Functional Investigation of SERCA-Regulatory Subunits: The "Regulins"

• Author / Creator

  Bak, Jessi J.

• Calcium homeostasis is essential and central to a variety of cellular processes from cell life and death pathways to metabolism to muscle contraction. One component that is essential to intracellular calcium regulation is known as the sarco(endo)plasmic reticulum Ca-ATPase, or SERCA. SERCA resides in the sarcoplasmic reticulum (SR) membrane and is responsible for pumping calcium ions across the SR membrane to be stored in the SR, the calcium storage organelle. SERCA is expressed in almost all cell types, is involved in many physiological processes, and is also regulated by a variety of single-pass transmembrane peptides, which mostly act to lower SERCA’s apparent calcium affinity. The structure and function of two regulatory peptides in relation to SERCA has been well studied over the years, and excitingly, these studies can now be expanded to a family of peptide regulators. Recent bioinformatic investigations have shed light on new SERCA-regulators expressed throughout the body collectively referred to as the “regulins.” In this family there is myoregulin (MLN) in skeletal muscle, ubiquitously expressed another-regulin (ALN), endoregulin (ELN) in endothelial and epithelial tissue, and DWORF (Dwarf open reading frame) in cardiac muscle. Interestingly, Ca-ATPase regulation is not isolated to mammals and a variety of invertebrate regulators, known as sarcolambans (SLB), have also been identified and found to regulate the invertebrate Ca-ATPase. While initial studies have been published to show physiological roles and their effect on SERCA, specific kinetic details of these effects have not been established. Thus, one goal of the work presented here was to characterize the effect of MLN and ALN on the maximal activity and apparent calcium affinity of SERCA in a controlled, in vitro system. The second goal of this study was to determine if sequence variation among regulators, SLB peptides in this case, could influence SERCA regulation. To do this, purified SERCA, regulatory peptide, and lipids were reconstituted to form isolated proteoliposomes from which SERCA activity could be measured through a coupled-enzyme ATPase assay. Since this was the first time these regulators were put in this system, a series of experiments to confirm the incorporation and orientation of these peptides in proteoliposomes was done. From this, it was found that MLN incorporated into proteoliposomes in the correct orientation and acted to lower the maximal activity of SERCA. ALN also lowered the maximal activity in addition to lowering the apparent calcium affinity of SERCA. The four SLB peptides used in this study all had distinct effects indicating that sequence variation does have a role in the way SERCA is regulated. The work presented here is the first to elucidate the specific kinetic effect of newly identified regulatory peptides of SERCA.

• Subjects / Keywords

  • Calcium
  • Muscle
  • SERCA
  • Biochemistry

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-je9c-ze37

• License

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