Project Summary Excessive lipid accumulation is central to the pathogenesis of prevalent metabolic diseases including fatty liver. Substantial evidence supports a critical role for endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in driving lipid accumulation and fatty liver development. However, the precise link from the ER stress-UPR to ectopic fat storage remains elusive. In our preliminary studies, we identified ubiquitin-like protein 5 (UBL5) as a novel ER stress responder, undergoing rapid depletion in response to ER stress in mammalian cell lines and in livers of mice. The ER stress-induced UBL5 depletion was mediated by the ubiquitin-independent proteasome system (UIPS). The PKR-like ER kinase (PERK) arm of the UPR pathway was responsible and sufficient for inducing UBL5 breakdown. RNA-Seq analysis revealed distinctly up- and down-regulated pathways in UBL5-depleted cells. Most strikingly, UBL5 knockdown upregulated lipogenic programs and simultaneously downregulated the fatty acid oxidation pathway. Functional analyses confirmed that UBL5 silencing promoted de novo lipid synthesis and attenuated fatty acid oxidation, placing UBL5 at a unique nexus to control fat storage by transcriptionally affecting the principal lipid anabolic and catabolic processes in opposite directions. Consistent with this, UBL5-silenced cells and liver tissues showed excessive lipid droplets and dramatic increase in cellular triacylglycerol content. These results reveal a novel function of UBL5 in suppression of lipid accumulation and a regulatory/signaling mechanism to control UBL5 protein stability and activity by the UPR- PERK arm. Based on these observations, UBL5 offers a candidate link from ER stress to abnormal lipid accumulatio, the hallmark of NAFLD. In further support of the pathophysiological relevance of UBL5, our additional preliminary data showed that UBL5 protein was significantly reduced in fatty livers of mice fed high-fat diet (HFD) and in liver biopsies of NAFLD patients. We have also developed ubl5 conditional knockout and liver-specific transgenic models to explore the biological functions of UBL5 in vivo. We hypothesize that UBL5 is a fat suppressor whose function is disrupted by recurring or chronic ER stress-UPR-PERK activation to promote lipid storage and fatty liver development. The lipid burden itself could aggravate the cellular ER stress, further depleting UBL5 protein and its fat-suppressive function in a vicious cycle toward the full phenotype of inflammatory non-alcoholic steatohepatitis (NASH). We will examine the hypothesis by establishing a general role and underlying mechanisms for UBL5 in suppression of lipid accumulation (Aim 1), identifying the key components of the UIPS machinery involved in UBL5 degradation and the communication network from PERK activation to UBL5 destabilization (Aim 2); and determining the biological relevance of the endogenous UBL5 to the development and progression of NAFLD ...