Biology subjects

Evavold, C. L.

Publications and source records attributed to Evavold, C. L..

4 recordsLinked to original sources

bioRxiv2026

Cholesterol constrains pore formation by mammalian gasdermins

Pyroptosis is a lytic form of cell death executed by gasdermin pores. Through a CRISPR screen to uncover macrophage genes required for gasdermin D (GSDMD) pore activity, we identify the endosome adaptor Rabgef1 as a positive regulator of pore formation. Rabgef1 deficiency preserves NT-GSDMD membrane binding but prevents NT-GSDMD oligomerization and lytic death. Mechanistic analyses revealed that Rabgef1 deficiency is associated with an overabundance of cholesterol in the plasma membrane. Cholesterol depletion or redistribution through multiple approaches restores GSDMD oligomerization and pyroptosis. The Rabgef1-mediated control of membrane cholesterol and pore formation was found to be a general regulator of the gasdermin family. In cancer cells, organoids, and tumors, cholesterol reduction sensitizes resistant cells to GSDME pyroptosis driven by synthetic mRNAs, chemogenetic induction, and chemotherapy treatment. These results establish membrane cholesterol content as an endogenous checkpoint for gasdermin-mediated pyroptotic cell death during the execution phase, with implications for metabolic disease and cancer immunotherapy.

immunology↗

bioRxiv2022

Correction of age-associated defects in dendritic cell functions enables CD4+ T cells to eradicate tumors in the elderly

Defective host defenses later in life are associated with changes in immune system activity. The means to correct immune defects to ensure immunity in the elderly are undefined. In this study, we found that CD8+ T cells, which are necessary for anti-tumor immunity in young mice, are not required to eradicate the same cancers later in life. Rather, CD4+ T cells drive anti-tumor immunity in elderly mice. The generation of anti-tumor CD4+ T cells requires multiple dendritic cell (DC) activities that are elicited by immune agonists known as hyperactivators, but not by adjuvants that model those used clinically. DC hyperactivators correct age-associated defects in DC migration and T cell co-stimulation while enabling NLRP3 inflammasome activities within living cells. These combined activities enable DCs to induce TH1-skewed T cells that persist into old age and eliminate implantable tumors. These results raise the possibility of correcting age-associated immune defects through DC manipulation.

immunology↗

bioRxiv2022

The pore-forming protein gasdermin D is a cellular redox sensor

Reactive oxygen species (ROS) affect inflammation and immunity in a multitude of ways, in particular in the context of the signaling pathways that determine cell fate. Inflammasomes are multiprotein cytoplasmic complexes whose pyroptosis-inducing activities are controlled by ROS. Our knowledge of how ROS mediates inflammasome activities is largely based on studies of the initiating events in these pathways. Herein, we show that ROS controls the terminal events in the pyroptosis pathways. We found that ROS oxidizes the protein gasdermin D (GSDMD) and promotes its assembly into a death-inducing pore forming complex. Mechanistically, ROS enhances GSDMD-mediated pyroptosis in an intrinsic manner, likely through oxidative modification of a specific cysteine residue (C192). Diverse ROS sources promote GSDMD oxidation, ranging from homeostatic control via the Ragulator-Rag complex, to inducible control via diverse microbial products and environmental toxins. These findings expand the steps in the inflammasome pathway that are controlled by ROS and suggest that GSDMD operates as a pyroptosis-inducing redox sensor.

immunology↗

bioRxiv2020

Downstream of gasdermin D cleavage, a Ragulator-Rag-mTORC1 pathway promotes pore formation and pyroptosis

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins are recognized to promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that was designed to identify regulators of pyroptosis that act downstream of GSDMD cleavage. This screen identified several components of the Ragulator-Rag complex, which is known for its metabolic control of mTOR. Mechanistic studies revealed that Ragulator-Rag is not necessary for GSDMD localization to the plasma membrane, but is necessary for pore formation and mitochondrial inactivation. Downstream of Ragulator-Rag is mTORC1, which we found to promote pyroptosis in response to diverse natural stimuli, including infection. GSDMD therefore requires a Ragulator-Rag-mTORC1 pathway in order to form pores and execute pyroptosis.

immunology↗