Mouse prion protein (PrP) segment 100 to 104 regulates conversion of PrP(C) to PrP(Sc) in prion-infected neuroblastoma cells.

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).

Synthetic fibril peptide promotes clearance of scrapie prion protein by lysosomal degradation.

Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.

Interacting targets of the farnesyl of transducin gamma-subunit.

G protein gamma-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Talpha/Tbetagamma). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tbetagamma analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tgamma. POG-modified Tbetagamma was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tbetagamma. Photoaffinity labeling experiment with the photoreactive Tbetagamma analogue in its free form indicated that the POG moiety of Tgamma interacted with Tbeta. In the trimeric Talpha/Tbetagamma complex, the POG moiety was cross-linked with Talpha in addition to concurrent affinity labeling of Tbeta. When photoreactive Tbetagamma was reconstituted with Talpha and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Talpha and Tbeta but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tgamma plays an active role in protein-protein and protein-membrane interactions and suggest that the farnesyl-PE/PS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*.

Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.

BACKGROUND: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. RESULTS: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. CONCLUSION: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

Prevention of prion propagation by dehydrocholesterol reductase inhibitors in cultured cells and a therapeutic trial in mice.

In prion diseases, the normal cellular form of prion protein (PrP(C)) is converted into the disease-associated isoforms (PrP(Sc)) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrP(Sc) in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrP(Sc) from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 microM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.

Effects of beta-sheet breaker peptide polymers on scrapie-infected mouse neuroblastoma cells and their affinities to prion protein fragment PrP(81-145).

The effects of Soto's 'beta-sheet breaker peptide' and its polymer on PrPSc formation in ScN2a cells were investigated. Surface plasmon resonance study indicated that direct binding between PrP(81-145) and the 'beta-sheet breaker peptide' is not specific and may not play a major role in the inhibition of PrPSc formation.