Michael Greenberg - US grants

FMRFamide (Phe-Met-Arg-Phe-NH2) is a neuropeptide isolated from clam ganglia; it is stored in granules, and released upon depolarization. FMRFamide is a potent molluscan pharmacon: it is cardioexcitatory or inhibitory depending on species; it causes contractures of various muscles and affects neuronal activity. FMRFamide is only one example of a set of heterogenous, but structurally and functionally homologous, molluscan neuropeptides. Moreover, it is hypothesized that FMRFamide-like peptides are widely distributed among animals, including vertebrates, and may have had a common origin with the opioid peptides in protein evolution. This project focuses on the phyletic distribution and mechanisms of action of FMRFamide. First, three peptides, already identified in the brains of the gastropods, Busycon contrarium and Helix aspersa, will be further purified by high performance liquid chromatography, and their amino acid compositions and sequences determined. The extraction and purification steps will be monitored by bioassays specific for FMRFamide (Busycon radula protactor muscle) or of the opioid peptides (guinea-pig ileum and mouse vas deferens). FMRFamide-like peptides will also be sought in other tissues: porcine adrenal medulla and sympathetic ganglia; and the central ganglia from other invertebrate phyla. As these peptides are characterized, their subcellular localization will be determined, and their release by potassium depolarization will be demonstrated. A particle-bound peptidase, coverting N-terminal extended precursors to FMRFamide, has been proposed; an attempt will be made to demonstrate it. FMRFamide receptors will be characterized by structure-activity studies on particular molluscan hearts and muscles and on rabbit vascular smooth muscle; the selected preparations are sensitive to the peptide; and their responses exemplify the range of its effects. The inotropic effects of FMRFamide will be correlated with its actions on cyclic nucleotide levels. The sucrose gap technique will be used to correlate electrical and mechanical responses.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

Biochemical instrumentation will be acquired for use in research projects in a broad range of basic biological disciplines, including developmental biology, microbiology, protein chemistry, cell biology, biochemistry, cell physiology, molecular biology, and biotechnology. The equipment, which includes microchemical instrumentation for DNA, RNA, and protein chemistry, and ultracentrifuges and related equipment for basic biochemical manipulations, will be part of a shared facility. Access to modern equipment is critical for progress in contemporary biology and in biotechnology-related research.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

Funds for a ultracentrifuge and gel analyzer for Whitney Lab, University of Florida, are requested. All the research efforts will be directed toward aquatic systems and vary over a broad range of subjects. Recently, molecular genetics has been added to the research portfolio at the lab. Among the species studied will be clams, shrimps, and jellyfish.

The electrical stimulation of neurons during the development and maintenance of the nervous system has both immediate and longer term effects on cellular physiology. The long term neuronal responses include alterations in neuronal sprotting, and changes in neurotransmitter, transmitter receptor and ion channel protein production. These cellular changes appear to require the activation of new gene expression, and may be fundamental to processes such as neural development, and information storage. The long-range objectives of the proposed research are to understand the mechanisms by which electrical stimulation controls the expression of genes in neurons, and the function of these electrical stimulation controls the expression of genes in neurons, and the function of these electrically-regulated genes during the development, and maintenance of the nervous system. Recent studies have identified a class of "immediate early genes" that encode mRNAs whose transcription is activated rapidly as a response to electrical stimulation. Many of these genes encode transcription factors that have been hypothesized to control the neuronal cell response to trans-synaptic stimulation. Experiments are proposed that will elucidate the biochemical pathway by which membrane depolarizing agents induce the transcription of two members of the immediate early gene family c-fos and nur/77. Activation of these genes in the pheochromocytoma cell line PC12 by electrical stimulation requires an influx of calcium from the extracellular medium. Depolarization-activation of c-fos is controlled by a calcium response element (CaRE) within the c- fos promoter that binds the transcription factor CREB. Membrane depolarization activates the phosphorylation of CREB raising the possibility that electrical stimulation of gene expression in neurons is mediated by the phosphorylation-activation of a specific transcription factor. The specific aims of the proposed research are: (1) to determine whether the sites of phosphorylation on CREB are critical for transcriptional activation; (2) to characterize the protein kinases that mediate the depolarization response; (3) to identify novel CaREs within the immediate early gene promoters, and CaRE binding proteins, that control alternative pathways for electrical stimulation of gene expression; (4) to test the hypothesis that varying the duration, strength, or repetitive nature of the electrical stimulation alters the pattern of activation of immediate early genes. Given the function of these genes in the process of normal cell growth and differentiation it is likely that alterations in the developmental process that lead to cancer, neurodegenerative diseases, or seizure disorders will involve changes in the regulation of function of the immediate early genes.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

