Abstract
BackgroundVascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.Methodology and Principal FindingsThe present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.Conclusions/SignificanceColocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes.
Highlights
The composition of the eukaryotic cytoskeleton consists mainly of three discrete fibrous structures—actin microfilaments, microtubules and intermediate filaments
We examined the effects of low and high doses of thrombin and compared it to the signaling pathways elicited by anti-HLA class I antibodies (MZ unpublished data)
We hypothesized that the isolation and proteomic characterization of the cytoskeletal proteins in HLA class I stimulated endothelial cells (ECs) could lead to a better understanding of the mechanisms underlying HLA class I induced stress fibers
Summary
The composition of the eukaryotic cytoskeleton consists mainly of three discrete fibrous structures—actin microfilaments, microtubules and intermediate filaments These structures provide the framework to maintain cell shape and polarity and contribute to other fundamental cellular functions such as motility, organelle transport and division [1]. These cellular processes are carried out through the actions of multiple molecules, which comprise functional modules [2]. Crosslinking of HLA class I molecules by anti-HLA antibodies triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood
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