Biological characteristics of Chinese hamster ovary cells transfected with bovine Prnp

A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.

Envolvimento da proteína prion celular e seus ligantes nos mecanismos de plasticidade neuronal e neuroproteção / Involvement of cellular prion protein and its ligands in the mechanisms of neuronal plasticity and neuroprotection

Atualmente muitos grupos de pesquisa estão empenhados em desvendar a função da forma celular da proteína prion, PrPc. PrPc é uma isoforma normal da proteína prion infecciosa (PrPsc-proteína prion scrapie) relacionada às Encefalopatias Espongiformes Transmissíveis (TSEs), genericamente designadas por doenças priônicas. Uma das maneiras de esclarecer o papel de PrPc é investigar moléculas ligantes e associá-las a fenômenos biológicos. As funções propostas para PrPc vão desde atividade semelhante a superóxido dismutase, proteção contra estresse oxidativo, diferenciação neuronal à sinalização. Em 1997, descrevemos um receptor/ligante para PrPc utilizando o princípio da hidropaticidade complementar. No presente trabalho, apresentamos o isolamento e identificação deste ligante de PrPc como sendo STI1 (?Stress inducible protein 1?), uma co-chaperonina. In vitro, a STI1 interage com PrPc de maneira específica, saturável e com alta afinidade (Kd=10-7M). Ex vivo, a interação entre PrPc e STI1, possui efeito neuroprotetor ao ativar a via de PKA (Proteína quinase dependente de AMPc), além disso, promove crescimento neurítico através da via de MAPK (Proteína quinase ativada por mitógeno) em neurônios do sistema nervoso central. Paralelamente, mostramos que PrPc atua como um ligante de proteínas de matriz extracelular: vitronectina e laminina. A interação entre vitronectina e PrPc leva ao crescimento axonal de células do sistema nervoso periférico. Ao interagir com laminina, PrPc induz formação e manutenção de neuritos e se mostra importante para os mecanimos de consolidação da memória de curta e longa duração, sendo que este processo requer a ativação de vias clássicas de sinalização (PKA/MAPK). Assim, a caracterização das interações PrPc-STI1, PrPc-Vn e PrPc-Ln representa contribuições importantes para a elucidação do papel biológico de PrPc...(AU)

owadays, many research groups are interested in unraveling the role of the cellular prion protein, PrPc, which is the normal isoform of protein associated with the Transmissible Spongiform Encephalopaties (TSEs), generically designated prion diseases. Several biological roles for PrPc have been proposed. In 1997, our group described a PrPc receptor/ligand based on the complementary hydropathy theory. Herein, we showed the identification of the PrPc receptor/ligand as STI1, or Stress inducible protein 1. In vitro studies demonstrated that STI1 is a specific, saturable and high affinity ligand for PrPc (Kd=10-7M). Ex vivo, PrPc-STI1 interaction promoted neurite outgrowth through MAPK (Mitogen activated protein kinase) and showed neuroprotective effects by activating PKA (cAMP-dependent protein kinase) in central nervous system neurons. We also demonstrated that PrPc act as an extracellular matrix protein receptor for vitronectin (Vn) and laminin (Ln). The interaction between Vn and PrPc led to axonal outgrowth in peripheral nervous system. Upon its interaction with Ln, PrPc induced formation and maintenance of neurites and participated in short and long term memory consolidation mechanisms by activating classical signaling pathways (PKA/MAPK). Thus, the characterization of PrPc-STI1, PrPcVn and PrPc-Ln interactions represents important contributions for the elucidation of PrPc physiological roles (AU)

Biosynthesis of the prion proteins in scrapie-infected cells in culture

Prions are small proteinaceous particles that transmit scrapie and other fatal encephalopathies of humans and animals, and that appear to be devoided of nucleic acids. The only known-and perhaps the sole-component of the scrapie prion is an abnormal host-encoded protein, the scrapie prion protein PrPSc. The biosynthesis of this pathological protein in the host cell, which is thus of paramount importance to prion replication, is still poorly understood. We are studying the biosynthesis and degradation of the scrapie prion protein PrPSc and of its normal isoform PrPC in scrapie-infected rodent cells in culture. PrPC is anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI) moiety. In scrapie-infected mouse neuroblastoma N2a cells, PrPSc is formed post-translationally, probably from plasma membrane PrPC, in an unknown subcellular compartment that is readily accessible from the plasma membrane. Transport along the secretory pathway is necessary for PrPSc synthesis. In contrast to PrPC, PrPSc accumulates intracellularly, primarily in secondary lysosomes. The subcellular compartment(s) in which PrPSc is formed remain to be determined