Abstract
The cellular prion protein (PrP(c)) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrP(c) yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrP(c) in cell survival.
Highlights
We recently established that PrPc overexpression led to an increased staurosporine-evoked cell death [13] in transfected or inducible cell lines and showed that this cellular response was associated with an activation of caspase-3 linked to a transcriptional and post-transcriptional control of the p53 transcription factor [14]
PrPc is mainly endoproteolyzed on the N-terminal side, at the 110/111 peptidyl bond, to produce a 17-kDa C-terminal fragment C1 tethered to the plasma membrane [17, 18]
Very few works have examined the putative functions of C1 and C2, and their potential as a regulator of cell death, a function harbored by the parent protein
Summary
The PRI-308 monoclonal antibody was raised against a synthetic peptide corresponding to the human 106 –126 sequence. SAF32 and SAF61 monoclonal antibodies are directed against PrP 79 –92 residues and 142–160, respectively. Anti-human p53 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti--tubulin monoclonal antibody and staurosporine were purchased from Sigma (St. Quentin-Fallavier, France). The 3F4-tagged PrPc coding vector (3F4MoPrPc) drives the expression of mouse PrPc in which 2 methionines in positions 109 and 112 of the human sequence replace lysine 108 and valine 111 in the mouse PrPc cDNA, thereby allowing its detection by human-specific PRI-308). To obtain C1 and C2 coding vectors, 24 – 89 and 24 –110 deletions were created in 3F4tagged PrPc cDNA using a QuikChangeTM site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) following the manufacturer’s specifications.
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