Abstract
Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPC and PrPSc are identical, but their conformations are rather different; PrPC rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPC and PrPSc. Here we discuss three issues; (1) Protein conformation modifying activity working on collectly-folded proteins such as PrPC, (2) Intracellular PrPC trafficking, and (3) Mitochondria-mediated apoptosis in transgenic mice overexpressing PrPC.
(1) To examine the protein unfolding activities against correctly-folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone, Unfoldin, from S.cerevisiae. The purified protein formed homo-oligomer consisting of 10-12 subunits arranged in a grapple-like structure ∼10 nm in diameter with a ∼2 nm opening. Unfoldin adopted an “open state” in the presence of ATP and a “closed state” in its absence. Only the “open state” captured F-actin and facilitated the formation of the protease-susceptible circular form of F-actin. This property was promoted by ATP binding but not its hydrolysis. Overexpression of Unfoldin induced multiple buds in yeast cells, and its depletion interfered with cleavage furrow formation during cytokinesis. Finally, the protease susceptibility of F-actin purified from the bud neck decreased in a strain lacking Unfoldin.
(2) By utilizing double-labeled fluorescent PrPC, we revealed that the NH2-terminal and COOH-terminal PrPC fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells and HpL3-4, a hippocampal cell line established from prnp gene-ablated mice. The NH2-terminal PrPC fragment, which predominantly localized in the intracellular compartments, congregated in the cytosol after the treatment with a microtubules depolymerizer (nocodazole). With a real-time imaging of fluorescent PrPC (GFP-PrPC) in living cells, GFP-PrPC exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140-180 nm/sec, and a retrograde movement inwardly at a speed of 1.0-1.2 μm/sec. The anterograde and retrograde movements of GFP-PrPC were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Furthermore, anti-Kinesin antibody (α-Kinesin) blocked its anterograde motility, whereas anti-Dynein antibody (α-Dynein) blocked its retrograde motility. These data suggested the kinesin family (KIF4)-driven anterograde and the dynein-driven retrograde movement of GFP-PrPC. Mapping of the interacting domains of PrPC identified amino acid residues indispensable for interactions with kinesin family (KIF4): NH2-terminal mouse residues 53-91 and dynein: NH2-terminal residues 23-33, respectively.
(3) Transgenic mice harboring a high-copy-number of wild-type mouse PrPC are known to develop a spontaneous neurological dysfonction in an age-dependent manner, even without inoculation of PrPSc. Here we demonstrate mitochondria-mediated apoptosis in aged transgenic mice overexpressing wild-type PrPC. These mice remained healthy, and no neuropathological evidence was observed in their brains when examined. The aged mice (Tg(MoPrP)4053/FVB), however, exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasome activity, while younger littermates did not. Such aberrant mitochondrial localization of PrPC was accompanied by cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal pyramidal cells. Simultaneously, co-immunoprecipitation of PrPC with anti-apoptotic protein, BCL-2, were observed in the mitochondria. When N2a culture cells expressing wild-type PrPC were used in combination with proteasome inhibitors as a cell culture model, and a novel mitochondria-mediated apoptotic pathway was identified; a novel 14-3-3 isotype transports PrPC to a mitochondrial outer membrane receptor; PrPc thereafter binds mitochondrial BCL-2 and subsequently induces mitochondria-mediated apoptosis. The apoptosis requires PrP residues 122-139, and the same system is involved in the apoptosis of N2a ceils expressing PrP residues 1-144, a heritable human prion disease model with Y145STOP.
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