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Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domaincontaining protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-β-IFN-α in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-β-IFN-α-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acylbiotin exchange assay and click chemistry, we show that TNF-β-IFN-α treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury. [ABSTRACT FROM AUTHOR]