José G. Castaño

Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain

In Huntingtons disease (HD), as in the rest of CAG triplet‐repeat disorders, the expanded polyglutamine (polyQ)‐containing proteins form intraneuronal fibrillar aggregates that are gathered into inclusion bodies (IBs). Since IBs contain ubiquitin and proteasome subunits, it was proposed that inhibition of proteasome activity might underlie pathogenesis of polyQ disorders. Recent in vitro enzymatic studies revealed the inability of eukaryotic proteasomes to digest expanded polyQ, thus suggesting that occasional failure of polyQ to exit the proteasome may interfere with its proteolytic function. However, it has also recently been found that in vitro assembled aggregates made of synthetic polyQ fail to inhibit proteasome activity. Because synthetic polyQ aggregates lack the post‐translational modifications found inside affected neurons, such as poly ubiquitylation, we decided to study the effect of mutant huntingtin (htt) aggregates isolated from the Tet/HD94 mouse model and from human HD brain tissue. Here, we show that isolated ubiquitylated filamentous htt aggregates, extracted from IBs by a previously reported method, selectively inhibited the in vitro peptidase activity of the 26S but not of the 20S proteasome in a non‐competitive manner. In good agreement, immuno‐electron microscopy revealed a direct interaction of htt filaments with the 19S ubiquitin‐interacting regulatory caps of the 26S proteasome. Here, we also report a new method for isolation of IBs based on magnetic sorting. Interestingly, isolated IBs did not modify proteasome activity. Our results therefore show that mutant htt filamentous aggregates can inhibit proteasome activity, but only when not recruited into IBs, thus strengthening the notion that IB formation is protective by neutralizing toxicity of dispersed filamentous htt aggregates.

Mechanism of direct degradation of IκBα by 20S proteasome

IκBα regulates activation of the transcription factor NF‐κB. NF‐κB is activated in response to several stimuli, i.e. proinflamatory cytokines, infections, and physical stress. This signal dependent pathway involves IκBα phosphorylation, ubiquitylation, and degradation by 26S proteasome. A signal independent (basal) turnover of IκBα has also been described. Here, we show that IκBα can be directly degraded by 20S proteasomes. Deletion constructs of IκBα allow us to the determine that N‐terminal (ΔN 1–70) and C‐terminal regions (ΔC 280–327, removing the PEST region) of IκBα are not required for IκBα degradation, while a further C‐terminal deletion including part of the arm repeats (ΔC2 245–327) almost completely suppress the degradation by 20S proteasome. Binding and competition experiments demonstrate that the degradation of IκBα involves specific interactions with α2(C3) subunit of the proteasome. Finally, p65/relA (not itself a substrate for 20S proteasome) inhibits the degradation of IκBα by the proteasome. These results recapitulate in vitro the main characteristics of signal independent (basal) turnover of IκBα demonstrated in intact cells.

α-Synuclein expression levels do not significantly affect proteasome function and expression in mice and stably transfected PC12 cell lines

α-Synuclein (α-syn) is a small protein of unknown function that is found aggregated in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease and other synucleinopathies. Mutations in the α-syn gene and a triplication of its gene locus have been identified in early onset familial Parkinson disease. α-Syn turnover can be mediated by the proteasome pathway. A survey of published data may lead to the suggestion that overexpression of α-syn wild type, and/or their variants (A53T and A30P), may produce a decrease in proteasome activity and function, contributing to α-syn aggregation. To investigate the relationship between synuclein expression and proteasome function we have studied proteasome peptidase activities and proteasome subunit expression (α, β-constitutive, and inducible) in mice either lacking α-syn (knock-out mice) or transgenic for human α-syn A30P (under control of PrP promoter, at a time when no clear gliosis can be observed). Similar studies are presented in PC12 cells overexpressing enhanced yellow fluorescent protein fusion constructs of human wild type, A30P, and A53T α-syn. In these cell lines we have also analyzed the assembly of 20 S proteasome complex and the degradation rate of a well known substrate of the proteasome pathway, Iκbα. Overall the data obtained led us to the conclusion that α-synuclein expression levels by themselves have no significant effect on proteasome peptidase activity, subunit expression, and proteasome complex assembly and function. These results strengthen the suggestion that other mechanisms resulting in synuclein aggregation (not simply expression levels) may be the key to understand the possible effect of aggregated synuclein on proteasome function.

