Proteasome isoforms in human thymi and mouse models.

The thymus is the organ where functional and self-tolerant T cells are selected through processes of positive and negative selection before migrating to the periphery. The antigenic peptides presented on MHC class I molecules of thymic epithelial cells (TECs) in the cortex and medulla of the thymus are key players in these processes. It has been theorized that these cells express different proteasome isoforms, which generate MHC class I immunopeptidomes with features that differentiate cortex and medulla, and hence positive and negative CD8+ T cell selection. This theory is largely based on mouse models and does not consider the large variety of noncanonical antigenic peptides that could be produced by proteasomes and presented on MHC class I molecules. Here, we review the multi-omics, biochemical and cellular studies carried out on mouse models and human thymi to investigate their content of proteasome isoforms, briefly summarize the implication that noncanonical antigenic peptide presentation in the thymus could have on CD8+ T cell repertoire and put these aspects in the larger framework of anatomical and immunological differences between these two species

Poly-Ub-Substrate-Degradative Activity of 26S Proteasome Is Not Impaired in the Aging Rat Brain

<div><p>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.</p></div

Detection and separation of proteasomes by non-denaturing polyacrylamide gel electrophoresis and glycerol gradient centrifugation.

<p>Panel A. Detection of chymotrypsin-like activity in polyacrylamide gels after non-denaturing electrophoresis by substrate overlay technique. Lanes 26S and 20S each contain 2 µg of purified erythrocyte 26S and 20S proteasome, respectively. Lanes Young and Aged contain 170 µg of tissue extracts each of cerebrum from young and aged rats; an aliquot of pooled fractions of peak Ι and ΙΙ as well as ΙΙΙ obtained after glycerol gradient centrifugation of rat cerebrum were run in lanes Ι+ΙΙ and ΙΙΙ, respectively. Panel B. Extracts of brain tissue from young (open circles) and aged (filled circles) rats were separated by centrifugation in a glycerol gradient (40–10%) and then fractionated. Each fraction was tested for its content of chymotrypsin-like activity. Panel C. Chymotrypsin-like activity in the four peaks (I, II, III, IV) obtained by glycerol gradient centrifugation was measured in the presence (black columns) and absence (white columns) of the 50 µM lactacystin. Panel D. 30 µg each of 20S proteasome (filled circles) and 26S proteasome (open circles) purified from human erythrocytes were subjected to glycerol gradient centrifugation under the same conditions as described in panel B and their chymotrypsin-like activity was measured.</p

Tissue weight, total protein and proteasome content.

<p>Means ± SEM and p values are given for comparison of young and aged rats. Number of animals per age group used were 5 for cerebrum, 7 for cerebellum, and 4 for hippocampus.</p

2D-PAGE electrophoresis of 20S proteasome purified from cerebellum of young and aged rats.

<p>Panel A. 30 µg of purified proteasome was applied to each gel, which were stained with Coomassie. Proteasome subunits were assigned according to our earlier investigations with proteasomes from rat liver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064042#pone.0064042-Schmidt1" target="_blank">[31]</a>. The location of subunits β1i, β2i and β5i are indicated by a bar. Panel B–E. Standard- (β1 and β5) and immuno-subunits (β1i and β5i) in 26S proteasomes isolated from cerebrum (panel C), cerebellum (panel D), and hippocampus (panel E) from young and aged rats were detected by immunoblot analysis after SDS-PAGE. About 2–5 µg of 26S proteasome was subjected to the electrophoresis gels. The specificity of the antibodies was tested with 0.5 µg 20S proteasomes purified from rat spleen and muscle (panel B). As we are not aware of an antibody specific for rat proteasome β2 and β2i subunits, these proteins were not analysed here. As a loading control subunit α1 was identified in panel B–E and in panel C–E; the ratio of the signals (pixel intensity) of the immunosubunits β1i and β5i were calculated against α1 after their densitometric quantification by use of the ImageJ software.</p

Specific hydrolytic activities towards fluorogenic peptide substrates of 20S and 26S proteasomes purified from different brain parts of young and aged rats.

<p>From cerebrum, cerebellum, and hippocampus of young (white columns) and aged (black columns) rats 20S and 26S proteasomes were separated by glycerol gradient centrifugation. The proteasome containing fractions were pooled and their proteasome content determined by immunoelectrophoresis. Chymotrypsin- (chtr), trypsin- (tryp) and caspase-like (casp) proteasome activities were measured and shown as pmol substrate hydrolysis/min x µg proteasome. Values are given as means ± SEM (n = 5) and data obtained for young and aged rats were compared by Students t-test. p-values indicating statistically significant differences are indicated (*, p<0.05; **, p<0.01).</p

Degradation of Ub₅Muc₄ and poly-Ub-GST-UbcH5 by 26S proteasomes purified from cerebrum and cerebellum.

<p>Panel A–D. 70 nM of purified 26S proteasomes from young and aged animals were incubated with 600 nM Ub₅Muc₄ and incubated at 37°C. At the times indicated aliquots of the reaction mixture were removed and subjected to SDS-PAGE. Afterwards Ub₅Muc₄ was detected on immunoblots with an antibody raised against Muc_950–958 peptide. Panel A shows a representative result of the experiments performed. Proteasome subunit α1 was monitored as a loading control with antibody IB5. For quantitative determination of the degradation process, the amount of Ub₅Muc₄ on the blots was measured by densitometry. Results obtained are indicated for the digestion processes with 26S proteasomes from cerebrum (panel B) and cerebellum (panel C) of young (open circles) and aged (filled circles) rats. Data shown are means ± SEM of 3–5 animals. Values of the degradation rates were compared between young and aged animals and statistically different values (*, p<0.05) are indicated. Panel D. 26S proteasome (30 nM) purified from cerebellum (circles) and cerebrum (squares) of young (white symbols) and aged (black symbols) rats were preincubated with or without 300 nM Ub₅Muc₄ for 15 min at 37°C before 100 µM Suc-LLVY-MCA was added and fluorogenic peptide hydrolysis was measured at the indicated time points. The data (means of two animals) shown are the difference of hydrolytic activity measured after preincubation with and without Ub₅Muc_4. Panel E–H. Purified 26S proteasome (30 nM) from young and aged animals was incubated with 800 nM poly-Ub-GST-UbcH5 at 37°C. After 30 and 60 minutes aliquots of the reaction mixtures were removed and subjected to SDS-PAGE, blotted and UbcH5 was detected on immunoblots using the Ubch5 antibody. Panel E shows a representative result of the experiments performed. Proteasome was monitored as a loading control by using the 20S proteasome antiserum 37. For quantitative determination of the degradation process, the amount of poly-Ub-GST-UbcH5 on the blots was measured by densitometry. Results obtained are indicated for the digestion processes with 26S proteasomes from cerebrum (panel F) and cerebellum (panel G) of young (open circles) and aged (filled circles) rats. Data shown are mean values ± S.D. of 2 animals. Panel H. 26S proteasome purified from cerebellum (circles) and cerebrum (squares) of young (white symbol) and aged (black symbol) rats were preincubated with or without poly-Ub-GST-UbcH5 (same ratio as detailed for panel E–G) for 15 min at 37°C before 100 µM Suc-LLVY-MCA was added and fluorogenic peptide hydrolysis was measured at the indicated time points. Data shown are representative of two independent experiments.</p

Content of 20S and 26S proteasome in cerebrum, cerebellum, and hippocampus of young and aged rats.

<p>Means ± SEM and p values are given for comparison of young and aged rats. Number of animals per age group used were 5 for cerebrum, 7 for cerebellum, and 4 for hippocampus.</p

Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing.

If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow-and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation