Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - TAMPTING (The biogenesis of tail-anchored membrane proteins: knowledge and exploitation)

TAMPting is a Marie Curie ITN project funded by the 7th European Union framework programme from November 2013 to November 2017. This four-year project brings together five academic partners and three SMEs at locations across Europe and Israel. The primary objective of the TAMPting network is to provide an interdisciplinary platform of research and training that enables the application and exploitation of tail-anchored membrane proteins, and defines the molecular mechanisms underlying their unique biological properties.

Our project has two overarching goals: firstly, understanding how tail-anchored membrane proteins are incorporated into a variety of target membranes; secondly, exploiting tail-anchored membrane proteins to improve liposome based drug delivery and create artificial membranes.

Project 1 = Creating synthetic membranes using tail-anchored (TA) protein chimeras. This project has established a robust platform for the spontaneous insertion of TA proteins into artificial membrane systems. Tasks already successfully achieved include the design, expression and purification of recombinant TA proteins, their incorporation into cargo filled liposomes and the analysis of the uptake of TA-protein decorated liposomes using cell culture models in combination with fluorescence activated cell sorting and confocal microscopy. It is envisaged that the future exploitation of such TA proteins will enhance liposome mediated drug delivery and the production of synthetic membranes.

Project 2 = BAG6/SGTA mediated quality control of mislocalized membrane proteins. This project has provided an understanding of potential links between tail-anchored protein biogenesis, the quality control of mislocalized membrane proteins and the molecular pathology of protein folding diseases. Examples of some of the tasks performed are the creation of stable mammalian cell lines expressing mislocalized membrane protein substrates and investigating the interactome of key quality control factors. Outcomes will include an understanding of the effects of quality control on substrate stability and ubiquitination status, and the cellular consequences of these changes.

Project 3 = Intracellular targeting of TA proteins. This project has addressed the mechanisms that underlie the specificity of tail-anchored protein delivery to different subcellular compartments within a eukaryotic cell. Examples of tasks performed include determination, by confocal microscopy and cell fractionation, of the intracellular distribution of TA proteins in cells silenced for components of the TRC40 pathway and in vitro targeting assays using recombinant TA proteins. Expected outcomes include the creation of an optimised cell-free system for studying TA protein biogenesis and an understanding of how tail-anchored proteins are selectively delivered to either the endoplasmic reticulum or the mitochondrial outer membrane.

Project 4 = Spontaneous TA protein insertion. This project is central to the creation of a ubiquitous platform that employs TA proteins to enhance liposome mediated drug technology by incorporating additional biological effectors. Examples of the tasks performed include the development of novel assays for studying the spontaneous membrane insertion of TA proteins and the application of protein “ligation” technology to create a flexible TA based scaffold to which different effector domains, such as biologically active peptides, can be attached.

Project 5 = Tail-anchored protein biogenesis in E. coli. This project has identified bona fide prokaryotic TA proteins and is in the process of defining the cellular components that mediate their biogenesis. Examples of the tasks performed include the expression of candidate TA proteins and the analysis of their subcellular localisation using cell fractionation approaches in combination with fluorescence microscopy studies of GFP-tagged versions of TA proteins. Expected outcomes include a catalogue of known/predicted E. coli TA proteins and new insights into the molecular mechanisms that underlie their biogenesis.

Project 6 = Mycobacterial tail-anchored proteins: a druggable target? This project has identified candidate mycobacterial TA proteins and is using both biochemical and genetic approaches in order to identify key mediators of their biogenesis in Mycobacteria. Examples of the tasks performed include the bioinformatics based identification of candidate TA proteins, biochemical studies of components implicated in their production and the creation and/or exploitation of conditional mutants of Mycobacterium smegmatis to study TA protein biogenesis in vivo. Expected outcomes include an understanding of TA protein biogenesis in Mycobacteria and the identification of potentially druggable Mycobacterial targets.

Project 7 = Biogenesis of mitochondrial tail-anchored proteins. This project has provided detailed insights into the biogenesis and integration of TA proteins at the mitochondrial outer membrane (MOM). Examples of the tasks performed include identification of true TA proteins resident in the MOM, developing assays for TA protein insertion into the MOM and studying the effect of specific components on the production of TA proteins in comparison with other membrane proteins. Expected outcomes include the identification of cellular factors that promote mitochondrial TA protein biogenesis and an understanding of their mode of action.

Project 8 = Cytosolic delivery factors for mitochondrial tail-anchored proteins. This project has identified cytosolic factors that are implicated in the early stages of mitochondrial TA-protein biogenesis. Examples of the tasks performed include the generation of TA protein specific antibodies and development of biochemical assays to monitor the membrane-integration of TA-proteins both in vivo and in vitro. Expected outcomes include an understanding of the molecular basis for the selective delivery of TA proteins to defined subcellular organelles.

Project 9 = Redefining the function of TRC40. This revised project is investigating the role of mammalian TRC40 as a molecular chaperone and the physiological consequences of such a function. Examples of tasks performed include biochemical investigations of TRC40, in silico and experimental studies of its tissue distribution and the analysis of potential substrates. Expected outcomes include the determination of whether TRC40 performs a chaperone like function that contributes to quality control and cellular proteostasis.

Project 10 = Role of WRB in cardiac function. This project is investigating the role of the TRC40-mediated ER delivery pathway for TA protein biogenesis using a cardiac myocyte model. Examples of the tasks performed include the isolation and analysis of cardiomyocytes from mice with depleted levels of the WRB subunit of the TRC40 receptor and the characterisation of WRB-depleted hearts. This project will address the physiological importance of the TRC40-receptor dependent targeting pathway in an animal model.

Project 11 = Customising liposomes with TA protein chimeras. This project is core to developing a TA protein based platform the customization of liposomes that are suitable for drug delivery. Examples of tasks performed to date include preparation of the preparation and characterization of drug loaded liposomes, optimization of the incorporation of TA proteins, and preliminary analysis of uptake using cultured cells. This project will establish the efficacy of using TA protein to enhance liposome uptake.

Project 12 = Recombinant TA protein chimera production: This project aims at optimizing E. coli based production and subsequent purification of recombinant TA-protein chimeras. The approaches used are also suitable for the production of other types of membrane protein that can be used for e.g. antibody production and functional studies. Expected outcomes include the production of TA chimeras for use in liposome manufacture and the identification of factors involved in TA biogenesis.

Project 13 = Novel antibodies for key cellular components. This project continues to deliver a range of new antibodies that recognise a variety of TA proteins as well as additional antibodies against key cellular components that facilitate their biogenesis. Examples of the tasks performed include the design and production of suitable immunogens comprising recombinant proteins and synthetic peptides, the production and affinity purification of antibodies and the creation of eukaryotic expression vectors for GFP-tagged fusion proteins to enable the characterization of new antibodies.

Reported by

United Kingdom


Life Sciences
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