Identification of interacting proteins of Yes-Associated Protein (Yap) in mammary epithelial cells exposed to laminin-rich extracellular matrix

Author(s)

Antonio Carlos Manucci

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2019-03-01

Examining board members

Alexandre Bruni Cardoso; Nathalie Cella; Ana Carolina Baptista Moreno Martin; Flavia Vischi Winck

• Advisor: Alexandre Bruni Cardoso

Abstract

Extracellular matrix (ECM)-signaling is crucial for determination of epithelial cell fate and behavior in the mammary gland. However, little is known about the molecular mechanisms involved in these processes. The Hippo pathway, a signaling cascade involved in the regulation of several cellular processes, including organ size, seems to be an important candidate as a mediator of this signaling. Our preliminary results indicate that the tissue architecture and the basement membrane, an ECM component of epithelia and other tissues, influence the location, level and activity of YAP, an effector of the Hippo pathway. In this context, the goal of this work was to identify the proteins that interact with Yap (ortholog of YAP in mouse) in mammary epithelial cells in response to the basement membrane. We used EpH4 cells, a nontumoral murine mammary cell, in a functional differentiation and acini-forming in tridimensional (3D) culture assay. Treatment of 3D multicellular structures pre-formed on nonadhesive plates with a laminin-rich extracellular matrix (lrECM) altered the subcellular localization and pattern of Yap, as well as gene expression of Hippo pathway proteins and Yap targets, but did not altered the expression of the pathway members at the protein level. Coimmunoprecipitation (CoIP) followed by mass spectrometry analysis identified a differential set of proteins interacting with Yap in cytoplasmic fractions of EpH4 cells in the absence or presence of lrECM in an ECM-overlay culture model. An analysis performed with the KEGG Pathways database revealed that putative Yap interactors in non-treated cells participate in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to lrECM Yap interactors are associated to metabolic processes and are mainly key-proteins of metabolism of lipids and carbohydrates. A search in interaction networks platform STRING did not identify previous works that showing the interaction of Yap with these proteins. Vizit platform indicated the participation of Yap in processes related to the synthesis and activity of lipids and hormones, which reinforces the evidences that Yap can play a novel poorly explored role. To obtain complementary results to CoIP, we devised the proximity-dependent biotinylation identification (BioID) assay on embryonic renal cells of 293FT cell line. Pulldown-isolated proteins were identified by mass spectrometry and an analysis performed with Gene Ontology database revealed that putative Yap interactors are Hippo pathway-related proteins, which reinforces the robustness of the assay. We intend to transpose this system to the EpH4 cells. We expect that, together, these results will guide us in future projects to understand the signaling mechanisms of ECM in mammary gland morphogenesis and differentiation. (AU)

• FAPESP's process: 16/09561-3 - Identification of the partner proteins of Yes-Associated Protein (YAP) in mammary gland epithelial cells exposed to reconstituted basement membrane
• Grantee: Antonio Carlos Manucci Pereira Júnior
• Support Opportunities: Scholarships in Brazil - Master