in an impaired activity of proteasomes going along with an accumulation of high-molecular mass ubiquitin-conjugated proteins, the preferred substrates of 26S proteasomes. The in vivo susceptibility of the three proteasome activities resembles that found in vitro with chymotrypsin-like>caspase-like>trypsin-like activities. Since inhibition of the proteasome system might affect fundamental basic cellular processes but on the other side might also prevent the degradation of a proteinacous cargo, we suggest that this proteasome inhibitory activity should be taken into account when oligo-arginines are being considered for use as vectors for the intracellular delivery of pharmaceuticals.

The 20S proteasome isolated from Alzheimer's disease brain shows post-translational modifications but unchanged proteolytic activity.

Chronic neurodegenerative diseases are characterized by the accumulation of aggregated protein species, and functional impairment of the ubiquitin proteasome system has been hypothesized to contribute to neuronal cell loss. Decreased proteolytic activity of the 20S proteasome has been shown postmortem in crude brain lysates from Alzheimer's disease (AD) patients. In the present study, we demonstrate, however, that catalytic activity of the 20S proteasome increases during chromatographic purification from AD brains as compared with age-matched controls. By two-dimensional difference gel electrophoresis we detected pI shifts in several proteasome subunits in AD samples pointing to differential post-translational modifications. Moreover, we identified N-terminal acetylation and dephosphorylation of subunit alpha7 in AD by tandem mass spectrometry. Thus, reduced peptidase activity in AD brain extracts is not an intrinsic property of the 20S proteasome, but may be resulting from the presence of endogenous inhibitory proteins or substrates. Post-translational modifications of non-catalytic subunits in situ may contribute to the trend towards enhanced hydrolytic activity of the isolated 20S proteasome after removal of the endogenous inhibitors.

Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches.

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.