Programmed cell death (apoptosis) is a process whereby individual cells are terminated to benefit the organism. However, many cancers inhibit apoptotic pathways to allow cell proliferation, while degenerative diseases may up-regulate apoptosis to kill cells prematurely. In the intrinsic cell death pathway, cytochrome c is released from mitochondria and interacts with Apaf-1 in the presence dATP. This triggers apoptosome assembly and the resulting platform recruits and activates procaspase-9. This holo-enzyrne then activates executioner procaspases which kill the cell. Significantly, Apaf-1 assembly has recently been implicated as a possible contributing factor to major depression syndrome (MDD). In Drosophila, a similar cell killing machine is formed by Dark, an Apaf-1 Related Killer which sequentially activates the procaspases Drone andDrICE.
In Specific Aims 1 and 2,we will determine high resolution, 3-dimensional structures of the Apaf-1 and Dark apoptosomes. In addition, structures will be obtained of """"""""holo-enzymes"""""""" with prodomains from their apical procaspases. In these studies, electron cryo-microscopyand single particle methods, X- ray crystallography and homology modeling will be used to create atomic models. These studies will provide a detailed picture of human and Drosophila apoptosomes and will also reveal conformational changes that occur when procaspases bind. Thus, Specific Aims 1 and 2 will and give a clearer understanding of how these large platformsassemble and function in the intrinsic cell deathpathway. In pro-inflammatory pathways, the Apaf-1 related protein NOD2 senses bacteria and assembles a signaling platform. Througha series of steps, this complexactivates the transcription factor NFK0, which up-regulates genes involved in innate or adaptive immunity. Defects in NOD2 are associated with chronic inflammation of the small intestine (Crohn's disease) and with Blau's syndrome.
In Specific Aim 3, a NOD2signaling platformwill be assembled and the first 3-dimensional structure of this complex will be determined. In the long term, these studies will help us to understand how NOD2 functions in the innate immune response to bacteria in the gastro-intestinal tract Boston University School of Medicine (BUSM) Boston, Mass. PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/ProgramDirector (Last, First, Middle): Akey, Christopher W. KEY PERSONNEL. See instructions. Use continuation pages as needed to providethe required information in the formatshownbelow. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Akey, Christopher W. BUSM PI Head, James F. BUSM Co-Investigator OTHER SIGNIFICANTCONTRIBUTORS Name Wang, Xiaodong Shi, Yigong Nunez, Gabriel Jung, Yong-Keun Yu, Xinchao Yuan, Shujun Organization Role onProject University of Texas Collaborator Princeton Collaborator University of Michigan Collaborator Gwangju Inst. Science/Technology Collaborator BUSM Graduate student BUSM Graduate student Human Embryonic Stem Cells ^ No QYes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://Stemcells.nlh.gov/registrv/index.asp. Usecontinuation pages as needed. If a specific tine cannot be referenced at this time, include astatement that one from the Registry will be used. Cell Line Disclosure Permission Statement Applicable to SBIR/STTR Only. SeeSBIR/STTR Instructions. Yes DNo PHS 398 (Rev.09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Akey, Christopher W. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ^ Description,
| Kim, Seung Joong; Fernandez-Martinez, Javier; Nudelman, Ilona et al. (2018) Integrative structure and functional anatomy of a nuclear pore complex. Nature 555:475-482 |
| Dorstyn, Loretta; Akey, Christopher W; Kumar, Sharad (2018) New insights into apoptosome structure and function. Cell Death Differ 25:1194-1208 |
| Mendez, Deanna L; Akey, Ildikó V; Akey, Christopher W et al. (2017) Oxidized or Reduced Cytochrome c and Axial Ligand Variants All Form the Apoptosome in Vitro. Biochemistry 56:2766-2769 |
| Cheng, Tat Cheung; Akey, Ildikó V; Yuan, Shujun et al. (2017) A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation. Structure 25:40-52 |
| Cheng, Tat Cheung; Hong, Chuan; Akey, Ildikó V et al. (2016) A near atomic structure of the active human apoptosome. Elife 5: |
| Yuan, Shujun; Akey, Christopher W (2013) Apoptosome structure, assembly, and procaspase activation. Structure 21:501-15 |
| D'Brot, Alejandro; Chen, Po; Vaishnav, Mahesh et al. (2013) Tango7 directs cellular remodeling by the Drosophila apoptosome. Genes Dev 27:1650-5 |
| Yuan, Shujun; Topf, Maya; Reubold, Thomas F et al. (2013) Changes in Apaf-1 conformation that drive apoptosome assembly. Biochemistry 52:2319-27 |
| Yuan, Shujun; Yu, Xinchao; Topf, Maya et al. (2011) Structure of the Drosophila apoptosome at 6.9 å resolution. Structure 19:128-40 |
| Yuan, Shujun; Yu, Xinchao; Asara, John M et al. (2011) The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3. Structure 19:1084-96 |
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