Biology subjects

Autry, J. M.

Publications and source records attributed to Autry, J. M..

3 recordsLinked to original sources

bioRxiv2019

Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

We have analyzed gene transcription, protein expression, and enzymatic activity of the Ca2+-transporting ATPase (SERCA) in horse gluteal muscle. Horses are bred for peak athletic performance but exhibit a high incidence of exertional rhabdomyolysis, with myosolic Ca2+ suggested as a correlative linkage. To assess Ca2+ regulation in horse gluteus, we developed an improved protocol for isolating horse sarcoplasmic reticulum (SR) vesicles. RNA-seq and immunoblotting determined that the ATP2A1 gene (protein product SERCA1) is the predominant Ca2+-ATPase expressed in horse gluteus, as in rabbit muscle. Gene expression was assessed for four regulatory peptides of SERCA, finding that sarcolipin (SLN) is the predominant regulatory peptide transcript expressed in horse gluteus, as in rabbit muscle. Surprisingly, the RNA transcription ratio of SLN-to-ATP2A1 in horse gluteus is an order of magnitude higher than in rabbit muscle, but conversely, the protein expression ratio of SLN-to-SERCA1 in horse gluteus is an order of magnitude lower than in rabbit. Thus, the SLN gene is not translated to a stable protein in horse gluteus, yet the supra-high level of SLN RNA suggests a non-coding role. Gel-stain analysis revealed that horse SR expresses calsequestrin (CASQ) protein abundantly, with a CASQ-to-SERCA ratio [~]3-fold greater than rabbit SR. The Ca2+ transport rate of horse SR vesicles is [~]2-fold greater than rabbit SR, suggesting horse myocytes have enhanced luminal Ca2+ stores that increase intracellular Ca2+ release and muscular performance. The absence of SLN inhibition of SERCA and the abundant expression of CASQ may potentiate horse muscle contractility and susceptibility to exertional rhabdomyolysis.

biochemistry↗

bioRxiv2019

Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry

We have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activity in vitro, yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the same a- and d-arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on the e-arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that the e-arm and hydrophobic residues in the adjacent b-arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN-SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.

biophysics↗

bioRxiv2019

SERCA2a-activating PLB mutant improves calcium handling in dilated cardiomyopathy model of hiPSC-CMs

There is increasing momentum toward the development of gene therapy for heart failure (HF), cardiomyopathy, and other progressive cardiac diseases that correlate with impaired calcium (Ca2+) transport and reduced contractility. We have used FRET between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (its ventricular peptide inhibitor) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLBM) that were engineered to compete with the binding of inhibitory wild type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by utilizing the reserve adrenergic capacity of PLB to enhance baseline cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of L31A/I40A-PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of L31A/I40A-PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used an hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of L31A/I40A-PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Based on these results, PLBM transgene expression is a promising gene therapy strategy for cardiomyopathies associated with impaired Ca2+ transport and decreased contractility.

biophysics↗