Melvin I. Simon, Ph.D. - US grants

Bacteria have a complex information processing system that allows them to respond to changes in specific chemicals in their environment by modifying the pattern of flagella rotation and thus regulating their motility. Many of the genes that encode specific proteins in this pathway have been identified, sequenced, and their gene products overproduced. A family of transmembrane proteins plays a central role in this process. They interact with specific ligands and transduce these interactions into signals that affect flagellar rotation. They are analogous in structure and function to cell surface receptors found in a variety of other prokaryotes as well as in complex eukaryotic systems. We plan to continue to focus our attention on this family of signal transducing molecules in order to determine how they function. We will use a variety of techniques to specifically mutagenize these genes and study the effects of mutated gene products on the function of the transducers and their interaction with other components of the chemotaxis pathway. We will develop techniques for studying the function of these proteins in vitro and methods for measuring the coupling between ligand binding events, signalling and the reversible methylation of the transducer. We hope to understand how these molecules transmit information across the cell membrane and transduce this information into a change in the chemistry of the cells.

All cells and organisms detect chemical and physical changes in their environment via cell surface receptors. Information collected by these receptors regarding the concentration of hormones, growth factors or sensory stimuli is transduced into intracellular changes in regulatory molecules that control cell growth and differentiated cell function. We have teen studying the GTP-binding protein family. The G-proteins are generally composed of alpha, beta and gamma subunits, and to serve to couple receptors to a variety of changes in intracellular metabolism. Our work has led to the partial characterization of a family of genes that encode the G-proteins. In continuing this work we will determine the extent of diversity in the G-protein gene family in mammals by isolating cDNA clones corresponding to G-protein subunits in various tissues. We will test the hypothesis that there are specialized G-proteins in highly differentiated cells that couple to specific receptor and effectors. We plan to generate variant G-protein cDNAs by in vitro mutagenesis. Gene product expression and overproduction systems will be developed to study the effects of specific modifications in the amino acid sequence of the G- protein subunits on signal transduction activity. The experiments will focus on understanding the structural basis for specificity in the interaction of G-proteins with receptors and effectors and the regulation of G-protein function. In vivo studies will be designed to understand the nature of the interaction between different G-proteins and the intracellular integration of signals. We will also define the mechanisms that control specific G-protein gene expression in differentiated cells.

nervous system; genetically modified animals; aging;

In the course of our work on the control of flagellar formation we showed that flagelin gene expression was controlled by DNA inversion mediated by Hin Recombinase. Our detailed studies of the mechanisms of this reaction have revealed that it involves the formation of nucleo-protein complexes that include multiple proteins associated with specific DNA binding sites. The assembly of similar structures involving elaborated DNA-protein interactions has been implicated in the mechanisms involved in specific gene expression, viral integration and transposition. Hin-Recombinase offers a relatively simple model in which to study the role of "enhancers" and other "factors" in the formation of the complex DNA-protein structures required to achieve efficient, site specific, directional recombination. Our immediate goal is to understand in detail the nature of the Hin Recombinase reaction, i.e. the mechanism of Hin-DNA interaction, the activation of the Hin-DNA cleavage reaction, the nature of the interaction of Hin with the enhancer complex and the mechanism involved in Hin-DNA inversion. We will use extensive mutagenesis, studies of invivo function and in vitro reconstitution with modified protein and DNA sites to understand how the recombinase and enhancer function during the course of the reaction.

All cells have the ability to detect and respond appropriately to small changes in the concentration of a variety of chemical signals. In the microbial world, bacterial behavior, sporulation, pathogenesis, and gene regulation all incorporate a series of steps that involve signal transduction. We have shown that in bacterial chemotaxis a complex information processing system includes a transmembrane receptor protein which is capable of binding ligand, transmitting information across the membrane and initiating an citation and an adaptation process. Excitation involves the activation or inactivation of a protein kinase and the regulation of phosphorylated levels of the CheY protein. CheY regulates the "effector" element in this system, a "switch" which in turn controls the direction of flagellar rotation. The adaptation mechanism involves the modification of the receptor and the modulation of its ability to influence kinase activity. Homologues of the chemotaxis kinase and regulator proteins are found in many other microbial systems and the entire information processing circuit is analogous to signal transduction systems in eukaryotic organisms. This grant focuses on the continued detailed study of the components of the chemotaxis system and how the properties of the information processing system are derived from the interactions between their components. We will probe the nature of the transmembrane regions of the receptor using mutagenesis and crosslinking to understand the process of transmembrane transmission of information. Functional domains of the transmembrane receptor will be overproduced and their interactions with the kinase coupling protein and other components of the chemotaxis system will be studied in a reconstitution system. We will also reconstitute the effector portion of the pathway and study the interaction of the CheY protein with the flagellar "switch". Details of these processes will be directly applicable to understanding other homologous systems in bacteria and eventually will allow us to understand general "design" strategies involved in biological information processing systems.

The retina provides an ideal system in which to examine the nature of the mechanisms involved in late onset degenerative diseases that may be related to aging. A great deal is known about the biochemistry involved in the photoreceptor cascade that converts light to electrical signals and there is a great deal known about the structure of the retina and its development and differentiation. By using transgenic animals in the past four years we have developed lines of animals that express dominant mutant forms of components of the phototransduction cascade and studied the effects of these mutations on photoreceptor function and on retinal degeneration. These systems provide us with an opportunity to examine the effects of aging on comprised retinal systems and compare these models with late onset retinal degeneration disease. In continuing this project, we will generate more sophisticated mouse models by using homologous recombination to disrupt and replace genes encoding specific major components of the phototransduction cascade and by using cell type specific promoters to generate reporter systems that respond to growth and differentiation factors in the retina. In addition to defining the role of the components of the phototransduction cascade in maintaining cells in the retina, we will also study the effects of growth factors on retinal cell differentiation and maintenance. We will use transplantation and transgenic techniques to generate animals that have cells that secrete different growth factors at different stages during retinal development and to study the effects of these factors on the degeneration and differentiation. In addition, we will use transgenic mice and the reporter group s that we developed in order to transplant cells into the retinas of new born and eventually mature animals in order to try to ameliorate the effects of retinal degeneration.

