Obesity causes chronic “sterile” inflammation, which is implicated in insulin resistance and other metabolic complications. NLRP3 (NLR family pyrin domain containing 3) is an intracellular sensor for various “danger” signals from microbial infection and metabolic stress. Activation of NLRP3 inflammasomes elicits host defense responses by promoting caspase 1-dependent release of IL-1β and other pro-inflammatory cytokines. Intriguingly, activation of NLRP3 inflammasomes is also implicated in “sterile” inflammation and insulin resistance in diet-induced obesity (DIO), yet the “danger” signals that lead to NLRP3 activation in obesity remains elusive. Prions are misfolded proteins that are capable of self-transmitting their misfolded shape onto normal variants of the same protein. Recent progress in the field has identified more than 240 non-infectious prion-like proteins in mammals. Although a few of these proteins are well implicated in the pathogenesis of neurodegenerative diseases, surprisingly little information is known about majority of the prion-like proteins’ potential involvement in human health and disease. Here, we propose to investigate a novel pathway by which a putative prion-like protein regulates NLRP3 activation in DIO. This pathway is mediated by the ALCAT1 enzyme, the first acyl-CoA dependent lysocardiolipin acyltransferase previously identified by us. Our groundbreaking work in the field shows that ALCAT1 promotes the development of age-related metabolic diseases by catalyzing pathological remodeling of cardiolipin (CL) with very long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). Enrichment of DHA renders CL highly sensitive to oxidative damage (CL-Ox) by reactive oxygen species (ROS), leading to CL depletion and mitochondrial dysfunction in metabolic diseases. Remarkably, our preliminary studies also identified a pivotal role of ALCAT1 in linking DIO to NLRP3 activation as a putative prion-like protein, including 1) ALCAT1 forms prion-like protein aggregates in response to oxidative stress, which is mediated by a prion-like domain at the N-terminus; 2) ALCAT1 expression in adipose tissue is dramatically upregulated by DIO; and 3)Targeted deletion of ALCAT1 in adipocytes significantly attenuates NLRP3 activation by preventing macrophage infiltration in white adipose tissue. Together, these exciting findings let us to hypothesize that ALCAT1 promotes NLRP3 activation in DIO as a putative prion-like protein, which will be tested by three Specific Aims: AIM 1 will determine whether ALCAT1 promotes NLRP3 activation through mitochondrial dysfunction; AIM 2 will identify molecular mechanisms by which ALCAT1 promotes NLRP3 activation as a prion-like protein; and AIM 3 will assess whether ALCAT1 links cellular senescence in adipose tissue to chronic inflammation in DIO mice. Successful completion of the proposed studies will not only validate ALCAT1 as a novel drug target of chronic inflammation, but also provide ...