Deciphering the activation mechanisms and immune functions of GSDMC - Project Summary The Gasdermin (Gsdm) family consists of six paralogous genes encoding GSDMA, GSDMB, GSDMC, GSDMD, GSDME and F (GSDMF also known as PJVK or DFNB59). Gsdm proteins play a crucial role in innate immunity, particularly in inflammation and the initiation of pyroptosis, a form of programmed necrotic cell death. Unlike many other immune modulators, transcriptional regulation of Gsdm family proteins is not sufficient to execute their biological functions. Gsdms A-E share highly conserved N-terminal (N-ter) and C-terminal (C-ter) domains separated by a variable linker. The C-term exhibits self-inhibition by completely masking the N-ter hydrophobic pocket that binds lipids. Thus, even if upregulated transcriptionally, Gsdm cannot be functional until it is cleaved by a protease to release the N-ter from the self-inhibited C-term. Thus, the key for understanding Gsdm biology is the identification of the protease. This work is based on our robust unpublished data where we identified a new protease that can directly cleave both human and murine GSDMC. Rooting from this discovery, we further identified three unique features of cleaved and activated GSDMCN-ter not observed in other Gsdm family members in terms of its molecular properties, cellular functions and in vivo phenotypes. (1) In contrast to other protease-processed Gsdm family members, such as GSDM-A, -B, -D and -E, the N-terminus of GSDMC (GSDMCN-ter), processed by the newly identified protease, did NOT effectively promote pyroptosis. (2) This is because GSDMCN-ter does not localize to the plasma membrane but to other subcellular organelles. (3) GSDMCN-ter has an immune regulatory role in animal models via amplifying type-2 immune responses, unrelated to pore formation at the plasma membrane (e.g. IL-1 family member cytokine release) and pyroptosis. Accordingly, we have planned three aims to understand the mechanisms controlling these features. In Aim 1, we will analyze the non-conserved amino acids between GSDMCN-ter and other Gsdms proteins that explain the pyroptosis deficiency. We will further determine the lipid binding profile of GSDMCN-ter and try to understand how it differs from that of other Gsdms proteins. In Aim 2, we will systematically investigate the intracellular membrane structures that can be targeted by GSDMCN- ter and assess the biological consequences after GSDMCN-ter targeting. Last, in Aim 3 we will use two animal models to determine how GSDMCN-ter amplifies type 2 immune responses. In summary, Aim 1 and 2 will unveil novel mechanisms of how GSDMC functions different from other Gsdms, as well as its new roles in cell biology (not effectively promoting pyroptosis). For Aim 3, with our unique animal models, we will reveal new mechanisms of actions for GSDMC as well as therapeutic strategies to boost type 2 immunity.
