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Site-direct mutations on the metaloenzyme Cu,Zn-superoxide dismutase (SOD1): structural and functional aspects

Grant number: 12/24604-0
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): April 01, 2013
Effective date (End): March 31, 2017
Field of knowledge:Physical Sciences and Mathematics - Chemistry
Principal Investigator:Giselle Cerchiaro
Grantee:Tânia Maria Manieri
Home Institution: Centro de Ciências Naturais e Humanas (CCNH). Universidade Federal do ABC (UFABC). Ministério da Educação (Brasil). Santo André , SP, Brazil

Abstract

Mechanisms involving Free-radical and oxidant species are complexes to understand, specially those which involves high redox activity transition metals ions as copper, or those that are part of cell function, as zinc, both present at metaloenzyme Cu,Zn-Superoxide Dismutase (SOD1). It is known that many process involving SOD1 aggregation e peroxidase activity remain in literature discussion even more than 40 years from its discovery and characterization. In this research area the role of mutation on the enzyme related to cases of familial Amyotrophic Lateral Sclerosis (ALS) begins to be elucidated and mutations related to zinc coordination are more intriguing because they seem to cause a more pathogenic ALS. In this way, in this PhD project we intend to study some site-direct mutations on SOD1, as a continuation of master project (FAPESP, 2010/12817-3, ended in 03/2013), taking special attention to zinc site, to verify how the enzyme acts catalytic and structurally. Also, we will study the influence of those mutations in several aspects: I) SOD1 peroxidase activity; II) SOD1 protein aggregation in mammalian cells. There will be 5 steps of work in order to conclude the studies above mentioned: 1) build of mutations by PCR; 2) expression and purification of mutated hSOD1 (hSOD1mut); 3) catalytic activity studies of hSOD1mut and structural studies by electron paramagnetic resonance (EPR); 4) peroxidase activity and protein aggregations assays when hSOD1mut are inside of mammalian cells, by genic expression; 5) Crystallization of hSOD1mut. (AU)