In vertebrates, the first line of defense against pathogen invasion involves the recognition of molecular markers unique to the invading organism known as pathogen associated molecular patterns (PAMPs). Retinoic Acid-Inducible Gene I (RIG-I) is an intracellular pattern recognition receptor (PRR) primarily involved in initiating the host defense response upon infection by a set of viruses, including Influenza, Dengue, and Ebola. RIG-I acts by binding to non-self, double-stranded RNA present in the cytoplasm, and inducing an innate immune signaling cascade subsequent to its recognition.
The RIG-I protein comprises three major domain groups, including N-terminal tandem Caspase Activation and Recruitment Domains (CARDs) that engage immune signaling, a central DExD/H-box ATPase core, and a C-terminal Domain (CTD) that provides ligand specificity. RIG-I distinguishes between self and non-self nucleic acids by recognizing chemical features not found in endogenous cytoplasmic RNA. In particular, RIG-I exhibits a high affinity for blunt ended duplex RNA termini containing a 5' triphosphate moiety. Ligand recognition by RIG-I facilitates the formation of an ATPase active site pocket within the DExD/H-box core, enabling the protein to bind and hydrolyze ATP. The process of ligand binding, ATP binding, and enzymatic hydrolysis results in the activation of CARD-mediated signaling by RIG-I. This work is concerned with elucidating the molecular details regarding the transition by RIG-I from an autorepressed to signaling competent state.
As a preliminary step, I collaborated with Dr. Andrew Kohlway to characterize the minimal ligand for RIG-I activation (Chapter 2). I next performed experiments aimed at determining the role of ATP binding and hydrolysis in RIG-I signaling. To identify the molecular events modulated by interactions with ATP, I produced a series of mutants designed to perturb the ability of RIG-I to bind and/or hydrolyze this substrate. Using a cell-based assay, I show that the signaling activity of ATPase-mutant RIG-I exhibits ligand length dependence. Specifically, ATPase deficient mutants are capable of WT-like signaling in the context of the minimal ligand identified in Chapter 2, but lose this capability when challenged by ligands bearing longer internal duplexes. Through biochemical analysis I show that the signaling behavior of RIG-I cannot be directly correlated to its enzymatic efficiency. Rather, the nucleotide found at the active site influences the ability of the protein to adopt and maintain conformations required for CARD release and signaling (Chapter 3).
The second aim of my thesis was to investigate the role of a novel a-helical motif in RIG-I annotated the Pincer domain. This domain represents a major architectural departure from simpler DExD/H-box ATPase folds. Its arrangement is intriguing in the context of intramolecular signaling dynamics, as it provides a physical link between three disparate folds of the protein. The Pincer consists of two a-helices following the C-terminus of the second Rec-A-like fold of the helicase core (HEL2) that stack back across the more N-terminal Rec-A-like fold (HEL1). The Pincer is also mechanically tethered to the CTD through a proline-rich linker following the second helix. I show here that the Pincer domain acts in mediating the enzymatic and signaling activities of RIG-I through the transduction of ligand binding to catalytic activity. I identify a series of mutations that additively decouple the pincer motif from HEL1, and show that this results in impaired signaling. Further, I utilize a series of biochemical and biophysical analyses to show that the pincer motif allosterically modulates the catalytic efficiency of RIG-I ATPase activity, while simultaneously assisting in proper ligand recognition (Chapter 4).
Because RIG-I represents a major checkpoint in viral RNA recognition and induction of the innate immune machinery, it represents a promising target for therapeutics capable of enhancing or inhibiting the natural inflammatory response. Using assays developed in my biochemical investigation of RIG-I, I leveraged our molecular understanding of RIG-I to develop a high throughput screen (HTS) to identify small molecules that modulate RIG-I activity. I performed a pilot screen of approximately 11,000 compounds and developed a pipeline for lead optimization in collaboration with Mark Forsberg (Chapter 5).
|Advisor:||Pyle, Anna Marie|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 77/06(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Biophysics, Immunology|
|Keywords:||ATPase, Innate Immunity, Pattern Recognition Receptor, RIG-I Like Receptor, RNA Helcase|
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