Joydip Das - US grants

[unreadable] DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse cause major health problems worldwide. Defining the target(s) and elucidating the mechanism of its action at the molecular level is necessary to develop effective prevention. The overall goal of this proposal is to identify alcohol binding sites on a signal transducing protein, protein kinase C epsilon (PKCe), and to determine the secondary structure of these sites. Evidence indicates acute alcohol exposure modulates PKC activity and alters subcellular distribution of individual PKC isoenzymes, but chronic exposure to ethanol leads to an elevation of PKC expression and/or function. Conversely, alteration in the expression of PKC isoforms influences alcohol consumption and behavioral responses to alcohol. While in vivo studies [unreadable] with PKC null mice showed decreased alcohol consumption compared to the wild type, encouraging preliminary in vitro data suggest ethanol inhibits PKC activity. The specific hypothesis to be tested is that there is a site for alcohols in the C1 domain of PKC. The first aim of this proposal is to identify alcohol binding by using fluorescent PKC activators for its allosteric interaction with alcohols and by the use of novel photoreactive alcohols to precisely determine the contact points between the PKCeC1 and alcohols. Upon covalent attachment to the amino acid residues within the binding site(s), the labeled protein will be proteolytically cleaved followed by amino acid sequencing of the generated peptide by mass spectrometry to identify the alcohol site(s) on PKC. The second aim is to characterize the identified binding site(s) by mutational analysis and high resolution X-ray crystallography. These will lead to the final aim of generating PKC mutants with altered alcohol sensitivity which could be exploited in studies in cells or mice to test for the direct involvement of PKC in ethanol's action. The significance of my studies is to establish and characterize alcohol binding site in PKC and to develop mutants with altered alcohol sensitivity which will contribute to understanding the mechanism of alcohol action. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse is a major cause of death (annually 75,000-100,000 deaths in the US) with an economic burden of 184 billion dollars/year in the US. Defining the target(s) and elucidating the molecular mechanism of its action is needed for effective intervention. The objective of this proposal is to define the molecular mechanisms by which alcohols exert their action on intracellular signal transduction pathways in brain. Alcohols are known to alter the expression and activity of Protein Kinase Cs (PKC), a family of kinases mainly expressed in the brain. While PKC epsilon knock-outs showed significant decrease in alcohol consumption and increase in tolerance to ethanol compared to wild type mice, the PKC gamma knock-outs showed significant increase in alcohol consumption and decrease in tolerance. PKC[unreadable] and PKC3 knock-outs also showed opposite properties in regulating responses to GABAA (3-amino butyric acid) receptors. GABAA is a ligand-gated ion channel in brain and believed to be an important target of alcohol. Structurally, PKC3 has its regulatory C1 domain (combination of C1A and C1B) at the N terminus followed by the regulatory C2 domain. On the other hand, PKC[unreadable] has its C2 domain at the N terminus followed by the C1 domain. The C1A and C1B subdomains of epsilon and gamma differ significantly in terms of their ligand binding properties. The central hypothesis to be tested is that the structural and ligand binding differences in the regulatory domains of PKC[unreadable] and PKC3 are responsible for their differential behavioral response to alcohol. The first aim of this proposal is to determine how alcohols regulate PKC[unreadable] and PKC3 activity and if there is an alcohol binding site in their regulatory domains. In order to identify alcohol binding site we will use novel photoactive diazirine analog of alcohols to photolabel the PKC domain/subdomains followed by identification of the labeled residues by mass spectrometry. The second aim is to generate PKC chimeras by swapping the regulatory domains/subdomains between PKC[unreadable] and PKC3 and characterize their alcohol binding properties. This will be achieved by measuring the effect of alcohols on the activities of the chimeric proteins. The third aim is to investigate the effect of alcohols on GABAA-PKC interactions by measuring GABAA current electrophysiologically. The proposal will enhance our current understanding on the molecular mechanism of alcohol action and will provide strategy for designing new therapeutics against alcohol addiction. PUBLIC HEALTH RELEVANCE: Despite huge number of alcohol related deaths (annually 75000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol related diseases. To develop new medications and effective prevention it is necessary to define the target and mechanism of its action at the molecular level. The significance of the present study is to establish the role of a signal transducing protein in regulating alcohol actions. This study will enhance the knowledge in developing medication for alcohol addiction.

