This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Section A:
Specific Aims The ultimate goal of research in my laboratory is to use various structural and biochemical techniques to understand the atomic basis of function of proteins of pathways like autophagy (a lysosomal targeting pathway for degradation of intracellular objects) and innate immune pathway;the cross-talk between these pathways;the role of these pathways/proteins in the defense against pathogens;and the inhibition of these proteins by different pathogens. One project focuses on investigating the structure, function and molecular mechanism of Beclin1, an essential autophagy effector. Our previous work, funded in part by an NIH R21 grant, investigated the molecular mechanism by which cellular and viral Bcl-2 proteins inhibit Beclin 1 and autophagy. We plan to continue our investigation of the Beclin 1 protein, including its interaction with other autophagy proteins and with pathogen-encoded proteins. Another project focuses on Rig-I, a key mediator of anti-viral innate immunity. We plan to investigate the atomic basis of the role of Rig-I in innate defenses by studying the intra-molecular, intra- pathway or cross-pathway interactions of the Rig-I CARD domains that transmit signals to trigger or repress various pathways. A prerequisite of all these studies is the availability of pure, homogeneous, recombinant protein. We are using COBRE funding to produce these reagents and obtain preliminary data to apply for other grants to further investigate these pathways.
Specific aims of the COBRE funding are to overexpress and purify the recombinant proteins listed below, in quantities suitable for subsequent structural and biochemical studies: + Aim 1: Different Beclin 1 domains. + Aim 2: Rig-I CARD domains. + Aim 3: CARD domains of IPS-1, its downstream signaling partner. + Aim 4: Repressor domains of Rig-I and LGP2 (a Rig-I homolog). + Aim 5: The essential autophagy effector Atg5, which interacts with the Rig-I and IPS-1 CARD domains.
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