Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.18/2704
Título: Cellular characterization of normal and mutant cystatin B
Autor: Duarte, Ana Joana
Ribeiro, Diogo
Chaves, João
Amaral, Olga
Palavras-chave: Genética Humana
Doenças Raras
Unverritch Lundborg
Cistatina B
Western Analysis
Doenças Genéticas
Data: 19-Dez-2014
Resumo: Unverricht- Lundborg disease (ULD or EPM1, MIM 254800), is a myoclonic epilepsy, caused by mutations in the cystatin B gene (CSTB gene) which lead to impaired function of cystatin B compromising its function. The normal protein is an endogenous inhibitor of cysteine proteinases and has several cellular localizations, it is found in the nucleus, cytosol and lysosome. Identification of a rare molecular mechanism causal of Unverricht Lundborg disease in a unique Portuguese patient triggered research in this field. Skin fibroblasts were obtained with informed consent from a homozygous patient with a rare genotype and from a normal control (anonymized). Fibroblast cell cultures were expanded using standard methods. Western Blotting (WB) and immunofluorescence (IF) experiments were carried out following previous described methods (Pinto et al, 2012). For cell fractionation analysis, cells were collected by scraping and suspending in ice-cold phosphate buffer saline (PBS) and cell fractions where obtained using the methods described elsewhere (Suzuki et al, 2010). Immunofluorescence experimental results revealed consistency with the western blot analysis. Although with a decrease of about 4 fold, in relation to normal, cystatin B is clearly present in the cells’ total fraction and in the nuclear fraction. However, in the cytoplasm the decrease seems to be higher which could suggest a subsequent lower protective anti-protease function compromising cellular and lysosomal integrity. Discussion and future perspectives In patient’s fibroblasts the protein quantity is diminished. Although the nuclear location seems to be preserved in the patient´s cells, the cytoplasmic/lysosomal fraction is clearly decreased.
Descrição: A.J.Duarte received a grant awarded by Genzyme/SPDM
Peer review: yes
URI: http://hdl.handle.net/10400.18/2704
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