טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentZhitnitsky Daniel
SubjectConserved Amino Acids in Trans-membrane Domains of PIB-Type
ATPases, and their Role in Metal Translocation
DepartmentDepartment of Medicine
Supervisor Professor Oded Lewinson
Full Thesis textFull thesis text - English Version


Abstract

P-type ATPases are a large and ubiquitous family of trans-membrane ATPases that utilize ATP hydrolysis to drive a diversity of transport processes. The PIB subgroup ( PIB-ATPases) are efflux pumps of transition metals  such as Cu, Ag, Zn2, Cd2 or Pb2. Cu and Zn2 are essential co-factors to many cellular functions, yet in high intracellular concentrations they are highly toxic. Ag, Cd2 and Pb2 have no known biological function and they are extremely toxic, even in low concentrations. PIB-Type ATPases maintain a delicate balance between essential import and toxic overload of both the essential and non-essential transition metals. Mutations in the human Cu-efflux P-type ATPases leads to severe pathologies. In addition, these efflux pumps are essential for virulence and pathogenesis of microbial pathogens, and there is also evidence implicating them with cancer drug resistance. Previous studies have suggested several amino acids, crucial for metal binding and selectivity, but the complete trans-membrane translocation pathway is yet to be characterized. The molecular mechanism of PIB-type ATPases is also still a matter of debate. Using site directed mutagenesis, In-vivo metal resistance assays and in-vitro ATPase assays, I have discovered several novel amino acids, likely involved in ion translocation and selectivity. The spatial distribution of these newly discovered residues suggest the trajectory of the trans-membrane metal translocation. In addition, based on my results, by introducing 7 amino acid substitutions I was able to convert the specificity of a Zn2/ Cd2 pump to Ag.