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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-28626

Oliveira-Martins, J B. Alpha-cleavage of the prion protein and the putative function of the prion protein in lymphocyte homeostasis. 2009, University of Zurich, Faculty of Science.

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Prion diseases are fatal neurodegenerative disorders that have the particularity of being caused by an infectious proteinaceous agent. The main player in this disease is a structural modification of the endogenous non-pathogenic prion protein (PrPC), termed PrPSc. Though PrPC is widely expressed in the body and highly conserved in mammals, its physiological function remains elusive. Moreover, Prnp-/- mice devoid of PrP have no strong evident phenotype, apart from being completely resistant to prion diseases. Interestingly, many PrPC mutants with deletions around the α-cleavage site of PrPC are highly toxic when expressed in transgenic mice and this toxicity is rescued by coexpression of wild-type PrPC. One of the main mysteries in the prion field is the elucidation of the molecular mechanisms that drive not only the toxicity of PrPSc, but also the pathology caused by these PrPC deletion mutants.
To understand the principles behind PrP-dependent toxicity, first a model was conceptualized, which aimed to integrate and unify the largest possible set of experimental evidences already available in the literature. The goal was to conceive a framework that congregates a putative function of PrPC, as well as the pathological features of prion diseases and of prionopathies caused by PrPC deletion mutants. From all possible models considered, the most parsimonic one established that α-cleavage of the PrPC central domain is crucial for regulating PrPC function and for explaining PrP-dependent toxicity. The model was also based on a main set of assumptions, which are that internalization of uncleaved PrPC contributes to cell activation, and that miss-regulation of this process results in cell over-activation and consequent cell death. Therefore, I decided to study these two paradigms, which are the α-cleavage, and the toxicity due to the putative cell hyper-activation derived from PrPC miss-regulation.
Concerning α-cleavage, the main grounds sustaining its importance are that both PrPSc and all toxic PrPC deletion mutants have a strong impairment on α-cleavage. In this project I tried to identify the PrPC domains neighbouring the α-cleavage site, which play a role in mediating this proteolysis. Assessment of the α-cleavage rate of a large number of PrPC mutants designed in this study revealed that neither substitution of the amino-acid residues at the cleavage site, replacement or inversion of the charged residues, mutation of the palindromic region, nor exchange of the hydrophobic amino-acids, had major effects on the degree of α-cleavage. Also, short deletions at the cleavage site had only a mild effect on PrPC proteolysis. However, an increase in the size of the deletion resulted in a gradual decrease of α-cleavage rate, which was virtually blocked in the deletion mutant PrPΔ(106-119). These results suggest that cleavage of PrPC is largely sequence independent, which is an uncommon feature that can find parallelism with the proteolytic plasticity of the γ-secretase.
To investigate the second paradigm, that PrPC is a modulator of cell activation, and that miss-regulation of its function can result in cell death, the impact of PrPC on lymphocyte activation and positive and negative selection of lymphocytes was assessed. This study suggested that PrPC is strongly up-regulated in activated T-cells, and that it is found at high quantities in activated lymphocytes, in cells undergoing positive selection, and in populations that traditionally have high reactivity to antigens, like regulatory T-cells and marginal zone B-cells. An alteration of the lymphocytic homeostasis in mice deficient of or overexpressing PrPC was also observed. This alteration could be explained by a deletion of the transgenic cells with high levels of PrPC during the negative selection of auto-reactive cells, and by an arrest in development due to anergy of Prnp-/- lymphocytes. In addition, it was shown that the costimulatory CD3 molecule is down-regulated in circulating T-cells from PrPC-overexpressing mice, probably in order to re-establish the activation threshold, which might have been decreased due to the high presence of PrPC. Finally, I showed that PrPC overexpression partially rescues the impairment on development of CD4 T-cells in mice devoid of MHC class II, which is essential for providing an activatory signal to these cells.
Taken together, here I propose a unifying model for PrP function and PrP-dependent toxicity, assessed the main assumption, and studied the principles governing α-cleavage, which is the main mechanism of this model.

Item Type:Dissertation
Referees:Aguzzi A, Oxenius A, Becher B
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Neuropathology
DDC:570 Life sciences; biology
610 Medicine & health
Deposited On:05 Feb 2010 15:39
Last Modified:26 Sep 2012 14:41
Number of Pages:111
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005928153

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