Integrated study of adaptor protein 2 interaction interfaces in bioinspired membrane-based systems

• Autor(a) principal: Pereira, Daniel António Cunha
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/29276
• topic: Clathrin-mediated endocytosis; Adaptor protein 2; PIP2,TGN38; CD4; Membrane biomimetics; Multi-technique based-approach; QCM-D

Endocytosis plays a central role in many essential processes for the cell life, including uptake of nutrients, cell signalingand membrane trafficking.Dysfunctional endocytic processes are linked to some physiopathological conditions like Alzheimer’s disease, cancerand viral infections. Clathrin-mediated endocytosis(CME)is the main endocytic pathway for the incorporation of a wide variety of molecules.Although many aspects of CME are well understood, some questions still need to be addressed and further explored.This research aimedto provide insight into the early steps of CME pathways, particularly, the sorting and cargo selection, and anchoring of adaptor protein 2 (AP2) into the membrane.To achieve this a simplistic approach was implemented, where the system was reducedinto its most fundamental components: PIP2-enriched membranes, AP2 and cargo molecules (TGN38 and CD4). To fully characterize the system a multi-technique based-approach comprising solid-state NMR and surface-sensitive techniques is proposed.In this work, different membrane biomimetic systemswereproduced and characterized by ellipsometry, small-angle neutron scattering (SANS)and quartz crystal microbalance with dissipation monitoring (QCM-D).The viscoelastic contributions of the incorporation of the different anchoring points intothesupported lipid bilayer(SLB)weremonitored by QCM-Dand shown tender effects for PIP2, while more pronounced effects were observed for the incorporation of cargo molecules.New results presented in thisworkrevealed that AP2 could bindintoSLBsfunctionalized withcargo molecules, both TGN38 and CD4,in the absence of PIP2 lipids. These observations suggest that AP2 might have different binding mechanismsaccording to the anchoring points available at the surface of the membrane.