Understanding Signal Transduction Mechanisms of Inflammasomes

Inflammasomes are a key component of the human innate immunity. They are supramolecular signaling platforms called to assemble in response to various intracellular assaults. While there are many different inflammasome receptors, they all share a hallmark of signal transduction by sequential filament assembly which eventually activates the caspase-1 protease; activated caspase-1 then executes cytokine maturation and cell death. In most cases, a single adaptor protein, ASC (Apoptosis-associated Speck-like protein contain a Caspase Activation and Recruitment Domain (CARD)), is tasked with bridging the upstream receptors with caspase-1. This is achieved by interacting with filamentous Pyrin domains (PYDs) which most receptors contain. However, this creates an intriguing recognition problem; the different PYDs have to be unique enough to prevent intermixing, but similar enough to recruit each other. The goal of this thesis is to understand the molecular interactions required for signal transduction to occur, as well as to understand how all of the components interact at a systems level. This work focuses on the dsDNA sensing AIM2-ASC inflammasome and the ASC-independent NAIP-NLRC4 inflammasome. Since polymerization is key to all inflammasomes, Förster Resonance Energy Transfer (FRET) kinetics assays were crucial to understanding inflammasome assembly and recruitment. The kinetics results allowed me to mathematically model the AIM2-ASC inflammasome and reveal they form a Positive Feedback Network (PFN) capable of ultrasensitive response. In order to gain understanding at the molecular level, I acquired two cryo-EM filament structures, AIM2PYD and NLRC4CARD. Using the structures, I conducted Monte Carlo simulations to reveal that the higher-order oligomeric state of upstream receptors maximizes the probability of assembling signal competent filament interfaces. The structures were also used for in silico screen to find sidechains responsible specifically for heterotypic interactions, revealing that only a small subset of residues is involved. However, these residues require to be in a pocket formed by upstream filamentation for recruitment to occur. Additionally, the Monte Carlo simulation further suggest that this creates a signaling funnel, where the first polymerization has the highest energy barrier to get through, which can only be passed by properly assembling an oligomer after sensing its correct ligand

Understanding Signal Transduction Mechanisms of Inflammasomes

Inflammasomes are a key component of the human innate immunity. They are supramolecular signaling platforms called to assemble in response to various intracellular assaults. While there are many different inflammasome receptors, they all share a hallmark of signal transduction by sequential filament assembly which eventually activates the caspase-1 protease; activated caspase-1 then executes cytokine maturation and cell death. In most cases, a single adaptor protein, ASC (Apoptosis-associated Speck-like protein contain a Caspase Activation and Recruitment Domain (CARD)), is tasked with bridging the upstream receptors with caspase-1. This is achieved by interacting with filamentous Pyrin domains (PYDs) which most receptors contain. However, this creates an intriguing recognition problem; the different PYDs have to be unique enough to prevent intermixing, but similar enough to recruit each other. The goal of this thesis is to understand the molecular interactions required for signal transduction to occur, as well as to understand how all of the components interact at a systems level. This work focuses on the dsDNA sensing AIM2-ASC inflammasome and the ASC-independent NAIP-NLRC4 inflammasome. Since polymerization is key to all inflammasomes, Förster Resonance Energy Transfer (FRET) kinetics assays were crucial to understanding inflammasome assembly and recruitment. The kinetics results allowed me to mathematically model the AIM2-ASC inflammasome and reveal they form a Positive Feedback Network (PFN) capable of ultrasensitive response. In order to gain understanding at the molecular level, I acquired two cryo-EM filament structures, AIM2PYD and NLRC4CARD. Using the structures, I conducted Monte Carlo simulations to reveal that the higher-order oligomeric state of upstream receptors maximizes the probability of assembling signal competent filament interfaces. The structures were also used for in silico screen to find sidechains responsible specifically for heterotypic interactions, revealing that only a small subset of residues is involved. However, these residues require to be in a pocket formed by upstream filamentation for recruitment to occur. Additionally, the Monte Carlo simulation further suggest that this creates a signaling funnel, where the first polymerization has the highest energy barrier to get through, which can only be passed by properly assembling an oligomer after sensing its correct ligand

Structural Biology of NOD-Like Receptors

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are a large family of intracellular immune receptors conserved in both animals and plants. Mammalian NLRs function as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP or DAMP perception activates NLRs which consequently recruit pro-caspase-1 directly or indirectly. These sequential events result in formation of large multimeric protein complexes termed inflammasomes that mediate caspase-1 activation for pyroptosis and cytokine secretion. Recent structural and biochemical studies provide significant insights into the acting mechanisms of NLR proteins. In this chapter, we review and discuss these studies concerning autoinhibition, ligand recognition, activation of NLRs, and assembly of NLR inflammasomes