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Use of GFP fusions to analyze the mechanisms involved in aggregation of human VAPB protein expressed in Saccharomyces Cerevisiae

Grant number: 17/04094-0
Support type:Scholarships abroad - Research Internship - Scientific Initiation
Effective date (Start): June 12, 2017
Effective date (End): August 31, 2017
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Luis Eduardo Soares Netto
Grantee:Eduardo Tassoni Tsuchida
Supervisor abroad: Judith Frydman
Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Local de pesquisa : Stanford University, United States  
Associated to the scholarship:14/24541-3 - Use of GFP fusions to analyze the mechanisms of toxicity of human VAPB expressed in Saccharomyces Cerevisiae, BP.IC

Abstract

VAPB is a transmembrane protein located in the endoplasmic reticulum (ER) due to the presence of a transmembrane domains (TMD) in its C-terminal region, besides a MSP (Major Sperm Protein) domain in its N-terminal region. The mutation of a proline by a serine at the position 56 of the MSP domain is involved with the familial type 8 of Amyotrophic Lateral Sclerosis (ALS). ALS is a neurodegenerative disorder that affects selectively motor neurons and 10 percent of the cases are familial. The mutated VAPB P56S prevents the cleavage and secretion of MSP and its interaction with ephrine receptors (EphR) and also makes the protein resistant to solubilization in non-ionic detergent and induces the formation of aggregates that sequester the wild-type form in a negative dominant effect. Aggregation is an early and physiological feature of protein quality control (PQC) associated with the maintenance of cellular proteostasis. Some chaperones are involved in the formation of aggregates and destination to degradation pathways, like small heat shock proteins (sHSP), and the Hsp42 is essential to formation of Q-bodies, while Hsp104, to IPOD. Thus, this project has the objective to understand the molecular mechanisms involved in the formation, localization and degradation of VAPB aggregates fused to GFP (GFP-VAPB) by fluorescence and confocal microscopy analysis.