of the Funded Grant: The goal of the proposed work is to understand the molecular mechanism of NAIP/NLRC4 inflammasome regulation. The NAIP/NLRC4 inflammasomes are defenders against cytosolic infections by bacteria such as Salmonella and Pseudomonas through recruitment and activation of pro-caspase-1, leading to the production of interleukin-1? (pro-IL-1?) and IL-18, and subsequent pyroptotic cell death. The NAIP/NLRC4 inflammasomes are comprised with three components, the bacterial ligands such as flagellin, the NAIPs, and the NLRC4 protein. NAIPs recognize different bacterial ligands and determine the specificity of NAIP/NLRC4 inflammasomes. Upon activation, ligand/NAIP complex activate NLRC4 through a huge conformational change on NLRC4 protein, which exposes the nucleation surface and leads to the polymerization of NLRC4 molecule. Even though the activation mechanism of NAIP/NLRC4 inflammasome is well studied, we still don't know how the NAIP protein stays inhibited before ligand invasion. In the study proposed here, we will illustrate the structural mechanism of NAIP inhibition through crystallography, cryo-EM studies and cellular assays and screen for small molecules that inhibit the inflammatory pathway. We anticipate that these studies will help us understand the molecular mechanism of auto-inflammatory diseases that involve abnormal activation of NAIP/NLRC4 complex.
The specific aims of this proposal are:
Aim 1. Elucidating the mechanism of ligand dependent activation of NAIP/NLRC4 inflammasomes. In this aim, I will use a combination of biochemical characterization, structure determination and mutational analysis in cells to address how bacterial ligands are recognized and how this recognition activates NAIP proteins and initiates NLRC4 oligomerization. Unlike the previous proposal of NAIP proteins as the sole receptor, I found that NLRC4 may also directly interact with ligands and act as a co-receptor. As such, simultaneous interactions of ligands with both a NAIP protein and NLRC4 likely facilitate cooperative ligand- induced conformational changes. I have reconstituted several NAIP/NLRC4 inflammasomes and am on the way for cryo-EM 3D reconstruction of these important complexes. Collectively, these studies will reveal the molecular basis for ligand specificity and ligand- induced conformational transitions.
Aim 2. Establishing the inhibitory mechanism of NAIP proteins. In this aim, I will investigate the auto-inhibited conformation of NAIP proteins using structure determination and mutational analysis in cells. I anticipate that before ligand stimulation, NAIP proteins may exist in a closed conformation with extensive intra-molecular interactions. Furthermore, the unique N-terminal BIR domains of NAIP proteins may play a role in this process. As preliminary data, I have overcome the difficulty in expressing and purifying these large, and aggregation-prone proteins (~160 kDa).
Aim 3. Identifying small molecule inhibitors of CARD-CARD interactions in inflammasomes. In this aim, I will use structure-based identification of pockets and computational screening, as well as high throughput biochemical screening to identify small molecules that interfere with CARD-CARD interactions in NLRC4, ASC and caspase-1. Initial screen conditions have been established, and identified hits will be tested and validated in cells.
Ivan will be working on elucidating the structural basis of the inflammatory receptor, NAIP proteins, which help understanding how the immune system defends against bacteria invasion and will provide new insights into drug development for the treatment of inflammation-related diseases and cancer.