Larry R. Jones - US grants

DESCRIPTION (provided by applicant): This renewal application describes ongoing studies on characterization of key proteins regulating Ca release from junctional sarcoplasmic reticulum (SR) in cardiac muscle. Four proteins will be studied, which are abundantly expressed in heart, co-localize to the junctional SR membrane, and form a complex. The four proteins investigated are junctin, triadin 1, calsequestrin, and the ryanodine receptor. The interactions stabilizing the complex of these four proteins at the junctional SR membrane will be resolved and the role of the complex in regulating Ca release elucidated. Junctin and triadin 1 are homologous, integral membrane proteins which bind to each other, to calsequestrin, and to the ryanodine receptor. They are responsible for anchoring calsequestrin to the ryanodine receptor at the lumenal face of the SR membrane. The sites of junctin and triadin 1 binding to each other, to calsequestrin, and to the ryanodine receptor will be localized, and the effects of junctin and triadin 1 on the channel activity of the ryanodine receptor will be defined, alone and in combination with calsequestrin. Junctin appears to promote junction formation with sarcolemmal membranes, and an attempt will be made to identify putative sarcolemmal docking protein(s) which may bind to the cytoplasmic domain of junctin. Calsequestrin is the major Ca-binding protein located in the lumen of the junctional SR of cardiac muscle, which stores the Ca required for Ca release. The sites of calsequestrin binding to junctin and to triadin 1 will be localized. Junctin and triadin 1 effects on Ca binding to calsequestrin will be characterized, and the effects of calsequestrin on the channel activity of the ryanodine receptor will be determined. Calsequestrin will be crystallized, and its 3-D structure determined in the presence and absence of Ca, complexed with and without the calsequestrin-binding domains of junctin and triadin 1. To assess the function(s) of junctin, triadin 1, and calsequestrin in intact myocytes and in live animals, the wild-type and mutated proteins will be overexpressed in transgenic mouse hearts. Effects of overexpression of the proteins on cardiac membrane biochemistry, ultrastructure, and intact cellular physiology will be investigated. Completion of these studies will increase our understanding of the role(s) of junctin, triadin 1, and calsequestrin in regulating the channel activity of the ryanodine receptor, in maintaining the molecular architecture at the junctional SR membrane, and in controlling the strength of the heartbeat. New animal models useful for investigation of cardiac hypertrophy and heart failure will result.

Phospholamban (PLB) is the principal membrane protein of the heart phosphorylated in response to P-adrenergic stimulation. The protein is localized to cardiac sarcoplasmic reticulum (SR), where it exists as a pentamer of small, tightly associated, 52-amino acid subunits. When dephosphorylated, PLB inhibits the Ca pump and Ca transport by cardiac SR. Phosphorylation of PLB at Ser16 and Thr17 disinhibits the Ca pump, stimulates active Ca sequestration, and increases the rate of myocardial relaxation during beta adrenergic activation. Precisely how this detailed regulation occurs is currently unknown. In this application we propose structure/function studies on PLB to define a molecular mechanism of action. Aim 1 will address protein structure in native SR vesicles. The membrane protein topology, pentameric organization, and molar stoichiometry with the Ca pump will be determined. These studies will lay the framework for expression studies correlating protein structure with function. In Aim 2, PLB will be expressed in atrial tumor cells, which contain Ca pumps, but no PLB. Both wild-type and mutated subunits will be expressed. Using this cellular reconstitution system, we will determine which amino acids stabilize the pentamer, and if the pentameric structure is required for Ca pump regulation. Whether the Ca channel activity of PLB is involved in this process will be investigated, and the mechanism of regulation of active Ca transport by the phosphorylation sites will be determined. In Aim 3, mg quantities of PLB will be expressed and purified from insect cells. The goal here is to produce sufficient material for detailed biochemical studies, including protein crystallization to determine the three dimensional structure. Co- expression of PLB with the Ca pump in insect cells will also be attempted. In Aim 4, expression of wild-type and mutant PLB subunits will be targeted to mouse atrium and ventricle in transgenic animals. By overexpressing wild-type PLB, the role of the PLB/Ca pump stoichiometry in controlling the rate of myocardial relaxation will be assessed. By expressing mutant PLB subunits in mouse hearts, we hope to identify transdominant mutations that disrupt the function of the wild- type pentamer. Mutations that destabilize the pentamer and/or alter the phosphorylation sites will be investigated. Thus, the studies will address the cellular and molecular physiology of PLB, from the purified protein level to the whole animal. By correlating experimental results from Aims 1-4 a complete picture of PLB structure and function will emerge. The results will be important for a precise understanding of catecholamine regulation of the heart.

