Screening Of New Binding-partners Of Prp~c By Yeast Two-hybrid System

Prion-related diseases, often called transmissible spongiform encephalopathies (TSE), affect many mammalian hosts including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and Kuru in humans, scrapie in sheep, chronic wasting disease in deer and elk, bovine spongiform encephalopathy in cattle and transmissible mink encephalopathy in minks. Accumulation of an abnormally folded isoform of the cellular prion protein PrPC, denoted as PrPSc, which represents the major component of infectious agent in brains.PrPC is a ubiquitous glycoprotein predominantly anchored at the plasma membrane via a glycosylphosphatidylinositol (GPI) moiety. It is abundantly expressed in neurons of the central nervous system (CNS) and is evolutionarily highly conserved.It has been speculated that PrPc is a portion of a multi-protein complex that modulates various cellular functions, including cell survival,cell signaling and cell adhesion, depending on both compositions in the complex and cell types. Identification of ligands or partners physiologically interacting with PrP may provide new insights into its physiological and pathological functions. As abundantly expressed in neurons, figuring out the putative neuronal proteins interacted with PrP is always a top priority. Therefore, in the present stydy, an adult human cDNA library was screened for new potential partners interacted with PrP by a yeast two-hybrid system. A novel protein, HS-1 associated protein X-1 (HAX-1), was identified to be able to bind with PrP strongly. The main results of our research are as follows:1. The full-length wildtype human PrP was fused to GAL4BD as a bait to screen an adult human brain two-hybrid cDNA library. Sequencing alignments on Website of NCBI identified 15 different targeting proteins, among which 15 positive colonies contained the sequences encoding the human HS-1 associated protein X-1 (HAX-1). The molecular interaction between PrP and HAX-1 was further confirmed in vitro and in neuronal or non-neuronal cells.2. To investigate the possible region requiring for the interaction between PrP and HAX-1, nine different truncated PrP constructs and eight different HAX-1 deleted constructs were generated and their binding abilities with full-length HAX-1 or PrP were assayed in yeast two-hybrid system. The results showed that the region for binding to HAX-1 restricts at the segment of residues aa 91-163 within PrP, while the minimal binding region for PrP locates at the fragment of aa 38-129 within HAX-1.3. SHSY-5Y and HEK 293T cells were transfected with expressing plasmids pEGFP-N1-HAX-1 and/or pcDNA3.1-PrP-PG5, and were fixed and monitored under a confocal microscopy after stained with the appropriate antibodies. The results showed obvious co-localization of HAX-1 and PrP in cytoplasm of the cells co-epxressing theos two proteins.4. To assess the effects of expressing HAX-1 and PrP on cell viability, plasmids expressing full-length HAX-1 and PrP, alone or together, were transiently transfected into cell lines 293T and SHSY-5Y. CCK-8 and MTT assays revealed that co-expression of HAX-1 and PrP led the cells acquiring remarkable stronger ability to resist the challenge of H2O2. This phenomenon was further confirmed by Annexinâ…¤-FITC binding assays.5. To see the effect of co-expressions of HAX-1 and PrP mutants with four- and nine-extra octarepeats (PrP-PG9 and PrP-PG14) on cell viability,293T and SHSY-5Y cells were transiently transfected with various plasmids. The cytotoxicity activities of genetic CJD (gCJD) associated PrP mutants PG9 and PG14 were not reversed, but enhanced, by co-expression of HAX1.Taking together, a yeast two-hybrid screening within an adult human brain cDNA library for potential PrP binding molecules was performed. A novel protein HAX-1 was identified to be able to bind with PrP specifically. The interaction between the two proteins has been further verified by GST pull-down and immunoprecipitation assays. The minimal binding regions were mapped to the segments of residues aa 91-163 within PrPC and residues aa 38-129 within HAX-1. Immunofluorescent assays of co-expressions of human PrP and HAX-1 in 293T and SHSY-5Y cells revealed marked co-localizations of those two proteins in cytoplasm. Moreover, the co-expression of HAX-1 and wild-type PrP (PG5) was found to enhance the cellular resistance to the challenge of H2O2. Contrarily, co-transfection of HAX-1 did not reverse, but aggravated the cytotoxicities of the gCJD associated PrP mutants with nine-(PG9) and fourteen-octarepeats (PG14).