Date of Completion

5-12-2019

Embargo Period

4-24-2019

Advisors

Dr. Kimberly Dodge-Kafka, Dr. Lixia Yue and Dr. Anne Delany

Field of Study

Biomedical Science

Degree

Master of Science

Open Access

Open Access

Abstract

Cardiovascular disease remains the number one cause of mortality worlwide. Heart failure occurs when the heart does not meet the body’s demand. Although significant medical advances have been made, 50% of heart failure patients still die within five years of prognosis, suggesting the need for more research. Alterations of inhibitor-1 (I-1) levels and activity have been associated with heart failure. I-1 PKA-mediated phosphorylation activates its inhibitory activity against PP1, which leads to calcium re-uptake into the sarcoplasmic reticulum (SR). Calcium mishandling plays an important role in cardiac diseases, such as heart failure. Since I-1 is at the top of this signaling pathway, attention has been giving into the function of I-1 in heart failure. AKAP18, a major cardiac scaffold, organizes a multimolecular complex comprised of I-1 and other signaling proteins, which allows the scaffold to regulate Ca2+ reuptake into the SR. Our lab was interested in looking into the binding between AKAP18 and I-1.

Our immunoblot analysis with AKAP18 siRNA showed that I-1 was not phosphorylated (by PKA) when AKAP18 was silenced. Previous worked from the lab narrowed the I-1 binding domain to the 1-75 segment of AKAP18 (unpublished work). Our lab looked into this further through protein pulldowns, which showed that I-1-GST immunoprecipitated with AKAP18g(1-75). Furthermore, I-1 and AKAP18g(1-75) are highly unstructured, which motivated us to study their binding structurally through NMR spectroscopy. Our 2D 1H-15N-Heteronuclear Single Quantum Coherence (HSQC) titration experiment showed that AKAP18g(1-75) remained disordered when titrated with I-1. But there were several chemical shifts perturbations in the HSQC spectrum, the most noticeable being a glycine residue we hypothesize to be Gly28. Chemical shift perturbations could be indicative of binding, suggesting the Gly28 might be part of the I-1 binding domain. Defining the AKAP18/I-1 binding domain can lead to the generation of reagents that disrupt their binding, which could help understand the role of AKAP18/I-1 binding in heart failure, and could open the door to new therapeutic approaches.

Major Advisor

Dr. Kimberly Dodge-Kafka

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