The G protein signal transduction system represents a source of important specific targets for a variety of therapeutic approaches ranging from the control of blood pressure, allergic response, kidney function, hormonal disorders to neurological disease and pain. It has been estimated that 30-40% of the pharmaceutical products sold in the United States target G protein mediated signal transduction system. We are analyzing the interactions in this complex information processing system at the level of purified proteins, the cellular level and the whole animal level. If we can connect the biochemical changes that are the result of specific protein modifications to the effects of those mutations on cellular function and eventually the effects of cellular function on complex physiology or changes in development and differentiation, we will have gone a long way toward an integral understanding of these systems. In the next grant period we intend to pursue these molecular biochemical and genetic approaches. We will analyze transgenic mice that we have constructed together with our collaborators that result in the disruption or deletion of all of the G/alpha subunits in the Gq signaling pathway as well as mice that we have constructed that have the G/alpha13 and G/alpha12 genes disrupted and animals with deletions of the PI-PLC beta2 and PI-PLC beta4 genes. We will try to determine the specific effects of these mutations on cell and tissue function. We will also examine the effects of mutants in tissue culture and delineate the functions of individual G protein pathways. We will study the modifications of the different components of the G proteins that result from the activation of specific signaling pathways and understand how these modifications affect and integrate different signal transducing pathways. Finally, we will continue our studies on the molecular modification of individual G protein components and measure the effect of modification on protein- protein interaction using plasmon resonance and a variety of other techniques to develop a quantitative picture of the elements necessary for the interaction of components in a specific pathway. These studies will provide a deeper insight into the function of two less well studied G protein families, the Gq and the G12 families. We hope eventually to be able to develop models of how specific signal transductions pathways process information and how these pathways interact and we expect that such models will be important in predicting and developing new approaches to ameliorating the effects of disease.

The mammalian visual system provides an excellent opportunity to understand how biological information processing systems operate in vivo and how they integrate with other cellular functions. Our long term goals are: (1) To understand the steps and biochemical mechanisms that operate in vivo to regulate and maintain the extraordinary performance characteristics of the phototransduction cascade in mammalian photoreceptors. (2) We want to understand how changes in the phototransduction circuit generate signals that induce cell death (apoptosis) and eventually retinal degeneration. A variety of age related, late onset retinal diseases are the result of apoptotic signaling and neural degeneration. Genetic modifications of components of the visual system provide models to study these effects and to evaluate gene therapy and pharmaceutical approaches designed to block apoptotic signaling in order to delay damage and prevent cell death in photoreceptors. In the next few years our laboratory will focus on developing multiple mutations and combinations of mutants and ectopic gene expression by breeding and by generating new genetic constructs designed to answer specific questions about the photocascade and about retinal degeneration. Some of our specific short term goals are: (1) To determine the nature of the factors that accelerate the GTPase activity of visual transducin. Is the RGS9 protein specifically involved in the rapid termination of the transducin mediated signal in vivo? Is RGS9 alone sufficient to account for this "turn off"? What is the nature of the molecular interactions of RGS with transducin? (2) To understand the molecular basis for rapid "turn off" and reactivation that we observe in Rhodopsin Kinase deficient photoreceptors. (3) We will focus on the mechanisms that are required to generate light induced signals that lead to apoptosis, particularly in Rhodopsin Kinase and Arrestin deficient mice; (4) We will try to determine the in vivo role of other gene products involved in the dephosphorylation and the recycling of Rhodopsin during the photocascade and examine how they might be involved in inducing apoptosis; (5) We will continue to study the effects of deletion or overexpression of genes that are involved in apoptotic signaling or in blocking apoptosis under conditions that ordinarily lead to retinal degeneration.

The goal of Systems Biology is the construction and experimental validation of mathematical models that explain and predict the behavior of biological systems. Systems Biology is a synergistic integration of theory, computation and experiment. Intuition is not reliable when confronted with the highly interconnected reaction networks that underlie biological processes.

The Second International Conference will be held in November 2001. It seeks to promote the growth and development of the field of Systems Biology. The goals include: establishing the foundations of the field; presenting new results; identifying promising directions; fostering communications; and helping to organize the community. A top priority is to adopt a sound foundation for theory, software and experiment. The meeting seeks to be a specific forum for Systems Biology.

This portion of the program project grant is focused on the molecular circuitry that underlies the function of the subsets of neurons that specifically express the Mrg family of GPCRs. The intracellular events triggered by ligand activation of receptor are ultimately reflected in the function and phenotype of the animal. Thus, we will further characterize the ligands that interact with the MrgA, MrgB, MrgC, and MrgD receptors and their homologues. We will establish the nature and molecular role of the specific macro-molecules that respond to and regulate receptor function in each of the different Mrg-expressing cell types. We will design and generate mutations and genetic constructs that change the activity of specific signaling molecules associated with the Mrg system. Together with our colleagues in this Program Project we will study the effects of these perturbations on molecular, cellular and animal phenotypes. The remarkable specificity of expression of the Mrg receptors in subsets of cells that have been associated with the perception of pain suggest that they play a role in neuropathic pain and associated disease. The comprehensive picture of the Mrg system provided by this program project grant will lead to a better understanding of pain perception and eventually to the development of novel treatments for disorders associated with pain.