Unphosphorylated phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), the enzyme that regulates cardiac muscle relaxation in humans. In its phosphorylated state, PLN (pS16-PLN, pT17-PLN, and pS16pT17-PLN) does not inhibit SERCA. Dysfunctions in SERCA:PLN interactions and in the PLN phosphorylation mechanism have been implicated in cardiac disease and targeting PLN is becoming a viable avenue for treating heart disease. Specifically, innovative genetic treatments using recombinant adeno-associated virus (rAAV) with S16E-PLN, a pseudo-phosphorylated form of PLN, have shown a remarkable efficacy in reducing the progression of cardiac failure in both small and large animals. The following thesis summarizes efforts to rationally design PLN mutants to tune SERCA function. Using a combination of NMR spectroscopy and biochemical assays, we have built a structure-dynamics-function correlation that shows PLN can be tuned to augment SERCA function by acting on the conformational coupling between the cytoplasmic and transmembrane domain and by pseudo-phosphorylation. Additionally, to better understand the role of mutation in PLN:SERCA interactions, we also investigated a mutant of PLN (R9C) known to be linked to hereditary dilated cardiomyopathy, showing that the mutation disrupts the pentamer-monomer equilibrium, and that these effects are exacerbated under oxidizing conditions. Insights to these issues will provide better paradigms with which to design therapeutic mutants of PLN for treatment of heart failure.
University of Minnesota Ph.D. dissertation. April 2012. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Gianluigi Veglia. 1 computer file (PDF); xiv, 161 pages.
Ha, Kim N..
Rational design of loss-of-function phospholamban mutants to tune SERCA function..
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