Pepe, Anna (2016) Role of non-integrin laminin receptor and prion-like protein Shadoo in the trafficking and folding of cellular prion protein. [Tesi di dottorato]

[img]
Preview
Text
Pepe_Anna_28.pdf

Download (2MB) | Preview
[error in script] [error in script]
Item Type: Tesi di dottorato
Lingua: English
Title: Role of non-integrin laminin receptor and prion-like protein Shadoo in the trafficking and folding of cellular prion protein
Creators:
CreatorsEmail
Pepe, Annaannapepe2103@gmail.com
Date: 31 March 2016
Number of Pages: 109
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Scuola di dottorato: Medicina molecolare
Dottorato: Genetica e medicina molecolare
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
nomeemail
Nitsch, Lucionitsch@unina.it
Tutor:
nomeemail
Sarnataro, DanielaUNSPECIFIED
Date: 31 March 2016
Number of Pages: 109
Uncontrolled Keywords: Shadoo; Laminin receptor; PrP
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/13 - Biologia applicata
Date Deposited: 13 Apr 2016 12:01
Last Modified: 03 May 2017 01:00
URI: http://www.fedoa.unina.it/id/eprint/11041

Abstract

Transmissible spongiform encephalopathies (TSE), also known as prion diseases, are fatal neurodegenerative disorders which can affect both in humans and animals. They occur as sporadic, infectious or genetic disorders (Prusiner et al.; 1998). A major feature of these diseases is the conversion of the non-pathogenic cellular prion protein (PrPC) into the pathogenic scrapie isoform (PrPSc). This isoform has a strong tendency to polymerize, forming amyloid aggregates and accumulating in the cells, thus resulting in neurodegeneration. The possible mechanisms of the prion conversion reaction into the PrPSc are still unknow; and could be involved other cellular factors which could trigger, enhance, or accelerate scrapie prion formation. An important role has been proposed for the 37/67 kDa laminin receptor in prion infections, since it has been shown to act as the receptor for both PrPC and PrPSc (Reiger et al.; 1999, Gauczynski et al.; 2001). Moreover, the 37/67 kDa laminin receptor is required for PrPSc propagation in cultured cells, thus suggesting that 37/67kDa LR-PrPC interaction is related to the pathogenesis of prion diseases. As a result, the inhibition of this receptor may be a strategic therapy against prion diseases. In the first part of my thesis we have investigated the relationship between 37/67kDa LR and PrPC in the presence of specific LR inhibitor compound. We have characterized the trafficking of 37/67kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67kDa LR as the almost exclusive isoform interacting with PrPC. Here, we show that the treatment with the 37/67kDa LR inhibitor, NSC47924, affects both the direct 37/67kDa LR-PrPC interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67kDa LR and then its degradation via-lysosomes. On the other hand, PrPC is stabilized on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrPC and 37/67kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases. Another important issue in prion diseases is the search for a cofactor involved in the conversion process of PrPC into PrPSc. Thus, it is important to evaluate the role of other genes and proteins in TSE pathogenesis and susceptibility. A very promising candidate is Shadoo, a glycosylated GPI-anchored protein like PrPC. It is a natively unstructured protein (Watts et al. 2007) that resembles aspects of the PrPC N-terminal and central region (Daude et al. 2010). Physiologically, Shadoo shows neuroprotective properties similar to those of PrPC (Watts et al. 2007): therefore, it is possible that Shadoo and PrPC may be functionally related. Shadoo is expressed in the mouse brain less widespread than PrPC and is prominent in two regions in which PrPC is notably absent, suggesting that it could provide a PrPC-like function into areas of the brain in which PrPC is deficient. The downregulation of Shadoo expression in the brain of prion-infected animals and the direct in vitro interaction between prion protein and Shadoo (Jiayu et al. 2010) suggest a relationship between Shadoo and prion replication. However, the contribution of this interaction to the evolution of prion pathologies or to the PrPC folding pathway remains uncertain (Ciric et al. 2015). Similar to PrPC, Shadoo recombinant protein is able to form amyloid assemblies in vitro (Watts et al. 2010): this feature, however, hasn't been verified in vivo yet. Our laboratory has demonstrated that a perturbation of the lipid rafts induces PrPC misfolding (Sarnataro et al. 2004), suggesting that they have a protective role in pathological conversion of PrPC. Therefore, in the second part of my thesis we have characterized Shadoo subcellular localization and folding properties in neuronal cells because we reasoned that, if Shadoo possesses the natural tendency to convert to amyloid-like forms in vitro, it should also be able to acquire scrapie-like characteristics in live cells also. We have found that Shadoo, under native conditions, is partially misfolded in neuronal cells. Moreover, lipid rafts perturbation and proteasomal block increased misfolding of the protein.

Actions (login required)

View Item View Item