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Use of GFP fusions to analyze the mechanisms of toxicity of human VAPB expressed in Saccharomyces Cerevisiae

Grant number: 14/24541-3
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2015
Effective date (End): March 19, 2018
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Luis Eduardo Soares Netto
Grantee:Eduardo Tassoni Tsuchida
Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:13/08028-1 - CEGH-CEL - Human Genome and Stem Cell Research Center, AP.CEPID
Associated scholarship(s):17/04094-0 - Use of GFP fusions to analyze the mechanisms involved in aggregation of human VAPB protein expressed in Saccharomyces Cerevisiae, BE.EP.IC

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. 5 to 10% of the total cases of ALS are familial and the remainder, sporadic. The human gene VAPB encodes the VAPB protein, which is located in the endoplasmic reticulum (ER) membrane and is probably involved in diverse cellular functions, including neurotransmission. This protein is associated to FALS 8 and presents an homologous protein in Saccharomyces cerevisiae, SCS2, which is also located in the endoplasmic reticulum. Only two point mutations in the human VAPB have been identified so far (VAPB P56S and VAPB T46I). There are evidences that the VAPB P56S mutation would generate a gain of toxic function with the formation of protein aggregates and induction of UPR (Unfolded Protein Response). The accumulation of unfolded proteins in the ER causes a condition known as ER stress, which induces multiple metabolic responses, as proteolysis by ERAD (Endoplasmic Reticulum Associated Degradation) and transcriptional activation of several genes involved in UPR, both processes related to ER quality control. Several neurodegenerative diseases have in common the formation of protein aggregates, such as Alzheimer's, Parkinson's and ALS and the baker's yeast S. cerevisiae is an appropriate model for studies of these and other neurodegenerative diseases. It is worth mentioning that recent studies point to an alternative hypothesis of haploinsufficiency, in which low levels of VAPB prevent the binding of secreted MSP domain to ephrin receptors, resulting in the death of motor neurons. Thus, in the laboratory are ongoing the construction of different plasmids expressing wild and mutant VAPB at different levels. Knowing that VAPB mutations result in ALS and that individuals with the same mutation in these genes may show different clinical features, we propose to study the susceptibility to ER stress and degradation pathways of VAPB as factors underlying this clinical heterogeneity.