Biomedical subjects

José G Castaño

Publications and source records attributed to José G Castaño.

9 recordsLinked to original sources

PubMed2006

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

In Huntington's 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.

Aged↗

PubMed2005

Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts.

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.

Amino Acid Motifs↗

PubMed2005

Mechanism of direct degradation of IkappaBalpha by 20S proteasome.

IkappaBalpha regulates activation of the transcription factor NF-kappaB. NF-kappaB is activated in response to several stimuli, i.e. proinflamatory cytokines, infections, and physical stress. This signal dependent pathway involves IkappaBalpha phosphorylation, ubiquitylation, and degradation by 26S proteasome. A signal independent (basal) turnover of IkappaBalpha has also been described. Here, we show that IkappaBalpha can be directly degraded by 20S proteasomes. Deletion constructs of IkappaBalpha allow us to the determine that N-terminal (DeltaN 1-70) and C-terminal regions (DeltaC 280-327, removing the PEST region) of IkappaBalpha are not required for IkappaBalpha degradation, while a further C-terminal deletion including part of the arm repeats (DeltaC2 245-327) almost completely suppress the degradation by 20S proteasome. Binding and competition experiments demonstrate that the degradation of IkappaBalpha involves specific interactions with alpha2(C3) subunit of the proteasome. Finally, p65/relA (not itself a substrate for 20S proteasome) inhibits the degradation of IkappaBalpha by the proteasome. These results recapitulate in vitro the main characteristics of signal independent (basal) turnover of IkappaBalpha demonstrated in intact cells.

Animals↗

PubMed2004

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

alpha-Synuclein (alpha-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 alpha-syn gene and a triplication of its gene locus have been identified in early onset familial Parkinson disease. alpha-Syn turnover can be mediated by the proteasome pathway. A survey of published data may lead to the suggestion that overexpression of alpha-syn wild type, and/or their variants (A53T and A30P), may produce a decrease in proteasome activity and function, contributing to alpha-syn aggregation. To investigate the relationship between synuclein expression and proteasome function we have studied proteasome peptidase activities and proteasome subunit expression (alpha, beta-constitutive, and inducible) in mice either lacking alpha-syn (knock-out mice) or transgenic for human alpha-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 alpha-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, Ikappabalpha. Overall the data obtained led us to the conclusion that alpha-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.

Animals↗

PubMed2004

Proteasomal expression, induction of immunoproteasome subunits, and local MHC class I presentation in myofibrillar myopathy and inclusion body myositis.

Inclusion body myositis (IBM) and myofibrillar myopathy (MM) are diseases characterized by the abnormal accumulation of proteins in muscle fibers, including desmin, alphaB-crystallin, gelsolin, actin, kinases, and phospho-tau, along with ubiquitin in muscle fibers, suggesting abnormal protein degradation as a possible cause of the surplus myopathy. Since the ubiquitin-proteasome system plays a crucial role in non-lysosomal protein degradation, the present study has examined by immunohistochemistry the expression of components of the catalytic core of 20S proteasomes and its regulators: 19S and PA28alpha/beta, and the expression of immunoproteasome subunits LMP2, LMP7, and MECL1 in 8 patients with MM and 10 patients with IBM. The patients with MM were from 6 unrelated families, 2 sporadic cases, I with autosomal recessive and 5 with autosomal dominant inheritance. One sporadic patient had a de novo R406W mutation in the desmin gene, and 1 patient with autosomal dominant MM had a single amino acid deletion at position 366 in the desmin gene. Increased immunoreactivity to 20S, 19S, and PA28alpha/beta colocalizing abnormal protein deposits, as revealed in consecutive serial sections, was seen in all cases with MM and IBM. In all cases, the subunits of the immunoproteasome LMP2, LMP7, and MECL1 colocalized with proteasomal immunoreactivity and abnormal protein accumulation. Immunohistochemistry revealed focal MHC class I immunoreactivity in the cytoplasmic membrane of muscle fibers in IBM and in association with protein aggregates in IBM, and to a lesser degree, in MM. The present findings provide a link between abnormal protein accumulation and altered proteasomal expression in IBM and MM.

Adenosine Triphosphatases↗

PubMed2003

Neuronal induction of the immunoproteasome in Huntington's disease.

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.

Aged↗

PubMed2003

The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity.

Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.

Amino Acid Sequence↗

PubMed2002

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.

Active Transport, Cell Nucleus↗

PubMed2002

The proteasome is a major autoantigen in multiple sclerosis.

Multiple sclerosis seems to be an autoimmune disease of unknown aetiology affecting the white matter of the CNS. It is generally accepted that the autoimmune response is directed against specific components of myelin. We show here that proteasome, a ubiquitous protease complex composed of 14 different subunits, is a target for autoantibodies (IgG and IgM classes) present in the serum (66%, 73 out of 110) and in the CSF (61%, 16 out of 26) of patients with multiple sclerosis. Using recombinant proteasomal subunits we demonstrate the presence of specific autoantibodies against subunits C2, C8, C9 and C5 in multiple sclerosis patients. Recombinant C2 constructs allow us to localize an immunodominant autoepitope recognized by the sera of multiple sclerosis patients within the C-terminal of C2 proteasomal subunit (251-DEPAEKADEPMEH-263). In addition, two constructs of the recombinant proteasomal subunits C2 and C8 were also used to study the proliferation of peripheral blood mononuclear cells from multiple sclerosis patients; 12 out of 30 (40%) multiple sclerosis patients show positive proliferation with one or both of these recombinant subunits. The high prevalence of anti-proteasome autoantibodies in multiple sclerosis sera compared with sera from patients with other chronic inflammatory conditions: systemic lupus erythematosus (35%, 35 out of 100), primary Sjogren's syndrome (16%, 5 out of 31), vasculitis (0 out of 20), sarcoidosis (7%, 1 out of 13) and Behcet's disease (19%, 4 out of 21) suggest that humoral autoreactivity to proteasome could be a useful test in multiple sclerosis patients that may be of help in the diagnosis and/or progression of this chronic inflammatory disease. Finally, these results suggest that some global abnormality in B and/or T cell function is also involved in the chronic inflammatory response observed in multiple sclerosis patients, as it is frequently observed in other human organ-specific autoimmune diseases.

Adult↗