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2.
Hum Mutat ; 41(6): 1131-1137, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32126152

RESUMO

Alexander disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander disease.


Assuntos
Doença de Alexander/genética , Proteína Glial Fibrilar Ácida/genética , Splicing de RNA , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Isoformas de Proteínas/genética , Adulto Jovem
3.
J Biol Chem ; 285(14): 10527-37, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20110364

RESUMO

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.


Assuntos
Doença de Alexander/metabolismo , Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Cadeia B de alfa-Cristalina/metabolismo , Doença de Alexander/patologia , Astrócitos/citologia , Western Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Imunofluorescência , Proteína Glial Fibrilar Ácida/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Mutação/genética , Ubiquitina/metabolismo
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