contributed equally to this work Prions are composed of an isoform of a normal sialoglycoprotein called PrP c , whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP c -binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from twodimensional protein gels followed by sequencing the speci®c spot, we have now identi®ed the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a speci®c and high af®nity manner with a K d of 10 ±7 M. The interaction sites were mapped to amino acids 113±128 from PrP c and 230±245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP c binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP c interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

No abstract

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

Prion diseases are caused by the conformational transition of the native alpha-helical cellular prion protein (PrPC) into a beta-sheet pathogenic isoform. However, the normal physiological function of PrPC remains elusive. We report herein that copper induces PrPC expression in primary hippocampal and cortical neurons. PrPC induced by copper has a normal glycosylation pattern, is proteinase K-sensitive and reaches the cell surface attached by a glycosyl phosphatidylinositol anchor. Immunofluorescence analysis revealed that copper induces PrPC levels in the cell surface and in an intracellular compartment that we identified as the Golgi complex. In addition, copper induced the activity of a reporter vector driven by the rat PrPC gene (Prnp) promoter stably transfected into PC12 cells, whereas no effect was observed in glial C6 clones. Also cadmium, but not zinc or manganese, upregulated Prnp promoter activity in PC12 clones. Progressive deletions of the promoter revealed that the region essential for copper modulation contains a putative metal responsive element. Although electrophoretic mobility shift assay demonstrated nuclear protein binding to this element, supershift analysis showed that this is not a binding site for the metal responsive transcription factor-1 (MTF-1). The MTF-1-independent transcriptional activation of Prnp is supported by the lack of Prnp promoter activation by zinc. These findings demonstrate that Prnp expression is upregulated by copper in neuronal cells by an MTF-1-independent mechanism, and suggest a metal-specific modulation of Prnp in neurons.

Conversion of the normal cellular prion protein (PrPc), whose physiological function is still under investigation, to an infectious form called prion is the cause of some neurodegenerative diseases. Therefore, the elucidation of PrPc gene regulation is important both to define a strategy to control the infection and to better understand PrPc function. We cloned the rat PrPc gene promoter region into a luciferase reporter vector, transfected C6 and PC-12 cells, and isolated clones with stable enzyme expression. The dependence of chromatin conformation on PrPc promoter activity was evaluated using the histone deacetylase inhibitor, trichostatin A, which was able to highly increase not only promoter activity but also PrPc mRNA and protein levels. The phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and cAMP poorly induced promoter activity; retinoic acid decreased it by 50%, whereas nerve growth factor and dexamethasone had no effect. When 12-O-tetradecanoylphorbol-13-acetate or cAMP but not retinoic acid was associated with trichostatin A, a potentiation of the primary effects was observed. These new data indicate that PrPc gene regulation is highly dependent on disruption of chromatin fiber assembly, which allows some ubiquitous transcription factors accession to specific DNA elements.