Biomedical subjects

Beatriz Alvarez-Castelao

Publications and source records attributed to Beatriz Alvarez-Castelao.

3 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.

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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.

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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.

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