|Membrane Traffic and Pathogenesis|
|HEAD||Dr Zurzolo Chiara / email@example.com|
|MEMBERS||Dr Vincenza Campana / Mr Philippe Casanova / Mlle Maria-Agata Catino / Dr Stephanie Lebreton/ Dr Zrinka Maijanovic / Mr Thomas Pocard / Mr Edwin Schiff /Dr Duncan Browman
One of the most important properties of epithelia is cell polarity, which enable cells to perform their vectorial activities. We study the mechanism of apical sorting of GPI-anchored proteins (GPI-APs) focusiong our attention on the role of membrane microdomains (or rafts), and protein and lipid segregation in this event. We are also studying the exocytic and endocytic transport of the prion protein, PrPC, and its infectious form PrPSc (scrapie), as well as pathological mutants associated with hereditary forms of TSEs. This will enable us to understand the role of intracellular trafficking and of association to rafts in the function and pathological conversion of this cellular GPI-AP.
1) Mechanism of GPI-anchored protein sorting to the plasma membrane
GPI-APs are sorted to the apical membrane in several epithelial cell lines and associate with rafts during their transport to the plasma membrane. Our knowledge of how this occurs is only rudimentary. In this project we are using both microscopic and biochemical approaches to analyze the role of raft domains in sorting and trafficking of GPI-anchored proteins and to characterize the molecular components of the machinery. We have shown that:
1. Rafts are not sufficient for sorting of GPI-APs, but oligomerization is required (Paladino, 2004)
2. Oligomerization is a specific requirement for GPI-AP sorting in different epithelia (Paladino, 2007)
3. The lipid composition of AP and BL detergent resistant domains is similar (Tivodar, 2006)
4. GPI-APs are sorted via a direct pathway to the apical domain of living cells (Paladino, 2006)
5. GPI-APs are organized in homoclusters forming choleseterol-dependent heteroclusters (submitted)
6. Distinct v-SNAREs regulate apical delivery in polarized epithelial cells (Pocard, 2007)
2) Intracellular trafficking of PrPC and its mutants: correlation with TSE pathogenesis
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders of humans and animals of either infectious, genetic or sporadic origin. They result from a post-translational alteration in the conformation of a host-encoded GPI-AP called PrPC into to the scrapie isoform PrPSc. This conformational transition is thought to be catalyzed by a specific physical interaction between endogenous PrPC and PrPSc, which is the principal component of the transmissible agent (or prion). The intracellular compartment where PrPC - PrPSc conversion occurs and how this process leads to neurological dysfunction are still unknown. Analyzing the intracellular trafficking of PrPC will be critical to understand how and where it is converted to PrPSc. We have analysed the biosynthethic/ degradative pathway, exocytic/endocytic trafficking of PrPC and of some mutants responsible for the hereditary diseases. We have shown that :
1) Raft-association of PrPC is nececessary for its correct folding in the ER (Sarnataro, 2004)
2) The raft environment may protect PrP mutants from conversion (Campana, 2005 and 2006, Sarnataro, 2006)
3) The proteasome is not involved in the degradation of PrP mutants (Campana, 2006).
4) Both raft-dependent and clathrin-dependent pathways are involved in PrPC endocytosis (submitted)
We are currently analysing the mechanisms of intercellular prion spreading and the site of conversion.
Keywords: Intracellular trafficking, prion , conversion site, GPI-anchored proteins, rafts, protein sorting, epithelial and neuronal cells
- Campana V., Caputo A., Sarnataro D., Paladino S., Tivodar S. and Zurzolo C. (2007) Characterization of the properties and trafficking of an anchorless form of the prion protein. The Journal of Biological Chemistry, 282 (31) : 22747-22755.
- Pocard T., Le Bivic A., Galli T. and Zurzolo C. (2007) Distinct v-SNAREs regulate direct and indirect apical delivery in polarized epithelial cells Journal of Cell Sciences, 120 : 3309-3320.
- Paladino S., Sarnataro D. and Zurzolo C. (2007) Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins. Traffic, 8, 251-258
- Campana, V., D. Sarnataro, C. Fasano, P. Casanova, S. Paladino, and C. Zurzolo. 2006. Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum.J Cell Sci 119:433-42
- Paladino, S., T. Pocard, M. A. Catino, and C. Zurzolo. 2006. GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells. J Cell Biol 172:1023-34.
Activity Reports 2007 - Institut Pasteur
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