Morphofunctional aspects of the interaction between Protein Disulfide Isomerase-A1 (PDIA1) and ENOLASE1 in VSMCs

Abstract

Interaction between Protein Disulfide Isomerase A1 (PDIA1) and enolase 1 (ENO1) has been reported in several interatome databases. In our current project we collected significant evidences that validate PDIA1-ENO1 interaction in vascular smooth muscle cells (VSMCs) including co-localization, co-immunoprecipitation and proximity ligation assays. However, the biological significance of this interaction remains unclear. Our NMR metabolomics data show that modulation of PDIA1 expression did not affect the metabolism of VSMCs, indicating that PDIA1-ENO1 interaction does not interfere with the canonical role of ENO1 in the glycolytic pathway. Using proximity ligation assay (PLA), we detected the interaction primarily associated with the tubular endoplasmic reticulum and invadopodia-like membrane processes. This is a meaningful clue to the biological role of this protein complex since both proteins have a minority pool in the plasmatic membrane and have a convergent function in the processes of migration, invasion, and matrix remodeling. ENO1 on the cell surface acts as a plasminogen receptor, mediating its conversion into plasmin through the action of the enzyme urokinase. Once formed, plasmin activates other proteases such as metalloproteinases and collagenases that act in the proteolysis of the extracellular matrix. On the other hand, it was recently demonstrated that PDIA3, other member of PDI family, co-localize with invadopodia and participates in ECM remodeling by reducing disulfide bridges in crosslink of matrix proteins, facilitating the activity of proteases. These findings provide evidence that supports our hypothesis that PDIA1-ENO1 association play a role in cellular in ECM remodeling and processes such as migration and cell invasion. However, a seminal question still unclear is how PDIA1 reaches the plasma membrane. Our laboratory has been working on this issue and has already excluded several intracellular pathways for protein trafficking and secretion. In addition, even more intriguing questions are how the PDIA1-ENO1 complex is formed since PDIA1 is an ER resident protein and ENO1 is essentially cytosolic? How does this preformed complex in the vicinity of the ER travel until it reaches the plasma membrane? What cellular structures support this transport? These are some of the questions we aim to answer using cutting-edge electronic microscopy techniques such as correlative light and electron microscopy (CLEM), ultrastructural immunostaining and high resolution live image to track the dynamics of PDIA1-ENO1 complex from the surface of the reticulum to the plasmatic membrane. In view of the many pathophysiological roles of extracellular PDIA1, our results are bound to have relevant basic and translational implications. (AU)