title: Degradation of cytokeratin intermediate filaments by calcium-activated proteases (calpains) in vitro: implications for formation of Mallory bodies.
abstract: The degradation of rat hepatic intermediate filament (IF) proteins cytokeratin A (CK-A, 55-kDa) and cytokeratin D (CK-D, 48-kDa) by purified rat liver calcium-activated proteases (calpains I and II) was evaluated in vitro. Calpain-mediated IF proteolysis was monitored by SDS-PAGE and Western blotting with antibodies to CK-A and CK-D and compared to microtubule protein actin. Both cytokeratins underwent rapid yet limited proteolysis by calpain I and II. Despite the conserved nature of cytokeratins and limited substrate specificity for calpains, distinct fragmentation patterns were obtained for calpain I) CK-A, 46- and 43-kDa/CK-D, 41-, and 39-kDa; and calpain II) CK-A, 46- and 43-kDa/CK-D 41-kDa. The 46-kDa CK-A fragment was the predominant fragment for both calpains. Two-dimensional electrophoresis (IEF/SDS-PAGE) of CK fragments revealed the presence of classic "staircase" patterns consistent with endogenous proteases. Furthermore, proteolytic fragments showed a 2-D electrophoretic shift to lower pI suggesting that the limited hydrolysis occurred within the N-terminal arginine-rich region of CK, a region believed essential for IF interactions in vivo. Thus, calpains may represent an initial step in the turnover of these stable and long-lived proteins and as such, may be relevant to diseases characterized by abnormal disruption and bundling of IF such as formation of Mallory bodies in alcoholic hepatitis.
authors.affiliation: Department of Laboratory Medicine, School of Medicine, University of Connecticut Health Center, Farmington 06030-2235, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Makowski
authors.forename: M L
authors.initials: ML
authors.lastname: Ramsby
mesh_terms: D000199:Actins / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000494:pharmacology*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D002479:Inclusion Bodies / Q000378:metabolism; D007382:Intermediate Filaments / Q000187:drug effects* / Q000378:metabolism; D007633:Keratins / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051381:Rats
chemical_list: D000199:Actins; D007633:Keratins; D002154:Calpain; D002118:Calcium