GABARAPs proteins modulate FVIII secretion in molecule-specific manner via energy related pathways

Little is known about the biogenesis of Factor VIII (FVIII), its control of expression, intracellular trafficking, secretion, and associated cellular energy requirements. In this project, we employed a Y2H approach in to identify new interacting proteins that may play a role in the above mentioned processes. As a result, we successfully isolated GABARAP, an ATG8 protein, as a potential interactor with the FVIII-B domain. While GABARAP did not co-immunoprecipitate with FVIII, we observed a weak interaction with full-length FVIII (FL-FVIII) but not with the B-domain-deleted FVIII (BDD-FVIII) using the BLITZ system. Additionally, there was a significant increase in the co-localization of GABARAP with FL-FVIII compared to BDD-FVIII. Furthermore, both transient knockdown of GABARAP through siRNA and stable knockout using CRISPR resulted in a decrease in FVIII secretion. Similar effects on FVIII secretion were observed for knockouts of MCFD2, LMAN1, and Calreticulin, while a contrasting effect, increasing secretion, was seen for knockouts of Calnexin, GABARAPL1, GABARAPL2, and ATG7. To explain the differential effects on FVIII secretion, we examined the expression profiles of the knockouts and the wild-type clones, revealing differences in several pathways, notably energy related oxidative phosphorylation and mitochondrial function. The residual cellular ATP content and cellular mitochondrial function showed a strong correlation with FVIII secretion levels. These findings establish a link between GABARAP proteins, the cellular energy status, and FVIII secretion.