Over the years, I've grown intellectually and demonstrated my potential to become an independent investigator. I have surrounded myself with mentors that have my best professional interests in mind. My work with Prof. Jack Johnson focused on an in vitro system of viruses for studies with primarily by electron microscopy. With Prof. Karin I would grain experience in cell biology studying interactions of transcription factors. I will develop a proficiency in Cryo-EM as I acquire experiences with the nuances associated with imaging transcription factor complexes. This will enable me to establish a single niche I need to pursue fundable research questions as a Structural Biologist, with significant contributions to the field of cell-mediated transcriptional regulation and viral infection. The goal of this project is to increase our understanding of how microbial pathogens lead to aberrant p53 and NLRP3 inflammasome assembly. The tumor suppressor p53 is a transcription factor that prevents cancer by promoting cell death. The NLRP3 inflammasome ? which controls the production of bioactive IL-1? and IL-18 via caspase-1, and p53 are key mediators of bacterial-induced inflammation and cancer. Both NLRP3 inflammasome and p53 are subject to substantial post-translational modifications which all influence their structure and function. Although there are crystal structures for isolated domains of p53, and partially for the N- terminal domain of NLRP3, it remains to be determined they form macromolecular complexes within the cell and in what way the functional mechanisms are different in cancer. The p53 N- and C- terminal regions are unstructured, which not only can cause intramolecular heterogeneity, but quaternary structures of the macromolecular complexes may also be heterogeneous. Thus, quaternary structure determination via crystallography would be extremely challenging, if not impossible. My long-term goal is to provide a physiological picture of microbial ? host interactions inside of human cells. The overall objective of this application, which is the next step toward attainment of this long-term goal, is to use Cryo-EM to characterize the NLRP3 inflammasome, and p53 tetramer macromolecular complexes in the presence of heterogeneity. With this technique, software can be used to purify various biologically relevant conformational or stoichiometric populations. I am interested in the native structure of these proteins and conformational changes that occur upon binding microbial infection. Herein we provide preliminary data towards elucidating the structure and domain organization for the full-length native p53 tetramer using electron microscopy. We will also use 3D Cryo-EM to characterize 3D interactions of NLRP3 inflammasome. The rationale for the proposed research is that once initial native structures of p53 and NLRP3 inflammasome proteins are known, pharmacological drugs can be designed to restore physiological functions of p53 and NLRP3.

Public Health Relevance

NLRP3 inflammasome and the tumor suppressor p53 are key mediators of in these diseases ? with greater incidence in economically disadvantaged Indian- and Mexican-American populations. We will apply 3D cryo- electron microscopy to study how bacterial pathogens induce aberrant p53 and NLRP3 inflammasome assembly in macrophage populations, leading to inflammation and cancer. This research supports the NIH goal to advance current inflammation-based and cancer treatments using unprecedented strategies to further healthy life and reduce illness.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Career Transition Award (K22)
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Microbiology and Infectious Diseases B Subcommittee (MID)
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Gondre-Lewis, Timothy A
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University of California Irvine
Schools of Arts and Sciences
United States
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