Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

Abstract

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.

Keywords: CHIP; Stub1; olfactory epithelium; prion; protein interaction; yeast two-hybrid.

Figure 1.

PrP^C interacts with 10 different ligands in cross-mating assay. Bait strain expressing PrP^C and the Lac-Z reporter gene were mated with target strains expressing the putative PrP^C interactors. X-gal was used to score positive interactions (blue color development). pBait and pTarget were used as a positive control for interaction, since they express known interaction partners. The empty bait vector (pGilda), which expresses only Lex A domain, was used as negative control.

Figure 2.

Stub1 is expressed in both olfactory epithelium (OE) and brain. (a) Protein extracts prepared from mouse brain and OE were analyzed by western blotting with anti-Stub1 (upper panel) antibodies. Anti-β-actin was used as loading control (lower panel). (b) Protein relative values were quantified using ImageJ Software® and normalized to β-actin. The results are representative of 4 independent experiments. Statistical analysis was conducted with GraphPad Prism (values were expressed as mean ±SE. Student's t test was used for statistical analysis and P<0.05 was considered significant).

Figure 3.

PrP^C interacts with Stub1. (a) A pull-down assay was performed using mouse brain extract incubated with His₆-PrP^C bound to Ni-NTA-agarose beads (+) or Ni-NTA-agarose alone (−). Pulled-down proteins were analyzed by western blotting (WB) employing anti-Stub1 antibody (upper panel). The same membranes subjected to pull-down assay were re-probed with anti-his-tag antibody to attest that recombinant PrP^C was recovered from the beads (lower panel). HEK293T cells protein extracts overexpressing GFP-PrP^C and Myc-Stub1 (b) or olfactory epithelium (OE) extracts (c) were immunoprecipitated with anti-PrP^C or pre-immune serum (PI). Co-precipitated proteins were analyzed using an anti-Stub1 antibody (upper panels). The same membranes were re-probed with anti-PrP^C (lower panels) to confirm that PrP^C was precipitated during IP-reaction.