Cellular Prion Protein (PrPC): Identification and Characterization of Novel Interacting Partners

Abstract

The cellular prion protein (PrPC) is highly conserved throughout the evolution of mammals. (What does this sentence mean? What do you mean by ``conserved´´? This protein was higly conserved during the evolution of mammals [pantera et al. 2009; Jiayu et al.2009] and the gene tree deduced from the PrP sequences largely agrees with the species tree, indicating that no major deviations occurred in the evolution of the prion gene in different placental lineages [Teun van Rheede et al. 2003].) However, the cellular function of this ubiquitous protein is still not clear. The accumulation of misfolded and aggregated forms of PrPC (known as PrPSc) causes transmissible neurodegenerative diseases. Despite increasing knowledge concerning the PrPSc, very little is known about the physiological characteristics of PrPC and its interaction with other cellular proteins. The present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. Human PrPC was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrPC along with its interacting proteins were affinity purified using STrEP-Tactin chromatography, in-gel digested and then identified by Q-TOF MS/MS analysis. Forty three proteins appeared to interact with PrPC in this neuronal cell line. Of these, fifteen were already known for their interaction with PrPC or PrPSc, while twenty eight new proteins were identified. All 43 (known and new) proteins which we identified as interacting partners) were structural constituents of the cytoskeleton. Some are involved in cell growth, some in metabolism and some in energy pathways. In addition, proteins that are important for cell homeostasis, cell communication, signal transduction, stress response and protein folding were also among the newly identified interacting partners of PrPC. Interactions of two novel interacting partners of the GTPase family (Rab7a and Arf1) which have a suggested role in vesicle trafficking as well as the cytoskeleton associated protein alpha-tubulin 1 were further investigated using confocal laser scanning microscopy and reverse co-immunoprecipitation. Both reverse immunoprecipitation and immunofluorescence results confirmed potential interactions of Rab7a, Arf1 and alpha-tubulin 1 with the PrPC. We used siRNA against the Rab7a gene to decrease the expression of Rab7a protein ( knockdown ), in PrPC expressing HpL3-4 and SH-SHY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrPC in Rab9 positive endosomal compartments. The PrPC which accumulated within these Rab9 positive late endosomes remained sensitive to proteinase K digestion. Furthermore, Arf1 deactivation by brefeldin A treatment down regulated PrPC expression and redistributed PrPC into the cytosolic ,whereas nocodazole treatment increased PrPC expression and redistributed PrPC into the cytosol. The work described demonstrated for the first time that Rab7a and Arf1 may interact with PrPC and may possibly be involved in the cellular trafficking and distribution of PrPC into microtubules. These results highlight the pivotal involvement of endosomal compartments in the trafficking and regulation of PrPC.