Characterization of the Roles of the Hax-1 Variants in Apoptosis

Abstract

HS-1-Associated-protein-X-1 (Hax-1) is a family of ubiquitously expressed proteins ranging in size from ~26 to ~35 kDa resulting from alternative splicing of the HAX1 gene. The prototypical Hax-1 variant 1 is a ~35 kDa protein, expressed in both humans and rodents. Hax-1 contains an NH2-terminal acidic box, followed by two predicted Bcl-2 homology domains, BH1 and BH2, a PEST motif, a predicted COOH-terminal transmembrane (TM) domain, and an Integrin β6 binding domain. Studies on Hax-1 have mainly focused on variant (v) 1, demonstrating its anti-apoptotic properties, which have been confirmed by its overexpression in HeLa cells, HEK293 cells, and cardiomyocytes promoting cell survival after exposure to different apoptotic stimuli. Hax-1 variant 1 has also been shown to be overexpressed in several diseases including psoriasis, melanoma, breast, and lung cancers. The presence of at least four Hax-1 variants expressed in rat heart both before and after insult was confirmed, some of which exhibited differential expression before and after induction of myocardial infarction (MI), with v2 being highly up-regulated post-insult. Contrary to anti-apoptotic rat and human v1, overexpression of rat v2 or human v4 (the human homologue of rat v2) in epithelial cells exacerbated cell death by 30% following H2O2 treatment, compared to control vector. Co-expression of rat v1 and v2 or human v1 and v4 neutralized the protective effects of rat and human v1, and the pro-apoptotic effects of rat v2 and human v4 by modulating cytochrome C release. This is, at least partly, mediated by the ability of Hax-1 proteins to form homotypic and heterotypic dimers with binding affinities in the nM range. The binding region supporting these interactions lies between amino acids 97-278, which are shared by nearly all Hax-1 proteins, indicating that additional factors regulate the preferential formation of Hax-1 homo- or hetero-dimers. Our studies are the first to show that Hax-1 is a family of anti- and pro-apoptotic regulators that may modulate cell survival and death through homo- or hetero-dimerization.