Study of AP1y2 and Alix function in the targeting of proteins for degradation in lysosomes or release in extracellular vesicles

Author(s)

Mara Elisama da Silva Januário

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-06-21

Examining board members

Luis Lamberti Pinto da Silva; Eurico de Arruda Neto; Juliano Coelho da Silveira; Ana Cláudia Trócoli Torrecilhas

• Advisor: Luis Lamberti Pinto da Silva

Abstract

Lysosomal degradation of endocytosed membrane proteins occurs through the targeting of these proteins to intraluminal vesicles (ILVs), formed in the multivesicular bodies (MVBs) lumen, and the subsequent fusion of MVBs with lysosomes. Despite its importance in the degradation of transmembrane proteins, MVBs have another important function, the production and release of extracellular vesicles (EVs). In this process, the MVBs do not fuse with lysosomes, but fuse with the plasma membrane resulting in the release of these vesicles that reside within MVBs to the extracellular environment. Several proteins regulate the targeting of cargo to MVBs. Studies that delineated the functions of AP1 in protein trafficking, focused on complexes containing the ?1 subunit, which mediates transport between trans-Golgi network (TGN) and endosomes. However, the human genome encodes a second isoform of this subunit, named ?2. Evidences from the literature, as well as results from our research group, indicate that AP1?2 regulates transport pathways that are distinct from the pathways classically attributed to AP1. By performing EGF-uptake assays under ?1 or ?2 knockdown (KD) conditions, it was observed that ?2 is required for degradation of internalized EGF, effect also observed for the EGF receptor (EGFR) using cell surface biotinylation assays. These results demonstrate that the lysosomal degradation of the EGFEGFR complexes via the canonical MVBs pathway requires the AP1?2 complex, but not AP1?1. In parallel with this study, we also analyzed the molecular mechanism of HIV-1 Nef targeting to MVBs associated with the EVs release. Nef is an important determinant in the modulation of the intracellular environment for efficient HIV replication and progression to AIDS. Nef is actively secreted via EVs and its release may lead to apoptosis of bystander acceptor cells. Moreover, Nef reduces the levels of CD4 and MHC-I molecules in EVs. Despite the importance of Nef release in EVs, the molecular mechanism used by Nef to be exported via EVs is unknown. Nef physically interacts with the ESCRT machinery accessory protein Alix, an important player in the process of ILVs formation and cargo selection. EVs released from HeLa cells and CD4+ T lymphocytes under Alix KD conditions demonstrated a significant IV reduction in Nef release via EVs. These results indicate that Nef requires Alix for its efficient release in EVs. (AU)