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism affect 4.5% of the United States population causing an economic burden of approximately 184 billion dollars/year. The ability to develop new pharmacotherapies to help fight the descent into alcohol dependence and recidivism requires an understanding of mechanisms of alcohol actions on the nervous system. It is particularly important to define the targets of ethanol binding as these may bring about the most complete therapeutic effect. Although alcohol is known to have distinct and profound effects on presynaptic function, the mechanisms underlying this large impact are essentially unknown. The long-term goal of this proposal is to define the molecular mechanism by which alcohol exerts its action on presynaptic function. The objective of this exploratory application is to examine the physiological and behavioral interactions between Munc13.1 protein and ethanol. Munc13.1 is a presynaptic active zone protein essential for neurotransmitter release in brain. In Caenorhabditis elegans, the homologous Unc13 protein is responsible for the sensitivity to volatile anesthetics. The C1 or diacylglycerol binding domain of the Munc13 family of proteins is structurally similar to that of protein kinase C (PKC) which regulates behavioral effects of alcohol and has alcohol binding site(s). Preliminary data demonstrate that ethanol will also bind to this C1 domain in Munc13.1. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13.1 C1 domain. The first aim of this proposal will examine the hypothesis that ethanol binding to the C1 domain of the Munc13.1 modifies the activity of this presynaptic protein. This will be accomplished by photolabeling and mass spectrometry to identify alcohol binding residues, and by elucidating the effects of this binding on Munc13.1 activity in membrane translocation assays. The second aim is to determine how a reduction in Dunc13 activity changes the behavioral and physiological responses to ethanol in Drosophila melanogaster. This invertebrate model system was chosen for its ability to rapidly and economically alter Dunc13 levels and to monitor the functional consequences. These consequences of the ethanol-Dunc13 interaction will be revealed by measuring ethanol preference, stimulation, and sedation in wild type and Dunc13 reduction of function animals. Moreover, the effect of the interaction will be examined using the synapto-pHluorin sensor to image vesicle release from a specific subset of GABAergic neurons in intoxicated and sober flies. These GABAergic neurons are critical modulators of the stimulatory and sedative effects of ethanol in Drosophila. The approach is innovative as it applies the strengths of ultrasensitive biochemistry and powerful neurogenetic techniques for the first time to dissect the function of an ethanol-receptor interaction. This proposal is significant as it is expected to uncover a novel primary mechanism for an effect of ethanol on presynaptic function. Ultimately, this understanding may lead to new drugs designed to disrupt the ethanol-unc13.1 interaction providing a valuable weapon in the fight for sobriety. PUBLIC HEALTH RELEVANCE: Despite the huge number of alcohol related deaths (annually ~75,000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol abuse and addiction. To develop new medications and effective intervention it is necessary to define the target and mechanism of alcohol action at the molecular level. The goal of the present study is to establish the role of a presynaptic protein in regulating alcohol actions within the brain and the significance of this study is that it may allow the development of new therapeutics based on this target protein to combat alcohol addiction.

DESCRIPTION (provided by applicant): Alcohol has pervasive impacts on presynaptic functions, yet the mechanisms underlying these considerable impacts are largely unknown. It is likely that these presynaptic mechanisms contribute significantly to the development of alcohol dependence. Identifying the molecular participants responsible for consummating ethanol's presynaptic impact is necessary to develop new targets to fight addiction and recidivism. It is particularly important to define the target of ethanol binding as this may bring about the most complete effect. The long-term goal of this application is to unfold the presynaptic mechanisms for ethanol action. The objective of this proposal is to describe the interaction between ethanol and Munc13-1 and define the effects of this interaction in presynaptic physiology and behavior. Munc13-1 is a conserved presynaptic active zone protein essential for neurotransmitter release in the brain. Preliminary data demonstrate that ethanol binds to the C1 diacylglycerol-binding domain of Munc13-1. A reduction in Dunc13, the Drosophila Unc13 homolog results in flies that are resistant to ethanol and have defects in tolerance and self-administration. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13 C1 domain. This hypothesis will be tested in 4 aims. In Aim 1, the atomic structure of ethanol bound to the C1 domain of Munc13-1 will be determined. In Aim 2, the effect of ethanol on Unc13 vesicle fusion will be measured in vitro. In Aim 3, wild type Munc13-1 and Munc13-1 with mutations that reduced ethanol affinity will be used to functionally complement the Dunc13 haploinsufficient behavioral phenotypes. Moreover, the effect of the ethanol-Munc13-1 interaction will be examined using the synapto- pHluorin sensor to image synaptic vesicle release in intoxicated and sober flies. These experiments will determine how ethanol binding to Munc13-1 alters the activity of this protein in vivo. In Aim 4, we determine if heterozygous Munc13-1KO/+ mice, similar to the Drosophila Dunc13 mutants, have defects in ethanol sensitivity and self-administration. The approach is innovative as it applies in a single project the strengths of atomic level resolution, in vitro biochemistry, and in vivo behavior and physiology, to understand the function of an ethanol-effector interaction. This proposal is significant as it will likely provide unequivocal information on the importance of a general mechanism by which ethanol impacts presynaptic function, and how this mechanism is critical in the process required for alcohol dependence. Ultimately, these results will provide precise structural information on where alcohol binds Munc13-1, and how this interaction alters Munc13-1 activity, how this interaction impacts ethanol sensitivity and self-administration. The results will enable the design and validation of small molecule inhibitors that could be used in the development of drugs to fight dependence and recidivism.