Autophagy : investigating the possible interaction between cochaperone BAG3 and the Ubiquitin-binding protein p62

Abstract

Introduction: Autophagy is an evolutionary conserved intracellular catabolic process which involves the degradation of a cell’s own components. The degradation happens in two (2)main pathways: the ubiquitin-proteasome system that degrades short-lived proteins, and the autophagy system that degrades long-lived proteins and damaged organelles. There are three known different pathways of autophagy being; (1) macroautophagy (2) microautophagy and (3) chaperone-mediated autophagy The ubiquitin-binding protein p62 (SQSTM1) is the scaffold protein that binds to proteins that are to undergo macroautophagy, while BAG3 is known for its involvement in chaperonemediated autophagy (CMA). Any possible interaction between Bag3 and p62 of which would indicate convergence of CMA and macroautophagy where p62 is a major factor. Aims: To investigate any interaction between BAG3 and p62 or their co-localization in HeLa cells. Methods: Protein-protein interaction using GST pulldown and visualization of interaction with Coomassie blue staining and western blot. The investigation of co-localization was done through fluorescence confocal microscopy Results: BAG3 does not co-localize with p62wt or p62 (R7A/D69A)/ p62 (R21A/D69A), but has some interaction and co-localization with p62 (d123-339) and p62 (124-440). This suggests that BAG3 interact with p62 in the region amino acids 339-440. This is the region containing LIR, KIR and UBA domains of p62. Surprisingly, we found interaction between the p62 deletions constructs p62 (d123-339) and p62 (124-440) but not with the wild type p62. This may lie in the fact that p62 forms polymers making the reaction sites unavailable, or that the small constructs of p62 do not fold properly and get bound to BAG3 for destination to CMA. A further investigation is required using in vitro translation and radioactive analysis of BAG3 and p62 interaction, and also in vivo co-immunoprecipitation could be performed.