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Hypoxia Inducible Factor 1 alpha (HIF1A) transcription factor as a model substrate to study selective protein loading into exosomes.
Portugal ( PorMSIC) - Nova University of Lisbon, Lisbon
CEDOC- CHRONIC DISEASES RESEARCH CENTER Rua Camara Pestana no 6, 6-A, Edificio CEDOC II, 1150-082 Lisboa, Portugal
Paulo Pereira, Joao Ferreira, Ana Soares
Type of Research Project
- Basic science
What is the background of the project?
Age-related damaged to different tissues or organs is often associated with a decrease in the supply of oxygen. At a cellular level, the transcription factor HIF1A is the major cellular sensor of deficient oxygen supply, activating genes involved in helping cells cope with hypoxia. Another common feature of ageing organisms is the reduced ability in sustaining efficient HIF1A stability. HIF1A protein levels are regulated by a number of different pathways involving both the lysosome and the proteasome. In our lab we have now gathered compelling evidences suggesting a new path for the regulation HIF1A, involving the selective packing of the transcription factor into small vesicles primed for secretion. These vesicles, called exosomes, are secreted by most cell types and contain RNA and proteins. Nevertheless, the mechanisms whereby cells regulate exosomal composition remain largely unknown. In this project, we will use the transcription factor HIF1alpha as a model substrate to study the exosomal loading of cytosolic proteins and their effect in receiving cells. We now propose a new model in which the LAMP2A-HSC70 (Lysosome-Associated Membrane Protein 2 - Heat Shock Protein 70) complex participates in exosomal cargo triage by mediating its translocation into these vesicles.
What is the aim of the project?
First we want to demonstrate that HIF1A is able to get selectively loaded into exosomes, through the LAMP2A-Hsc70 machinery. We also intend to show that exosomal HIF1A is delivered and transcriptionally active in exosome receiving cells.
What techniques and methods are used?
In this project we will use cell culture techniques to produce cell and exosomal samples. Exosomes will be isolate from cell culture media of wild-type and LAMP2A KO (Gene Knockout) cells through ultracentrifugation. We will subsequently use western blotting and immunocytochesmistry techniques to assess HIF1A levels in cells and exosomes and HIF1A subcellular localization. We will also incubate cells with HIF1A loaded exosomes and search for the presence and activity of the transcription factor in the receiving cells by immunocytochemistry and western blotting of HIF1A target genes or, alternatively, using a reporter gene.
What is the role of the student?
What are the tasks expected to be accomplished by the student?
All work will be done with supervision in order to give the student enough time to understand the procedures and learn from observation. But after learning the techniques, hopefully the student will know how to perform some experiments on his/her own. If so, the student will be able to perform various different techniques, such as cell culture techniques, western blotting and immunocytochemistry techniques. The student is also expected to critically discuss the results obtained.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
The student will be able to develop his/her knowledge about cell biology/molecular biology. The student will also learn all the techniques and methods mentioned in previous question.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system
- Ferreira JV; Fofo H; Bejarano E; Figueira Bento C; Ramalho JS; Girao H; Pereira P. CHIP/STUB1 is required for HIF-1A degradation by Chaperone-Mediated Autophagy. Autophagy 2013;9:1349-1366.
- Ferreira JV; Soares A; Ramalho JS; Pereira P; Girao H. K63 linked ubiquitin chain formation is a signal for HIF1A degradation by Chaperone-Mediated Autophagy. Sci Reports 2015; 5:10210
- Soares AR; Martins-Marques T; Ribeiro-Rodrigues T; Ferreira JV Catarino S; Pinho MJ; Zuzarte M; Anjo SI; Manadas B; Sluijter JPG; Pereira P; Girao H. Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells. Sci Reports 2015; 5:13243.
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