Antibodies against the COOH-terminal region of E. coli ClpP protease in patients with primary biliary cirrhosis

BACKGROUND/AIMS The presence of antibodies in sera from patients with autoimmune diseases is an important tool for diagnosis and for providing insights into the mechanisms leading to autoimmunity. The aim of this study was to characterize new reactive antigens in liver autoimmune diseases. METHODS Sera of patients with liver-related autoimmune (n=74) and non-liver-related autoimmune (n= 211) diseases, non-autoimmune liver diseases (n=18) and healthy controls (n=160) were evaluated for antibodies against E. coli ClpP protease (EClpP) and 20S proteasome by immunoblot analysis. RESULTS Antibodies against EClpP were detected in 15 of 50 patients with primary biliary cirrhosis, in only one of 100 patients with systemic lupus erythematosus, and in three healthy subjects (Chi-square 59.1, d.f. 2, p< 0.001). Antibodies to 20S proteasome were found in only 35 of 100 patients with systemic lupus erythematosus. All other sera from patients with autoimmune diseases, liver diseases other than primary biliary cirrhosis, and healthy controls were negative for both antigens. Both IgG and IgM classes of antibodies against EClpP were present in primary biliary cirrhosis patient sera with titers of 1/400-1/1000. By using recombinant techniques and peptide ELISA, the immunodominant EClpP epitope recognized by the sera from primary biliary cirrhosis patients was localized in the amino acid sequences 177-194 (QIERDTERDRFLSAPEAV) within the COOH-terminal of EClpP. Affinity-purification of these anti-EClpP antibodies and immunoabsorption experiments established that the antibodies are specific for the bacterial EClpP. CONCLUSIONS Bacterial ECIpP has been identified as a new antigen specifically reacting with sera from approximately one third of patients with primary biliary cirrhosis.

Reduced protein stability of human DJ-1/PARK7 L166P, linked to autosomal recessive Parkinson disease, is due to direct endoproteolytic cleavage by the proteasome

Parkinsons disease (PD) is characterized by dopaminergic dysfunction and degeneration. DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. In this study, we found that human DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A were stable proteins in cells, only the L166P mutant was unstable. In parallel, the former were not degraded and the L166P mutant was directly degraded in vitro by proteasome-mediated endoproteolytic cleavage. Furthermore, genetic evidence in fission yeast showed the direct involvement of proteasome in the degradation of human DJ-1 L166P and the corresponding L169P mutant of SPAC22E12.03c, the human orthologue of DJ-1 in Schizosaccharomyces Pombe, as their protein levels were increased at restrictive temperature in fission yeast (mts4 and pts1-732) harboring temperature sensitive mutations in proteasomal subunits. In total, our results provide evidence that direct proteasomal endoproteolytic cleavage of DJ-1 L166P is the mechanism of degradation contributing to the loss-of-function of the mutant protein, a property not shared by other DJ-1 missense mutants associated with PD.

Proteasome dynamics during cell cycle in rat Schwann cells

The proteasome is responsible for most of the protein degradation that takes place in the cytoplasm and nucleus. Immunofluorescence and electron microscopy are used to study proteasome dynamics during the cell cycle in rat Schwann cells. During interphase, the proteasome is present in the nucleus and cytoplasm and shows no colocalization with cytoskeletal components. Some cytoplasmic proteasomes always localize in the centrosome both in interphase and in mitotic cells and only associate with microtubules during mitosis. The proteasome exits the nucleus during prophase. In anaphase, the proteasome becomes prominent in the region between the two sets of migrating chromosomes and in association with interzonal microtubules and stem bodies. In telophase, the proteasome begins to reenter the nucleus and is prominent in the midbody region until the end of cytokinesis. The proteasome does not colocalize with actin or vimentin during mitosis, except for colocalization with actin in the sheet‐like lamellipodia, which serve as substrate attachments for the cell during mitosis. During S phase, nuclear proteasomes colocalize with foci of BrdU incorporation, but this association changes with time: maximal at early S phase and declining as S phase progresses to the end. These results are discussed in relation to the biochemical pathways involved in cell cycle progression. GLIA 38:313–328, 2002.

Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

The N-terminal region of Nurr1 (a.a 1-31) is essential for its efficient degradation by the ubiquitin proteasome pathway.

NURR1/NR4A2 is an orphan nuclear receptor that is critical for the development and maintenance of mesencephalic dopaminergic neurons and regulates transcription of genes involved in the function of dopaminergic neurons directly via specific NGFI-B response elements (NBRE).and substantial data support a possible role of Nurr1 in the pathogenesis of Parkinsons disease (PD). Here we show that Nurr1 is degraded by the ubiquitin-proteasome pathway and determined that N-terminal region (a.a 1–31) of Nurr1 is essential for an efficient targeting of Nurr1 to degradation in the cell. Nurr1 Δ1–31 has a much longer half-life, and as a consequence its steady-state protein levels were higher, than full-length Nurr1 in the cell. Nurr1 Δ1–31 was as potent as Nurr1 full length in transcriptional luciferase reporter assays after normalization with the corresponding steady-state protein expression levels, either in trans-activation of NBRE or trans-repression of iNOS (inducible NO synthase) reporters. These results suggest that Nurr1 Δ1–31, because of longer persistence in the cell, can be a good candidate for gene and cell therapies in the treatment of PD.

Epitope Mapping of Antibodies to Alpha-Synuclein in LRRK2 Mutation Carriers, Idiopathic Parkinson Disease Patients, and Healthy Controls

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1–60) or C-terminal (a.a. 109–140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls’ studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD pathology.

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.