Molecular chaperone HSP70 mediates proteostasis in endothelial cells response to hemodynamic force

Abstract

The molecular chaperone HSP70 is the most abundant chaperome member. In the vascular context there are only two know clients in endothelial cells (ECs): CLEC14a (C-type lectin domain family 14 member) and nucleolin, proteins associated to tumor angiogenesis. Loss-of-function experiments in static ECs promotes broader general effects like impairment of migration, tube formation, phosphorylation of eNOS and AKT in response to VEGF. The shear stress is the main hemodynamic force in vessel that controls endothelial phenotype. In this context, there is a lack of screening to quantify and identify specific HSP70 clients which depends of this chaperone to reach native folding. The results will allow deep knowledge in molecular basis of vascular physiopathology. Through this proposal, we aim to study: (1) identify newly synthesized HSP70 clients under shear stress physiological vs pathological through adaptation of BONCAT (bioorthogonal non-canonical amino-acid tagging) coupled to HSP70 immunoprecipitation and mass spectrometry, (2) evaluate formation of HSP70 high-molecular-weight chaperome complexes with Native PAGE and pull down it through chemical YK5-bait, (3) as a disease model and HSP70 loss of function, we will pursue our preliminary results with HSP70 low levels in ECs isolated of pulmonary arteries proximal of thrombus from chronic thromboembolic pulmonary hypertension (TEPH) patients. HSP70 decrease staining compared to normal vessel was also confirmed by immunohistochemistry in tissue adjacent to thrombus. Our hypothesis is that chronic stress like shear stress regulate proteome lead to endothelial cell disfunction. Balance failure in proteostasis regulated by chaperones is a crucial step to vascular appropriate response. Longitudinal temporal analysis of hemodynamic forces in different flow partner could give insight and potentially gives us new molecular target to HSP70 in a vascular context-disease. (AU)