Proximity‐Labeling by BioID Reveals Pleiotropic Role of Ski in Cardiac Fibrosis

Abstract

Despite recent improvements made in cardiac patient treatment and outcomes, current therapies only serve to alleviate the symptoms of cardiac fibrosis, rather than correct the disease itself. Our lab has previously established Ski as a potent inhibitor of TGF‐β signalling in cardiac myofibroblasts, the primary effectors of fibrosis; however our recent investigations suggest that Ski may inhibit a multitude of pro‐fibrotic pathways, including Hippo. We have specifically observed upon Ski overexpression in primary cardiac fibroblasts, a marked reduction in the expression of TAZ (or WWTR1), one of the main nuclear effectors of the Hippo pathway. To further expand the current knowledge on Ski's anti‐fibrotic properties, and provide insight into potential mechanisms of action, we mapped its interactome using enzyme‐catalyzed biotin proximity labelling (BioID2). Using the BioID2 vector, E. coli BirA* biotin ligase was fused to the N‐terminus of human Ski and the fusion protein was then expressed in fibroblasts in the presence of an excess of free biotin. Whole cell lysates were then subject to streptavidin‐mediated protein capture and potential Ski binding partners were identified by tandem time‐of‐flight mass spectrometry. The resulting candidates included several known Ski interactors (eg. RSmad2 and co‐Smad4), but also revealed novel interactors which potentially link Ski's anti‐fibrotic capacity and interaction with the Hippo signaling pathway. We also observed several interactions with cytoskeletal components, including those involved in actin dynamics and stress fiber formation. To confirm results from the affinity capture, candidate interactors were verified using immunoblotting. In addition, the expression of potential Ski interactors was examined in a rat model of cardiac fibrosis, post‐myocardial infarction. Our data suggest that Ski serves not only as a Smad‐dependent TGF‐β inhibitor, but also interacts with the Hippo, Wnt/β‐catenin, and FoxO signaling pathways‐‐all of which are suspected contributors to fibrosis. In addition, Ski's interaction with various points of cytoskeletal organization further indicates that its functions in the cell go beyond simple inhibitory protein‐protein interactions.Support or Funding InformationNM Landry's research has been funded by studentships from the Canadian Institutes of Health Research (CIHR), Research Manitoba, as well as the Bank of Montreal (BMO). IMC Dixon is grateful to the Heart & Stroke Foundation of Canada for a grant (G‐17‐10018631) in support of this research, as well as the St. Boniface Hospital and Research Foundation for their continued operating patronage.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.