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The functional characterization of AP-1 in the endolysosomal system and its relationship with the assembly of HIV-1

Grant number: 21/01182-1
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): July 01, 2021
Effective date (End): June 30, 2023
Field of knowledge:Biological Sciences - Morphology - Cytology and Cell Biology
Principal researcher:Luis Lamberti Pinto da Silva
Grantee:Lucas Alves Tavares
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The biogenesis of the endolysosomal system organelles is linked to the multiple transport routes starting from either the Golgi complex or the plasma membrane, which are responsible for delivering proteins and lipids to the compartments of this system. One family of proteins involved in protein transport within the endolysosomal pathway is the Adaptor Proteins (APs). APs are heterotetrameric complexes that mediate the selection of cargo proteins and the formation of transport vesicles, regulating the trafficking of proteins between different organelles in the late secretory pathway. AP-1 and AP-4 are located at the Trans-Golgi Network (TGN), AP-2 at the plasma membrane, AP-3 at early endosomes, and AP-5 at late endosomes. Each complex contains two large subunits (³ and ²1 for AP-1, ± and ²2 for AP-2, ´ and ²3 for AP-3, µ and ²4 for AP-4, ¶ and ²5 for AP-5), a medium subunit (¼1- ¼5) and a small subunit (Ã1- Ã5). AP-1, AP-2, and AP-3 are potentially heterogeneous due to the existence of multiple isoforms of the subunits encoded by different genes, including two ³ subunits (³1 and ³2), two ¼1 (¼1A and ¼1B) and three Ã1 (Ã1A, Ã1B and Ã1C) subunits for AP-1. The combination of different subunit isoforms can, in theory, generate at least twelve AP-1 complexes. However, few studies demonstrate whether these different combinations of AP isoforms are formed and if they have different functional properties. Previous results from our group indicate that the ³1 and ³2 subunits define AP-1 complexes with distinct functions in intracellular traffic. However, the transport routes controlled by these AP-1 variants have not yet been precisely defined. Thus, this work seeks to identify and characterize the role of ³2 and ³1, subunits of AP-1, in intracellular protein trafficking in the physiological context and a host-pathogen interaction context. First, we will elucidate the possible role of AP-1³2 in endosomal maturation via interaction with the phosphatidylinositol 3-kinase 2 alpha (PI3KC2±). For this purpose, we will verify whether the knockdown (KD) of AP-1³2 or PI3KC2± causes changes in the subcellular localization of these proteins through microscopy and subcellular fractionation. We will also map the interaction sites between PI3KC2± and AP-1³2 and check if they are essential for the distribution and function of these proteins in the cell. In a second moment, we will verify the possible role of AP-1³2 in the intracellular trafficking of Cu+ receptors (ATP7A and ATP7). In this context, we will verify whether ATP7A and ATP7B can interact with AP-1³2 and whether the KD of AP-1³2 compromises the transport of ATP7A and ATP7B from endosomes to TGN. As a third objective, we will investigate the possible role of AP-1 in the assembly and externalization of HIV-1 viral particles. Thus, we will monitor the intracellular trafficking of HIV-1 Env from the ER to the plasma membrane using the RUSH (Retention Using Selective Hooks) system, allowing us to study in an innovative way how it relates to HIV-1 Gag. We will also verify if AP-1³2 can interact with the cytosolic tail of Env (gp41CT) through in vitro protein-protein interaction assays. Finally, we will investigate if the expression of AP-1 subunits (AP-1³1, AP-1³2 or AP-1¼1A) is necessary for: 1) the incorporation of Env and Gag into HIV-1 viral particles; 2) correct location of Env in the plasma membrane and; 3) the production of infectious viral particles. Therefore, the proposed study can potentially contribute to understanding fundamental processes in the biogenesis of HIV-1 viral particles. Besides, we will try to elucidate aspects, still unknown, of how HIV-1 manipulates cellular factors to evade the immune system. Altogether, the proposed study can contribute to a better understanding of essential processes regulating protein trafficking in the endolysosomal system. (AU)

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