The biogenesis of organelles of the endo-lysosomal system is dependent of multiple transport pathways from the Golgi complex and plasma membrane, which are responsible for the delivery of proteins and lipids to the compartments of this system. Molecules that play key roles in intracellular trafficking within the endo-lysosomal system include Rab5 and Rab7, which are involved in the maturation of early endosomes to late endosomes; and Alix, LBPA and the ESCRT (Endosomal-Sorting Complexes Required for Transport) machinery that control the biogenesis of intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) and coordinate the selection of ILV cargo for lysosomal degradation. Another important group of proteins involved in the biogenesis and function of endosomes and lysosomes are the adaptor proteins (APs). APs are heterotetrameric complexes that mediate the selection of the cargo proteins and the formation of vesicle carriers that mediate transport between different organelles. AP-1 and AP-4 are localized at the trans-Golgi region, AP-2 at the plasma membrane, AP-3 at early endosomes, AP-5 at late endosomes; and select cargo from these locations. Each complex is formed by 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 subunits encoded by different genes, including two ³ subunits (³1 and ³2), two ¼1 (¼1A and ¼1B) and three Ã1 (Ã1A, Ã1B and Ã1C) for AP-1; two ± (±A and ±C) for AP-2; and two ²3 (²3A and ²3B), two ¼3 (¼3A and ¼3B) and two Ã3 (Ã3A and Ã3B) to 3-AP. The combination of different isoforms of the subunit can, in theory, generate at least twelve types of AP-1, four AP-2 and eight AP-3 complexes. However, there are few studies to show whether these different combinations of isoforms of APs are formed and/or have different functional properties. Thus, the present study aims to identify and characterize the role of ³2 and ³1, AP-1 subunits, in the intracellular protein trafficking within the endo-lysosomal system. In this context, we will initially develop ³2 and ³1 knockout cells by CRISPR/Cas9 technology and then analyze the role of ³2 and ³1 in the MVBs biogenesis. Toward this goal, we will verify the ratio between the number of endosomal structures positive for Rab5 or Rab7 in ³1 and ³2 knockout cells by indirect immunofluorescence, as well as the levels of ESCRTs components by western blot. In addition, we will try to understand the role for ³2 and ³1 in correct function of lysosome, by investigating possible alterations in trafficking of hydrolases and hydrolase receptors in ³2 and ³1 knockout cells. In addition we will verify the morphology of lysosomes and MVBs by electron microscopy through immunostaining of lysosomal membrane molecules. Our unpublished findings revealed that ³1 and ³2 play distinct roles in HIV-1 Nef-mediated downregulation of CD4 and HLA-A2. Here, we will investigate the roles played by ³-isoforms in these Nef-mediated alterations in protein trafficking, in the context of HIV-1 infection in primary T cells. Therefore, we will perform depletion ³1 and ³2 by shRNA in primary lymphocytes and analyze the total cell and the plasma membrane levels of CD4 and HLA-A2 in HIV-1 infected cells. A better understanding of these processes will reveal relevant aspects about how HIV-1 Nef subverts cellular factors to reduce the expression of key molecules of the immune system. In addition, the proposed study has the potential to contribute to the understanding of fundamental processes of protein trafficking involved in the maturation of MVBs and lysosomes biogenesis.
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