The overall goal of our studies is to understand the regulation, function, and disease association of the keratin intermediate filament (IF) cytoskeletal proteins in digestive organs. Keratins 8 and 18 (K8/K18) are the IFs of hepatocytes and their major function is cytoprotection from mechanical and nonmechanical stresses such as apoptosis. This function is consistent with the finding that specific K8 and K18 genetic variants predispose to liver disease progression. K8 and K18 are also the major constituents of Mallory body (MB) inclusions that are found in association with some liver diseases. Several transgenic animal studies demonstrated that K8 and a K8-greater-than-K18 protein ratio, coupled with a drug insult are all essential in order for MBs to form. MBs can be rapidly re-induced once initially formed but the mechanism of such rapid predisposition to reformation is unknown. Our proposal includes 4 aims to examine the pathogenesis of MBs. The first three aims test the hypothesis that keratin transamidation at specific amino acids is essential for MB formation. The 4th aim tests the hypothesis that chaperone function is altered during MB (re)formation. The proposed studies utilize cell culture and mouse MB models to help understand their pathogenesis and, ultimately, their importance in human disease. The 4 aims are: (i) Study MB formation in transglutaminase-2 (TG2) null and control mice using established in vivo models. This aim is based on our preliminary findings that TG2-null mice have a markedly blunted ability to form MBs. (ii) Identify keratin transamidation sites and their crosslinked partners in vivo/vitro and study their role in crosslinking and inclusion body formation in cell culture. This is based on the findings that K8/K18 are excellent TG2 substrates (K8>K18) in vitro, (iii) Generate transgenic mice that express transamidation-mutant keratins and test the effect of the mutations on MB formation and susceptibility to liver injury, (iv) Test the effect of MB-inducing injury on chaperone function and the potential role of chaperones as "memory proteins" in association with MB re-accumulation. This is based on our recent findings that MB formation correlates with chaperone dysfunction. Our proposed studies are likely to generate important new biologic and clinically relevant information regarding the pathogenesis of MBs. We anticipate that completion of this project will shed light on the specific keratin amino acids that allow MB formation and address if MBs are bystanders, protectors or promoters of injury. Our findings should also shed light on whether chaperone dysfunction can provide 'molecular memory" and as such contribute to rapid MB reformation. Our proposal represents a direct approach to fully understand the pathogenesis and significance of MBs that were first described in 1911 by Dr. Frank Mallory, and may also impact on other inclusions that are found in association with several other neurological and neuromuscular human diseases whose pathogenesis is unknown.