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Production and functional analysis of human neuronal cells genetically modified to regulate the ubiquitin ligase FBXO25 expression

Grant number: 11/21891-5
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): April 01, 2012
Effective date (End): July 31, 2013
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcelo Damário Gomes
Grantee:Adriana Oliveira Manfiolli
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


FBXO25 protein, identified in silico in 1999, is one of 80 F-box proteins that serve as specificity factors for a family of ubiqutin ligases composed of Skp1, Rbx1, Cullin1 and an F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. FBXO25 is able to form a functional SCF1 ubiquitin (Ub) ligase complex and FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FANDs) that is involved in nuclear ubiquitination. Interestingly, it was demonstrated that FANDs colocalizes with aggregates of polyglutamine (PoliQ) proteins characteristic of several neurodegenerative disorders such as Huntington's disease and several spinocerebelar ataxias. In addition, overexpression of SCF1 (FBXO25) prevented aggregation of polyQ-containing proteins in cultured cells prone to develop the abnormal accumulation of these proteins. Both Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) studies demonstrated that the predominant site of FBXO25 expression was the central nervous system and interestingly, an FBXO25 gene variant has been linked to a genetically inherited cerebral disorder. This way, in the present study we propose to produce human neuronal cell lines (SH-SY5Y) that overexpress a stable and inducible FBXO25, besides producing a knockout SH-SY5Y cell line for FBXO25. In these modified cell lines we will analyze the viability, cell growth and differentiation, as well as differentially regulated genes by DNA microarray. This cell model is very important to complement our studies about the FBXO25 role in preventing the aggregation of mutant huntintin during the development of Huntington's disease and enables us to evaluate the degradation of substrates of the SCF1 (FBXO25) complex previously identified.

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
TEIXEIRA, FELIPE R.; MANFIOLLI, ADRIANA O.; VIEIRA, NICHELLE A.; MEDEIROS, ANA CARLA; COELHO, PRISCILA DE O.; GUIMARAES, DIMITRIUS SANTIAGO; SCHECHTMAN, DEBORAH; GOMES, MARCELO D. FBXO25 regulates MAPK signaling pathway through inhibition of ERK1/2 phosphorylation. Archives of Biochemistry and Biophysics, v. 621, p. 38-45, MAY 1 2017. Web of Science Citations: 2.

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