The overall goal of this program project is to generate new knowledge concerning specific molecular mechanisms that modulate the major functions of cardiac and vascular muscle, including contractility, growth and cellular proliferation. The unifying subtheme selected for this competitive renewal is: Ca2+ and protein phosphorylation: interacting subcellular mechanisms that modulate contractile and growth properties of cardiac and vascular muscles. This group of investigators will pursue, through interactive and collaborative studies, four major objectives under the general subtheme: 1) To evaluate the role of selected cell cycle regulatory proteins in the induction or maintenance of cardiac cellular proliferation by targeted expression in transgenic animal models; 2) To delineate the mode of regulation of key protein phosphatases implicated in modulation of growth, cell cycling and contractile function of cardiac and vascular cells; 3) To determine the role of Ca2+ in cardiac muscle contraction and cellular proliferation via specific investigation of intracellular uptake and release mechanisms; and 4) To identify and characterize specific steps in the vascular cell cycle that are dependent upon irreversible protein modification via Ca2+-dependent proteolysis. Four projects and three core units have been assembled to address these objectives. Project 1 investigates Ca2+-dependent cysteine proteases in regulation of the vascular cell cycle. Project 2 explores the mechanisms of cardiomyocyte proliferation by expressing mutant growth regulatory proteins in transgenic mice. Project 3 analyzes Ca2+ transport mechanisms in cardiac sarcoplasmic reticulum via structural/functional analysis of the proteins, phospholamban and Ca 2+ ATPase. Project 4 studies the regulation of phosphoprotein phosphatases common to both cardiac and vascular tissues that modulate contractility and growth. Collectively, this Program Project applies the methodologies of protein biochemistry and molecular biology to investigate highly fundamental mechanisms of cellular regulation in cardiovascular tissues. The common use of transgenic animal models to explore protein function in a physiological milieu promises to yield novel observations at several levels including both growth and contractile function. The basic studies proposed have important implications to the health-related problems of heart failure, regeneration of cardiac muscle and modulation of vascular tissue growth.

The long term goal of this research is to elucidate the mechanism by which phospholamban (PLB) regulates the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR which is composed of five identical monomers. Dephosphorylated PLB inhibits the Ca pump and Ca transport by SR, suppressing basal myocardial contractility. Phosphorylation of PLB during beta-adrenergic stimulation of the heart reverses Ca pump inhibition, augmenting contractility. For this renewal period, we will test the novel hypothesis that the PLB monomer is the active species inhibiting the Ca pump in the SR membrane. To test this hypothesis, four Specific Aims are proposed, which will examine PLB structure and function from the purified protein level to the level of the live animal. In Aim 1, the role of the PLB monomer in SERCA2a regulation will be investigated using co-expression of PLB with SERCA2a in Sf21 insect cells. The goal is to correlate the monomeric propensities of PLB mutants with the degree of SERCA2a inhibition. Taking advantage of this high-level expression system, we will resolve the kinetic step(s) regulated by PLB in the ATPase reaction scheme, and correlate changes in ATPase kinetics with PLB's effect on the rotational mobility of SERCA2a in the membrane. Aim 2 proposes to identify the sites of PLB:SERCA2a interaction in the membrane. This will be done by co- reconstituting SERCA2a into liposomes along with bioengineered PLB containing covalently attached photoaffinity- and spin-label probes. SERCA2a peptide fragments tagged with PLB photoaffinity probes will be sequenced. Residues of spin-labeled PLB in contact with the Ca pump will be identified by EPR spectroscopy. Aim 3 examines the dynamic equilibrium between PLB pentamers and monomers. The hypothesis will be tested that phosphorylation of PLB stabilizes monomeric mutants of PLB will be overexpressed in transgenic mouse hearts to assess the effects of the monomer on cardiac performance. Overexpression of superinhibitory PLB monomers in myocardium should yield dominant phenotypes exhibiting strong depression of contractility, conceivably leading to new animal models of heart failure. A total picture of PLB structure and function will emerge from the studies proposed. New insights on catecholamine regulation of the strength of the heartbeat will result.

DESCRIPTION (provided by applicant): The long term goal of this research is to elucidate the molecular and biophysical mechanism by which phospholamban (PLB) inhibits the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR, which is composed of five identical monomers. Previously, we demonstrated the PLB monomer is responsible for binding to SERCA2a and inhibiting it. Now, we propose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzyme inhibition, and determine how the molecular interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and phosphorylation. Emphasis will be placed upon identifying amino acids in the inhibitory complex that interact directly, taking advantage of our newly developed chemical cross-linking method. In Aim 1, we will perform Cys-scanning mutagenesis of PLB to localize distinct sites along its primary structure that cross-link to endogenous Cys residues of SERCA2a. The cross-linked Cys residues of SERCA2a will be directly identified by protein purification/peptide sequencing. In Aim 2, Lys residues of SERCA2a that cross-link to distinct sites of PLB will be localized. By use of crosslinking agents as molecular rulers and combining results from Aims 1 and 2, we will develop an accurate 3-D model of the binding-complex formed between the PLB monomer and SERCA2a. In Aim 3, the effects of Ca concentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLB to SERCA2a will be investigated. The hypothesis tested is that PLB binds exclusively to the Ca-free form (E2) of SERCA2a, but only that E2 state that has bound ATP or ADP. In Aim 4, we will determine how Ca relieves PLB inhibition of SERCA2a. We hypothesize that PLB binds preferentially to E2, antagonizing Ca binding to SERCA2a, and that Ca binds preferentially to El, antagonizing PLB binding to SERCA2a. The Ca-binding site of SERCA2a responsible for dissociating PLB from the pump will be identified, and the effect of PLB on the Ca-binding affinity of SERCA2a will be quantified. In Aim 5, we will determine how phosphorylation of PLB by protein kinases relieves PLB inhibition. The hypothesis tested is that phosphorylation of PLB directly dissociates it from SERCA2a. Here we will also determine if the two phosphorylated residues of PLB, Ser 16 and Thr 17, interact directly with SERCA2a to aid in enzyme inhibition. PLB is a key regulator of myocardial contractile dynamics. By defining its molecular mechanism of action on the Ca pump, new insights on PLB regulation of the strength of the heartbeat will result, that may ultimately lead to the design of new drugs to treat heart failure.