| Summary: | © 2015 Dr. Ismail Sami Mahmoud In the complex systems of eukaryotic cells, cellular contents, materials and molecules need to be transferred between distinct membrane enclosed compartments in a regulated and specific manner. Intracellular trafficking pathways mediate the transport of cargoes to their destination. This thesis deals with two distinct trafficking pathways: the endocytic/recycling and Golgi trafficking pathways. The recycling pathway was my main focus for this study, where I sought to define the trafficking and recycling of the neonatal Fc receptor (FcRn) in relation to its ligand albumin. In the second part of this thesis, I identified interacting partners for the Golgi-localized small G protein Rab30, which has suggested new insights into the function of this small G protein in mediating Golgi trafficking. The field of engineered therapeutic proteins is making a major impact on clinical treatment. For example, therapeutic use of IgG antibodies products is fundamental for treatment of various diseases such as cancer. However, the short life span of many recombinant antibodies and protein products is a key limiting factor of their effectiveness in many clinical applications. Hence, there would be considerable benefits in designing of new approaches to extend the serum half-life of recombinant protiens. IgG and albumin are taken up by cells through fluid phase endocytosis destined for lysosomal degradation pathway. The neonatal Fc receptor (FcRn) binds ligands in acidified endosomal compartments where the low pH is permissive for binding, forming a complex which is diverted from the lysosome and directed into the recycling pathway. There is a considerable interest in exploiting FcRn for the development of therapeutic proteins with extended serum half-lives. Understanding the mechanisms behind FcRn recycling and intracellular ligand interactions is particularly important to utilize FcRn for extending the lifespan of recombinant proteins. However, the pathways of intracellular trafficking, and the specific sorting mechanisms and machinery that regulate movement of FcRn and FcRn-ligand complexes in cells are poorly defined. In particular how FcRn is sorted from early endosomes into the recycling pathway is still a puzzle. A main aim of this project was to analyse the intracellular trafficking of FcRn, to define potential sorting signals of FcRn that mediate the sorting to the endocytic/recycling pathway and also to analyse the intracellular transport of the FcRn ligand, albumin, in primary cells. In Chapter 3 of this thesis I explored three different cell-based systems to study the intracellular trafficking pathways of FcRn and its ligand, namely HeLa cells, endothelium cells (HMEC-1) and mouse bone marrow derived macrophages (BMDMs). By analysing the receptor trafficking at steady state and after internalization, I show that FcRn in HeLa cells traffics predominantly to the recycling endosome via the early endosome. I have also identified macropinocytosis as a major endocytic pathway for the uptake of human serum albumin in endothelium HMEC-1 and BMDM cells. In Chapter 4, experiments were performed to dissect potential sorting signals in the cytoplasmic tail of FcRn that could mediate the sorting of this receptor into the recycling endosomes. A series of mutant constructs were generated and analysed for their localization and trafficking into endocytic/recycling compartments. By analysing FcRn mutants and CD8/FcRn chimeric molecules, I have identified the sequence (GLPAPWISL) which is responsible for sorting FcRn to the recycling endosomes. I also present evidence to suggest that the distance between the transmembrane domain and endocytic motif could be crucial in mediating the internalization of this membrane protein. The Golgi serves as the major protein-sorting hub for the secretory pathway. The Rab family of small G-protein plays a key role in regulating the transport of cargo proteins, and a number of Rabs have been reported to be localized to the Golgi. Rab30 is a small G-protein that has been recently identified as a Golgi Rab that may influence Golgi trafficking; however its function remained poorly defined. Here, I have used proteomics to identify the interacting partners of Rab30. In Chapter 5, I have optimized pull down experiments of Rab30 in HeLa cells stably expressing the constitutive active Rab30-GFP (Rab30 (Q68L)-GFP) and analysed the complexes by mass spectrometry, and identified potential binding partners of Rab30 including ARF-1 and COPA, two main components of retrograde transport from the Golgi to the ER. These analysis, together with preliminary findings from functional assays, provide insights for a potential role of Rab30 in the retrograde transport between the Golgi apparatus and the ER.
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