The molluscan cardioexcitatory peptide FMRFamide (Phe-Met- Arg-Phe-NH2), together with four structurally similar peptides, constitute a closely related nuclear family which now includes among its membership two tetrapeptides and three heptapeptides. The tetra-and heptapeptides have different phyletic distributions among the molluscs, and have distinctive effects and receptors on muscles and nerves. Antisera to FMRFamide also cross-react widely with antigens in the nerve and endocrine cells of non- molluscan groups, both vertebrate and invertebrate. Some of this immunoreactivity is due to established peptides, but previously unknown peptides have also been discovered. These immunochemical results are manifestations of a large, heterogeneous, widespread, extended peptide family; it is characterized by an amidated C-terminal dipeptide of the form Arg-X-NH2 (where X is an aromatic residue). The first long-term goal of this project is to examine the tissue distributions, actions and physiological roles of the individual FMRFamide-related peptides (FaRPs) making up the model nuclear family in the pulmonate snail, Helix aspersa. The receptors complementary to the FaRPs will be characterized by studying the structure- activity relations (SAR) of these peptides on whole tissues, biochemical mechanisms, and radioligand-receptor binding properties. The variation in the levels and potency of the FaRPs, as a function of the activity state of the snails will also be examined. The second long-term goal is to determine the phyletic distribution, size, and limits of the extended family of FaRPs, and to define its relationship with the nuclear family in molluscs. New extra-molluscan FaRPs will be sought in central nervous extracts of vertebrates and key invertebrates. The effects, of both the nuclear and extended FaRPs, on vertebrate vascular smooth muscle will be tested and the receptors characterized by SAR. The relationship between the nuclear family of FaRPs and the extended family may reside in the homology of their receptors; the similarities between the peptides themselves are probably due to convergence.

DESCRIPTION (provided by applicant): The human genome contains 8 different isoforms of myosin I, making it the largest family of unconventional myosins expressed in humans. Myosin Ic is perhaps the best studied myosin I isoform due to its proposed roles in dynamic adaptation in hair cells, insulin stimulated GLUT-4 transport, compensatory excocytosis, and nuclear transcription. Despite its association with these cellular processes and disease such as congenital deafness, the molecular role of myo1c in the cell is unknown. It has been proposed that myosin Ic may act as a transporter, a force generator, or a strain-sensing tether. The ability of myosin Ic to function in these roles depends on its abilities to generate force and to modulate its biochemistry in response to load, with each of these roles placing very different and specific mechanical requirements on the myosin. Thus, by understanding the mechanics of myosin Ic, the molecular role of myosin Ic can be deduced. Despite the fact that myosin mechanics are at the heart of its cellular function, very little is known about the mechanics and load dependent mechanochemistry of myosin Ic. Furthermore, it has been proposed that calcium binding to calmodulins on the myosin Ic regulatory domain may play a central role in modulating its mechanics and response to load; however this notion has never been tested experimentally. Also, it is unknown whether calcium induced changes in myosin Ic mechanics and kinetics are relevant to cellular function since the response of myosin Ic to transient increases in calcium has never been studied. This research will address these gaps in our knowledge. Using a battery of single molecule techniques, the specific aims of this research are: 1. Determine the mechanical properties of the myosin Ic working stroke 2. Determine the load sensitivity of myosin Ic 3. Directly measure the effects of transient calcium on myosin Ic mechanics and kinetics All of these experiments will be conducted in the presence and the absence of calcium to test how calcium affects myosin kinetics and mechanics. Besides elucidating the molecular role that myosin Ic plays in the cell and how this role is regulated by calcium and force, this research will also shed light on the diversity within the myosin I superfamily. PUBLIC HEALTH RELEVANCE: Molecular motors within the human body are responsible for generating and responding to forces with malfunction of these motors leading to a wide array of diseases including deafness, hypertension, cardiac failure, and developmental defects. While the ability to respond to forces is central to the function of these motors, little is known about how this is accomplished. Using cutting-edge single molecule techniques, this research will examine force generation and tension sensing in of one of these motors, myosin Ic, helping us to understand both its cellular function and the role that tension sensing plays in molecular motors in both health and disease.

Project Summary/Abstract Familial cardiomyopathies are the leading cause of sudden death in people under 30. Two closely related cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are characterized by remodeling of the tissue in the ventricular wall, often accompanied by fibrosis, and myocyte disarray. These diseases are primarily caused by mutation of the proteins involved in generating or regulating power output by the heart. It has been proposed that HCM is caused by hypercontractility at the molecular level while DCM is caused by hypocontractility at the molecular level; however, it is not clear how mutations at the molecular level lead to alterations in the contractility and structure of heart cells and tissues. The goal of this research is to better understand the connections between molecular-based changes and the changes in contractility and structure at the cellular and tissue levels. To do this, we will harness biophysical, biochemical, cell biological, and tissue engineering techniques to measure contractility across these scales of organization, and then use mathematical modeling to connect these observations. We will examine two model mutations in troponin-T that cause either HCM or DCM. In Aim 1, we will dissect the molecular mechanism of two mutations in cardiac troponin-T, R92Q and ?K210, that cause hypercontractility and hypocontractility at the molecular level, respectively. In Aim 2, we will dissect the effects of these mutations on the structural and contractile properties of stem cell derived cardiomyocytes and engineered tissues. In Aim 3, we will examine whether these mutations affect the ability of cells and tissues to sense and respond to their mechanical environment. This bottom up approach will give unprecedented insights into the mechanisms of cardiac force production, mechanosensing by the heart, and the disease pathogenesis of familial cardiomyopathies.